CN118206651A - Antibodies targeting CXCL4 and their use in the diagnosis of depressive disorders - Google Patents
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Abstract
The present invention relates to an antibody targeting CXCL4, and the use of such an antibody for diagnosing depressive disorders. The invention also relates to a kit comprising such an antibody targeting CXCL4, as well as to methods and uses for diagnosing depressive disorders by means of such a kit. The antibody to CXCL4 of the present invention can detect CXCL4 protein in a sample with high specificity and sensitivity.
Description
The application is a divisional application of an application patent application of which the application date is 2022, 12 and 15, the application number is 202211617708.2, and the application name is CXCL4 targeting antibody and application of the antibody in diagnosis of depressive disorder.
Technical Field
The invention relates to diagnosis of depressive disorders in accurate mental disease research, in particular to diagnosis of depressive disorders. In particular, the invention relates to an antibody targeting CXCL4, and the use of such an antibody for diagnosing depressive disorders.
Background
Mental disease (MENTAL ILLNESS), also known as mental disorder, is a generic term with respect to all pathological mental activities, which is characterized by a syndrome characterized by clinically significant cognitive, affective regulation or behavioral disorders in an individual. Common mental disorders include a variety of conditions such as schizophrenia, mood disorders, anxiety disorders, depression disorders, and the like.
Currently, classification criteria for mental diseases include the american society of mental medicine based DSM system, the world health organization based ICD system, the chinese based CCMD system, etc. However, these systems are based on clinical symptom assessment, and lack objective biological diagnostic indicators, i.e. subjects with a set of similar symptoms are classified as the same disease category, and the set of similar symptoms may be caused by completely different biological/pathological factors. In contrast, subjects with different symptoms, although classified as different diseases according to clinical symptom assessment, may be caused by whether the symptoms are the same biological/pathological factors. This presents difficulties in the accurate treatment of mental disorders.
In view of its multiplicity and breadth, it is desirable to find and establish a defined biomarker or a set of defined biomarkers for depressive disorders in mental diseases and to develop a method for detecting such biomarkers that is fast, accurate and economically suitable for clinical application, thereby enabling objective diagnosis of depressive disorders, which is of great importance for molecular level diagnosis of clinically depressive disorders.
Disclosure of Invention
The present invention is based on mass spectrometry based deep proteomic analysis to find biomarkers for diagnosing depressive disorders. Through experiments, CXCL4 protein is identified to distinguish patients with depressive disorder from healthy people, and has the potential of assisting and distinguishing diagnosis of depressive disorder.
On the basis, the invention further develops a specific antibody aiming at CXCL4 protein, and the expression level of the CXCL4 protein in a subject sample is detected by the antibody, so as to assist and differential diagnosis of depressive disorder, and particularly to more accurately diagnose the depressive disorder subject which is difficult to diagnose by the current mental disorder diagnosis and classification system.
In one aspect, the invention relates to an isolated antibody or antigen-binding fragment portion thereof that specifically binds to a CXCL4 protein, wherein the isolated antibody or antigen-binding fragment portion thereof comprises a heavy chain variable region comprising HCDR1, HCDR2, HCDR3 sequences; and a light chain variable region comprising LCDR1, LCDR2, LCDR3 sequences wherein the antibody or antigen binding fragment portion thereof has a heavy chain variable region and a light chain variable region selected from at least one of the group consisting of:
1) The heavy chain variable region comprises (a) SEQ ID NO:68, (b) HCDR1 of SEQ ID NO:69, and (c) SEQ ID NO:70, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:24, (e) LCDR1 of SEQ ID NO:25, and (f) SEQ ID NO: LCDR3 of 26;
2) The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30;
3) The heavy chain variable region comprises (a) SEQ ID NO:76, (b) HCDR1 of SEQ ID NO:77, and (c) HCDR2 of SEQ ID NO:78, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:32, (e) LCDR1 of SEQ ID NO:33, and (f) SEQ ID NO:34 LCDR3;
4) The heavy chain variable region comprises (a) SEQ ID NO:80, (b) HCDR1 of SEQ ID NO:81, and (c) HCDR2 of SEQ ID NO:82, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:36, (e) LCDR1 of SEQ ID NO:37, and (f) LCDR2 of SEQ ID NO: LCDR3 of 38;
5) The heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42;
6) The heavy chain variable region comprises (a) SEQ ID NO:88, (b) HCDR1 of SEQ ID NO:89, and (c) HCDR2 of SEQ ID NO:90, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:44, (e) LCDR1 of SEQ ID NO:45, and (f) LCDR2 of SEQ ID NO:46 LCDR3;
7) The heavy chain variable region comprises (a) SEQ ID NO:92, (b) HCDR1 of SEQ ID NO:93, and (c) HCDR2 of SEQ ID NO:94, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:48, (e) LCDR1 of SEQ ID NO:49, and (f) LCDR2 of SEQ ID NO:50 LCDR3;
8) The heavy chain variable region comprises (a) SEQ ID NO:96 HCDR1, (b) SEQ ID NO:97, and (c) HCDR2 of SEQ ID NO:98, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:52, (e) LCDR1 of SEQ ID NO:53, and (f) SEQ ID NO:54 LCDR3;
9) The heavy chain variable region comprises (a) SEQ ID NO:100, (b) HCDR1 of SEQ ID NO:101, and (c) HCDR2 of SEQ ID NO:102, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:56, (e) LCDR1 of SEQ ID NO:57, and (f) SEQ ID NO: LCDR3 of 58;
10 The heavy chain variable region comprises (a) SEQ ID NO:104, (b) HCDR1 of SEQ ID NO:105, and (c) HCDR2 of SEQ ID NO:106, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:60, (e) LCDR1 of SEQ ID NO:61, and (f) SEQ ID NO: LCDR3 of 62; and
11 The heavy chain variable region comprises (a) SEQ ID NO:108, (b) HCDR1 of SEQ ID NO:109, and (c) HCDR2 of SEQ ID NO:110, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:64, (e) LCDR1 of SEQ ID NO:65, and (f) SEQ ID NO: LCDR3 of 66.
In one aspect, the invention relates to an isolated antibody or antigen-binding fragment portion thereof that specifically binds to a CXCL4 protein, wherein the isolated antibody or antigen-binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
1) The heavy chain comprises a heavy chain consisting of SEQ ID NO:140, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:140, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:140, and the amino acid sequence encoded by the nucleotide sequence of 140, and
The light chain comprises a sequence consisting of SEQ ID NO:139 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:139, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:139, and a nucleotide sequence encoded by the nucleotide sequence of 139;
2) The heavy chain comprises a heavy chain consisting of SEQ ID NO:142, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:142, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:142, and the amino acid sequence encoded by the nucleotide sequence of 142, and
The light chain comprises a sequence consisting of SEQ ID NO:141, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:141, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:141, and a nucleotide sequence encoding the same;
3) The heavy chain comprises a heavy chain consisting of SEQ ID NO:144, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:144, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:144, and the amino acid sequence encoded by the nucleotide sequence of 144, and
The light chain comprises a sequence consisting of SEQ ID NO:143 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:143, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:143, a nucleotide sequence encoding an amino acid sequence;
4) The heavy chain comprises a heavy chain consisting of SEQ ID NO:146 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:146, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:146, and the amino acid sequence encoded by the nucleotide sequence of 146, and
The light chain comprises a sequence consisting of SEQ ID NO:145 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:145, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:145, and an amino acid sequence encoded by the nucleotide sequence;
5) The heavy chain comprises a heavy chain consisting of SEQ ID NO:148, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:148, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:148, and an amino acid sequence encoded by the nucleotide sequence of 148, and
The light chain comprises a sequence consisting of SEQ ID NO:147 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:147, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:147, and the amino acid sequence encoded by the nucleotide sequence of 147;
6) The heavy chain comprises a heavy chain consisting of SEQ ID NO:150 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:150, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:150, and the amino acid sequence encoded by the nucleotide sequence of 150, and
The light chain comprises a sequence consisting of SEQ ID NO:149 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:149, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:149, and a nucleotide sequence encoding the same;
7) The heavy chain comprises a heavy chain consisting of SEQ ID NO:152, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:152, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:152, and an amino acid sequence encoded by the nucleotide sequence of 152, and
The light chain comprises a sequence consisting of SEQ ID NO:151 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:151, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:151, and a nucleotide sequence encoding the amino acid sequence;
8) The heavy chain comprises a heavy chain consisting of SEQ ID NO:154 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:154, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:154, and an amino acid sequence encoded by a nucleotide sequence of seq id no
The light chain comprises a sequence consisting of SEQ ID NO:153 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:153, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:153, and an amino acid sequence encoded by the nucleotide sequence of 153;
9) The heavy chain comprises a heavy chain consisting of SEQ ID NO:156, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:156, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:156, and an amino acid sequence encoded by a nucleotide sequence of 156, and
The light chain comprises a sequence consisting of SEQ ID NO:155, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:155, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:155, and an amino acid sequence encoded by the nucleotide sequence of 155;
10 The heavy chain comprises a sequence consisting of SEQ ID NO:158 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:158, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:158, and the amino acid sequence encoded by the nucleotide sequence of 158, and
The light chain comprises a sequence consisting of SEQ ID NO:157 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:157, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:157, and a nucleotide sequence encoding the same;
11 The heavy chain comprises a sequence consisting of SEQ ID NO:160 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:160, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:160, and the amino acid sequence encoded by the nucleotide sequence of 160, and
The light chain comprises a sequence consisting of SEQ ID NO:159 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:159, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:159, and a nucleotide sequence encoding the amino acid sequence of 159.
In one aspect, the invention relates to an antibody composition directed against CXCL4 protein comprising at least two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to CXCL4 protein,
Wherein the at least one isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
Wherein the at least one further isolated antibody or antigen binding fragment portion thereof is selected from one of the following 5 antibodies:
(1) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:68, (b) HCDR1 of SEQ ID NO:69, and (c) SEQ ID NO:70, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:24, (e) LCDR1 of SEQ ID NO:25, and (f) SEQ ID NO: LCDR3 of 26; or alternatively
The heavy chain variable region comprising SEQ ID NO:67 or an amino acid sequence identical to SEQ ID NO:67, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23 or amino acid sequence corresponding to SEQ ID NO:23, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain consisting of SEQ ID NO:122, and the light chain consisting of the amino acid sequence of SEQ ID NO:111, an amino acid sequence of seq id no;
(2) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42; or alternatively
The heavy chain variable region comprising SEQ ID NO:83 or amino acid sequence identical to SEQ ID NO:83, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence identical to SEQ ID NO:39, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO:115, and a sequence of amino acids;
(3) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:88, (b) HCDR1 of SEQ ID NO:89, and (c) HCDR2 of SEQ ID NO:90, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:44, (e) LCDR1 of SEQ ID NO:45, and (f) LCDR2 of SEQ ID NO:46 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:87 or amino acid sequence corresponding to SEQ ID NO:87, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:43 or amino acid sequence corresponding to SEQ ID NO:43, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:127, and the light chain consisting of the amino acid sequence of SEQ ID NO:116, an amino acid sequence of seq id no;
(4) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:92, (b) HCDR1 of SEQ ID NO:93, and (c) HCDR2 of SEQ ID NO:94, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:48, (e) LCDR1 of SEQ ID NO:49, and (f) LCDR2 of SEQ ID NO:50 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:91 or an amino acid sequence identical to SEQ ID NO:91, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:47 or amino acid sequence corresponding to SEQ ID NO:47, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:128, and the light chain consisting of the amino acid sequence of SEQ ID NO: 117; and
(5) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:108, (b) HCDR1 of SEQ ID NO:109, and (c) HCDR2 of SEQ ID NO:110, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:64, (e) LCDR1 of SEQ ID NO:65, and (f) SEQ ID NO:66 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:107 or amino acid sequence identical to SEQ ID NO:107, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:63 or amino acid sequence identical to SEQ ID NO:63, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:132, and the light chain consisting of the amino acid sequence of SEQ ID NO:121, and a fragment thereof.
In one aspect, the invention relates to an isolated nucleic acid molecule encoding an antibody or antigen binding fragment portion thereof of any one of the invention; or alternatively
The nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following combinations: SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22.
In one aspect, the invention relates to an expression vector comprising a nucleic acid molecule of the invention.
In one aspect, the invention relates to a host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention.
In one aspect, the invention relates to a hybridoma cell expressing an antibody or antigen-binding fragment portion thereof of the invention.
In one aspect, the invention relates to a method of producing an antibody or antigen-binding fragment portion thereof that targets human tumor cells that express CXCL4, comprising:
(i) Expression of a nucleic acid molecule according to the invention, and
(Ii) Isolating and purifying the portion of the antibody or antigen binding fragment thereof expressed by the nucleic acid molecule.
In one aspect, the invention relates to a kit, test strip or test strip for diagnosing a depressive disorder, comprising an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, wherein the subject is suspected to have a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, the antibody is selected from the antibody or antigen-binding fragment portion thereof of any of the invention, or the antibody is an antibody composition for a CXCL4 protein of the invention, the kit, test strip or test strip further comprising instructions or inserts indicating that up-regulation of the expression level of a CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder.
In one aspect, the invention relates to a kit, test strip or test paper card for diagnosing a depressive disorder, wherein the kit, test strip or test paper card comprises a portion for receiving a sample from a subject; and a moiety coated with an antibody capable of specifically binding to a CXCL4 protein, said antibody being selected from the group consisting of an antibody or antigen-binding fragment portion thereof of any one of the present invention, or said antibody being an antibody composition against a CXCL4 protein of the present invention, wherein said subject is suspected to have a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, and an up-regulation of the expression level of the CXCL4 protein compared to a reference value indicates that said subject has a depressive disorder.
In one aspect, the invention relates to a kit, strip or card for detecting the expression level of a CXCL4 protein, comprising an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, said antibody being selected from the group consisting of an antibody or antigen-binding fragment portion thereof of any one of the invention, or said antibody being an antibody composition directed against a CXCL4 protein of the invention, wherein said subject is suspected of having a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, said kit, strip or card further comprising instructions or inserts indicating that up-regulation of the expression level of a CXCL4 protein compared to a reference value indicates that said subject has a depressive disorder.
In one aspect, the invention relates to a method for diagnosing a depressive disorder, comprising determining the expression level of a CXCL4 protein in a sample from a subject and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the antibody or antigen-binding fragment portion thereof of any one of the invention, or the antibody is an antibody composition of the invention against a CXCL4 protein, wherein the subject is suspected of having a psychotic disorder but is difficult to determine as a depressive disorder, wherein an elevated expression level of the CXCL4 protein compared to the reference value is indicative of the subject suffering from a depressive disorder.
In one aspect, the invention relates to a method for detecting the expression level of a biomarker in vitro comprising: (a) Collecting a sample from a subject, wherein the subject is suspected of having a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, (b) determining the expression level of a CXCL4 protein in the sample and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the antibody or antigen-binding fragment portion thereof of any one of the invention, or the antibody is an antibody composition of the invention against a CXCL4 protein, wherein an elevated expression level of the CXCL4 protein compared to the reference value is indicative of the subject having a depressive disorder.
In one aspect, the invention relates to a method for determining the likelihood of a subject suffering from a depressive disorder, wherein the subject is suspected of suffering from a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, wherein the method comprises determining the expression level of a CXCL4 protein in a sample from the subject and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the antibody or antigen-binding fragment portion thereof of any of the invention, or the antibody is an antibody composition of the invention against a CXCL4 protein, wherein an elevated expression level of the CXCL4 protein compared to the reference value is indicative of a high likelihood of the subject suffering from a depressive disorder.
In one aspect, the invention relates to the use of an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, wherein the subject is suspected to have a psychotic disorder but is difficult to determine as a depressive disorder, wherein the antibody is selected from the antibody or antigen-binding fragment portion thereof of any of the invention, or the antibody is an antibody composition of the invention against a CXCL4 protein, wherein an up-regulation of the expression level of the CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder, in the preparation of a test strip, test paper card and/or kit for diagnosing a depressive disorder.
In one aspect, the invention relates to an immunoassay composition comprising a CXCL4 protein in a sample from a subject and an effective amount of an antibody (preferably a binding partner) for detecting the expression level of the CXCL4 protein, and/or comprising a conjugate formed from a CXCL4 protein in a sample from a subject and an effective amount of an antibody (preferably a binding partner) for detecting the expression level of the CXCL4 protein, wherein the subject is suspected of having a psychotic disorder but is difficult to determine as a depressive disorder, wherein the antibody is selected from the antibody or antigen-binding fragment portion thereof of any of the invention, or the antibody is an antibody composition for a CXCL4 protein of the invention, wherein an elevated expression level of the CXCL4 protein in the immunoassay composition compared to a reference value is detected to indicate that the subject has a depressive disorder.
In one aspect, the invention relates to a container comprising an immunoassay composition comprising a CXCL4 protein in a sample from a subject and an effective amount of an antibody (preferably a binding partner) for detecting the expression level of the CXCL4 protein, and/or comprising a conjugate formed by a CXCL4 protein in a sample from a subject and an effective amount of an antibody (preferably a binding partner) for detecting the expression level of the CXCL4 protein, wherein the subject is suspected of having a psychotic disorder but is difficult to determine as a depressive disorder, wherein the antibody is selected from the antibody or antigen-binding fragment portion thereof of any one of the invention, or the antibody is an antibody composition for a CXCL4 protein of the invention, wherein detection of an elevated expression level of the CXCL4 protein in the immunoassay composition in the container compared to a reference value indicates that the subject has a depressive disorder.
The antibody disclosed by the invention has excellent binding property to CXCL4 protein, can be used for detecting the expression level of CXCL4 protein, can realize high-specificity diagnosis of depressive disorder by combining the antibody and a chemiluminescent detection method, and provides objective basis for diagnosis of depressive disorder.
Drawings
FIG. 1-partial least squares discriminant analysis of mass spectral data;
FIG. 2-CXCL4 expression levels differ in depressed MDD and healthy HC groups.
DESCRIPTION OF THE SEQUENCES
The leftmost column in Table 1 below is the sequence number (SEQ ID NO:) in the present invention.
TABLE 1a nucleic acid coding sequences for the light and heavy chains of the antibodies of the invention (SEQ ID NO:)
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TABLE 1b amino acid sequences of the variable regions, the respective CDRs, the signal peptide, and the constant region of the light chain and heavy chain of the antibody of the present invention (SEQ ID NO:)
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Note that: the sequences marked in bold and underlined in the amino acid sequences in the tables are the corresponding CDR sequences
TABLE 1c amino acid sequences of the light and heavy chains of the antibodies of the invention
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Note that: the sequences marked in bold and underlined in the amino acid sequences in the tables are the corresponding CDR sequences
Detailed Description
CXCL4 protein biomarker
Herein, the term "biomarker", also referred to as "biomarker", refers to a measurable indicator reflecting the biological status of a subject. The biomarker may be any substance (e.g., a gene or protein) in the subject, so long as they are associated with a particular biological state (e.g., disease) of the subject being examined. The biomarker may be one marker or a set of two or more biomarkers. The biomarker in the present invention refers to a protein.
In this context, the names of biomarkers for diagnosing depressive disorders refer to the definitions in the UniProt protein database. In one embodiment, the following proteins are all referred to as proteins in the species homosapiens (Human).
The term "CXCL4" refers to the chemokine (C-X-C motif) ligand 4 protein, which is also known as platelet factor 4 (PF 4). CXCL4 is numbered P02776 in the UniProt protein database, see in particular https:// beta. UniProt. Org/uniprotkb/P02776/entry. CXCL4 and its full, acronyms and abbreviations are interchangeable herein.
In one embodiment, the CXCL4 protein is used as a biomarker for diagnosing depressive disorders.
(II) depressive disorder
In this context, the diagnostic criteria for the mentioned diseases are based on the manual for diagnosis and statistics of mental diseases by the american society of psychiatric, 4 th edition (abbreviated as "DSM-IV"), and specific diagnostic criteria can be seen at "DSM-IV-TR Guidebook",the essential companion of the diagnostic and statistical manual of mental disorders,fourth edition text revision,2004. according to the diagnostic criteria for DSM-IV, depressive disorders are assigned to mood disorders (mood disorders) of mental diseases. Mood disorders are manifested clinically as mood episodes (mood episodes), including major depressive episodes (major depressive episode), manic episodes (manic episode) and mixed manic episodes (mixed episode). Although clinical symptoms may be diagnostic members of a specific typing/subtype of mood disorders, they cannot be used independently for distinguishing/diagnosing subtypes of mood disorders or for further typing of mood disorders. According to the diagnostic criteria of DSM-IV, depressive disorders (major depression disorder or major depressive disorder, MDD, or major depression, MD) belong to one of the clinical subtypes of mood disorders. In the present invention, the terms "depressive disorder", "MDD" and "MD" are also used interchangeably.
It is noted that there are also diagnostic criteria for non-uniformity of depressive disorders. For example, DSM-IV classifies depressive disorders as clinical subtypes of mood disorders, but DSM-5 published 2013 has separated depressive disorders from mood disorders and has been a major class of diseases in parallel with schizophrenia, depressive disorders, etc. as mental diseases. As another example, international statistical Classification of disease and related health problems version 10 (ICD-10) classifies depressive disorders as clinical subtypes of mood disorders, and then ICD-11 published 2018 does not change this classification criteria, still classifying depressive disorders as clinical subtypes of mood disorders. In the present invention, the classification and diagnosis of depressive disorders mentioned are based on DSM-IV. In one embodiment, the subject of the invention has a clinical typing of depressive disorder according to the DSM-IV diagnostic criteria.
Herein, the term "differentially expressed proteins" (DIFFERNEITALLY EXPRESSSED PROTEINS, DEPs) refers to fold changes in protein expression of >2 or <0.5 after analysis of plasma samples, p <0.05 in a two-tailed t-test. For example, if the change in protein expression in a depressive disorder subject is compared to the change in protein expression in a healthy control, a protein with fold change >2 or <0.5 (p <0.05 in a two-tailed t-test; normalized to data) in a depressive disorder subject is considered a differentially expressed protein. In one embodiment, the invention identifies depressive disorders by detecting expression of CXCL4 protein by an antibody.
Herein, the terms "diagnosis (diagnose)", "diagnostic (diagnosed)", or "diagnostic (diagnosing)" refer to a method of identifying and/or determining whether a subject has a particular disease in detecting and/or identifying a case status of the subject.
The term "diagnosing a depressive disorder" refers to the diagnosis of a depressive disorder that can be made by detection of a biomarker found in the present invention, particularly for diagnosing a subject suspected of having a psychotic disorder in clinical manifestations but having difficulty in determining that the psychotic disorder is a depressive disorder. The term "diagnosing depressive disorder" also refers to making further diagnosis at the molecular level for a depressive disorder subject that has been initially diagnosed by advanced diagnostic criteria. The term "diagnosing depressive disorder" also refers to diagnosing a large random population, distinguishing depressive disorder subjects from healthy subjects.
In this context, diagnosis of a depressive disorder is made by comparing the expression level of a biomarker of the invention to a reference value, an increase in the expression level being indicative of the subject suffering from a depressive disorder. In one embodiment, the reference value is the expression level of the biomarker in a sample from a healthy subject or from a standard sample.
The term "sample" as used herein refers to a sample of tissue or body fluid isolated from a subject, including but not limited to, for example, whole blood or any blood fraction, plasma, serum, urine, cerebrospinal fluid, blood derivatives, blood cells, lymph. In particular, the sample may be in the form of a blood sample, a plasma sample or a serum sample obtained from a subject of the general population, a subject suspected of suffering from or suffering from a mental disorder or a depressive disorder.
Herein, the term "agent capable of detecting the expression level of a biomarker" refers to a substance or means/method that can be used to determine whether the expression level of a biomarker in a sample obtained from a subject is elevated, unchanged or reduced. Herein, the agent capable of detecting the expression level of the biomarker may be a binding partner (binding partner) capable of targeting and binding to the biomarker. For example, the binding partner may be an antibody or antigen binding fragment thereof capable of targeting and binding to a biomarker. The expression level of the biomarker is detected by a binding partner that targets the biomarker. For example, the expression level of a biomarker is detected by an antibody that is capable of specifically binding to the biomarker. For another example, the expression level of the biomarker is determined by mass spectrometry, or antibody-based immunoassay methods, such as competitive immunoassay, non-competitive immunoassay, enzyme-linked immunosorbent assay (ELISA), immunohistochemical assay, chemiluminescent assay, western blot, dot blot assay, and the like. In one embodiment, an antibody directed against a CXCL4 protein is used as a reagent to detect the expression level of a CXCL4 protein.
Herein, the term "subject" refers to a mammal, such as a human. In one embodiment, the subject is a subject, such as a human, suspected of having a psychotic disorder but difficult to determine that the psychotic disorder is a depressive disorder. In one embodiment, the subject is a depressive disorder that has been initially diagnosed by a prior diagnostic criteria, such as a human, in need of further diagnosis at the molecular level. In one embodiment, the subject is a randomized population of subjects, a population of which may include people with depressive disorders.
(III) antibodies to CXCL4
A. General definition
The term "antibody" refers to an immunoglobulin molecule that comprises at least one antigen recognition site and is capable of specifically binding an antigen. For example, the antibody may be a monoclonal antibody, a polyclonal antibody, an antibody fragment, a humanized antibody, a camelid antibody, a chimeric antibody, or the like. The antibodies may be further modified to bear a detectable label, for example, a label that can be detected by chemiluminescent means.
An "antibody fragment" or "antigen binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full length, but that comprises at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody binding sites) of the antibody that binds an antigen, and thus retains binding specificity as well as at least a portion of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as an antibody from which the antibody fragment was derived. Examples of antibody fragments include, but are not limited to, fab ', F (ab ') 2, single chain Fv (scFv), fv, dsFv, diabodies, fd and Fd ' fragments, and other fragments, including modified fragments (see, e.g.) ,Methods in Molecular Biology,Vol207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003);Chapter 1;p 3-25,Kipriyanov).
"Hypervariable region," "HV," "complementarity determining region," and "CDR" and "antibody CDR" are used interchangeably to refer to one of a plurality of portions within each variable region that together form an antigen binding site of an antibody. Each variable region domain contains 3 CDRs, designated CDR1, CDR2, and CDR3. For example, the light chain variable region domain comprises 3 CDRs, designated VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable region domain comprises 3 CDRs, designated VH CDR1, VH CDR2 and VH CDR3. CDRs are known to those skilled in the art and can be identified based on Kabat or Chothia numbering (see, e.g., ,Kabat,E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242, and Chothia, C.et al (1987) J.mol.biol.196:901-917). In the present invention, the abbreviations "VH CDR" and "HCDR" and "VL CDR" and "LCDR" have the same meaning.
A "constant region" domain is a domain in an antibody heavy or light chain that comprises an amino acid sequence that is relatively more conserved than the amino acid sequence of a variable region domain. In conventional full length antibody molecules, each light chain has a single light chain constant region (C L) domain, while each heavy chain comprises one or more heavy chain constant region (C H) domains, including C H1、CH2、CH and C H 4.
An "isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody (1) is not associated with components that accompany it in its natural state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system that differs from the naturally derived cell of the polypeptide will be "isolated" from its naturally associated components. The protein may also be isolated such that it is substantially free of naturally associated components, i.e., using protein purification techniques well known in the art.
An "isolated nucleic acid molecule" is a nucleic acid molecule that is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen binding fragments.
Sequence "identity" has art-recognized meanings and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity may be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (see, e.g., ：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987;and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991). although there are many methods of measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (carrello, H. & Lipman, d., SIAM J APPLIED MATH 48:1073 (1988)).
B. Antibodies to CXCL4
The present invention finds that CXCL4 protein is a protein that is differentially expressed in blood from healthy subjects and subjects with depressive disorder, and thus can be used as a biomarker for diagnosis of depressive disorder. On this basis, the present invention developed a series of antibodies with excellent binding ability to CXCL4 protein. By utilizing the excellent binding capacity of the antibody, CXCL4 protein in a sample of a subject can be effectively detected, and the detection has excellent sensitivity and specificity. In addition, the method for detecting the antibody is fast in speed and high in accuracy, and is suitable for the current clinical biochemical detection analysis instrument or digital detection platform, so that the method has good clinical application prospect. In addition, these highly specific antibodies of the invention are also useful for the study of CXCL4 in the biological arts, and can be used as detection or analysis reagents in the biological study of CXCL 4.
Accordingly, in a first aspect, the present invention provides an isolated antibody or antigen-binding fragment portion thereof that specifically binds to a CXCL4 protein, wherein said isolated antibody or antigen-binding fragment portion thereof comprises a heavy chain variable region comprising HCDR1, HCDR2, HCDR3 sequences; and a light chain variable region comprising LCDR1, LCDR2, LCDR3 sequences wherein the antibody or antigen binding fragment portion thereof has a heavy chain variable region and a light chain variable region selected from at least one of the group consisting of:
1) The heavy chain variable region comprises (a) SEQ ID NO:68, (b) HCDR1 of SEQ ID NO:69, and (c) SEQ ID NO:70, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:24, (e) LCDR1 of SEQ ID NO:25, and (f) SEQ ID NO: LCDR3 of 26;
2) The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30;
3) The heavy chain variable region comprises (a) SEQ ID NO:76, (b) HCDR1 of SEQ ID NO:77, and (c) HCDR2 of SEQ ID NO:78, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:32, (e) LCDR1 of SEQ ID NO:33, and (f) SEQ ID NO:34 LCDR3;
4) The heavy chain variable region comprises (a) SEQ ID NO:80, (b) HCDR1 of SEQ ID NO:81, and (c) HCDR2 of SEQ ID NO:82, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:36, (e) LCDR1 of SEQ ID NO:37, and (f) LCDR2 of SEQ ID NO: LCDR3 of 38;
5) The heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42;
6) The heavy chain variable region comprises (a) SEQ ID NO:88, (b) HCDR1 of SEQ ID NO:89, and (c) HCDR2 of SEQ ID NO:90, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:44, (e) LCDR1 of SEQ ID NO:45, and (f) LCDR2 of SEQ ID NO:46 LCDR3;
7) The heavy chain variable region comprises (a) SEQ ID NO:92, (b) HCDR1 of SEQ ID NO:93, and (c) HCDR2 of SEQ ID NO:94, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:48, (e) LCDR1 of SEQ ID NO:49, and (f) LCDR2 of SEQ ID NO:50 LCDR3;
8) The heavy chain variable region comprises (a) SEQ ID NO:96 HCDR1, (b) SEQ ID NO:97, and (c) HCDR2 of SEQ ID NO:98, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:52, (e) LCDR1 of SEQ ID NO:53, and (f) SEQ ID NO:54 LCDR3;
9) The heavy chain variable region comprises (a) SEQ ID NO:100, (b) HCDR1 of SEQ ID NO:101, and (c) HCDR2 of SEQ ID NO:102, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:56, (e) LCDR1 of SEQ ID NO:57, and (f) SEQ ID NO: LCDR3 of 58;
10 The heavy chain variable region comprises (a) SEQ ID NO:104, (b) HCDR1 of SEQ ID NO:105, and (c) HCDR2 of SEQ ID NO:106, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:60, (e) LCDR1 of SEQ ID NO:61, and (f) SEQ ID NO: LCDR3 of 62; and
11 The heavy chain variable region comprises (a) SEQ ID NO:108, (b) HCDR1 of SEQ ID NO:109, and (c) HCDR2 of SEQ ID NO:110, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:64, (e) LCDR1 of SEQ ID NO:65, and (f) SEQ ID NO: LCDR3 of 66.
In some embodiments, the isolated antibody or antigen binding fragment portion thereof comprises a light chain variable region having a heavy chain variable region selected from at least one of the group consisting of:
1) The heavy chain variable region comprising SEQ ID NO:67 or an amino acid sequence identical to SEQ ID NO:67, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23 or amino acid sequence corresponding to SEQ ID NO:23, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical;
2) The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical;
3) The heavy chain variable region comprising SEQ ID NO:75 or amino acid sequence corresponding to SEQ ID NO:75, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:31 or amino acid sequence corresponding to SEQ ID NO:31, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
4) The heavy chain variable region comprising SEQ ID NO:79 or amino acid sequence identical to SEQ ID NO:79, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:35 or an amino acid sequence identical to SEQ ID NO:35, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
5) The heavy chain variable region comprising SEQ ID NO:83 or amino acid sequence identical to SEQ ID NO:83, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence identical to SEQ ID NO:39, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
6) The heavy chain variable region comprising SEQ ID NO:87 or amino acid sequence corresponding to SEQ ID NO:87, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:43 or amino acid sequence corresponding to SEQ ID NO:43, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
7) The heavy chain variable region comprising SEQ ID NO:91 or an amino acid sequence identical to SEQ ID NO:91, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:47 or amino acid sequence corresponding to SEQ ID NO:47, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
8) The heavy chain variable region comprising SEQ ID NO:95 or an amino acid sequence identical to SEQ ID NO:95, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:51 or amino acid sequence corresponding to SEQ ID NO:51, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
9) The heavy chain variable region comprising SEQ ID NO:99 or amino acid sequence corresponding to SEQ ID NO:99, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:55 or amino acid sequence identical to SEQ ID NO:55, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
10 A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:103 or amino acid sequence identical to SEQ ID NO:103, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:59 or amino acid sequence identical to SEQ ID NO:59, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; and
11 A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:107 or amino acid sequence identical to SEQ ID NO:107, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:63 or amino acid sequence identical to SEQ ID NO:63, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
In some embodiments, the isolated antibody or antigen binding fragment portion thereof comprises a light chain variable region having a heavy chain variable region selected from at least one of the group consisting of:
1) The heavy chain variable region consisting of SEQ ID NO:67, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:23, and a sequence of amino acids;
2) The heavy chain variable region consisting of SEQ ID NO:71, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:27, an amino acid sequence of seq id no;
3) The heavy chain variable region consisting of SEQ ID NO:75, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:31, an amino acid sequence of seq id no;
4) The heavy chain variable region consisting of SEQ ID NO:79, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:35, and a sequence of amino acids;
5) The heavy chain variable region consisting of SEQ ID NO:83, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:39, and a sequence of amino acids;
6) The heavy chain variable region consisting of SEQ ID NO:87, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:43, an amino acid sequence of seq id no;
7) The heavy chain variable region consisting of SEQ ID NO:91, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:47, an amino acid sequence of seq id no;
8) The heavy chain variable region consisting of SEQ ID NO:95, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:51, an amino acid sequence of seq id no;
9) The heavy chain variable region consisting of SEQ ID NO:99, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:55, and a sequence of amino acids;
10 A heavy chain variable region consisting of SEQ ID NO:103, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:59, an amino acid sequence of seq id no; and
11A heavy chain variable region consisting of SEQ ID NO:107, and the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 63.
In some embodiments, the isolated antibody or antigen binding fragment portion thereof further comprises a heavy chain constant region and/or a light chain constant region,
The heavy chain constant region comprises SEQ ID NO:134 or an amino acid sequence identical to SEQ ID NO:134, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and
The light chain constant region comprises SEQ ID NO:133 or amino acid sequence identical to SEQ ID NO:133, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
In some embodiments, the isolated antibody or antigen binding fragment portion thereof further comprises a heavy chain constant region and/or a light chain constant region,
The heavy chain constant region consists of SEQ ID NO:134, and
The light chain constant region consists of SEQ ID NO:133, and a sequence of amino acids.
In some embodiments, the isolated antibody or antigen binding fragment portion thereof comprises a heavy chain and a light chain having at least one selected from the group consisting of:
1) The heavy chain comprising the amino acid sequence of SEQ ID NO:122 or amino acid sequence corresponding to SEQ ID NO:122, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:111 or amino acid sequence corresponding to SEQ ID NO:111, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
2) The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
3) The heavy chain comprising the amino acid sequence of SEQ ID NO:124 or amino acid sequence identical to SEQ ID NO:124, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain comprising the amino acid sequence of SEQ ID NO:113 or an amino acid sequence identical to SEQ ID NO:113, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
4) The heavy chain comprising the amino acid sequence of SEQ ID NO:125 or amino acid sequence identical to SEQ ID NO:125, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:114 or amino acid sequence corresponding to SEQ ID NO:114, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
5) The heavy chain comprising the amino acid sequence of SEQ ID NO:126 or amino acid sequence identical to SEQ ID NO:126, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:115 or amino acid sequence identical to SEQ ID NO:115, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
6) The heavy chain comprising the amino acid sequence of SEQ ID NO:127 or amino acid sequence identical to SEQ ID NO:127, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:116 or amino acid sequence corresponding to SEQ ID NO:116, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
7) The heavy chain comprising the amino acid sequence of SEQ ID NO:128 or an amino acid sequence identical to SEQ ID NO:128, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain comprising the amino acid sequence of SEQ ID NO:117 or an amino acid sequence identical to SEQ ID NO:117, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
8) The heavy chain comprising the amino acid sequence of SEQ ID NO:129 or amino acid sequence corresponding to SEQ ID NO:129, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain comprising the amino acid sequence of SEQ ID NO:118 or an amino acid sequence identical to SEQ ID NO:118, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
9) The heavy chain comprising the amino acid sequence of SEQ ID NO:130 or amino acid sequence corresponding to SEQ ID NO:130, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain comprising the amino acid sequence of SEQ ID NO:119 or an amino acid sequence identical to SEQ ID NO:119, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
10 A) the heavy chain comprising SEQ ID NO:131 or amino acid sequence identical to SEQ ID NO:131, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:120 or an amino acid sequence identical to SEQ ID NO:120, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
11 A) the heavy chain comprising SEQ ID NO:132 or amino acid sequence corresponding to SEQ ID NO:132, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:121 or an amino acid sequence identical to SEQ ID NO:121, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
In some embodiments, the isolated antibody or antigen binding fragment portion thereof consists of a heavy chain and a light chain selected from at least one of the group consisting of:
1) The heavy chain consisting of SEQ ID NO:122, and the light chain consisting of the amino acid sequence of SEQ ID NO:111, an amino acid sequence of seq id no;
2) The heavy chain consisting of SEQ ID NO:123, and said light chain consisting of the amino acid sequence of SEQ ID NO:112, an amino acid sequence of seq id no;
3) The heavy chain consisting of SEQ ID NO:124, and the light chain consisting of the amino acid sequence of SEQ ID NO:113, and a fragment thereof;
4) The heavy chain consisting of SEQ ID NO:125, and the light chain consisting of the amino acid sequence of SEQ ID NO:114, and a sequence of amino acids;
5) The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO:115, and a sequence of amino acids;
6) The heavy chain consisting of SEQ ID NO:127, and the light chain consisting of the amino acid sequence of SEQ ID NO:116, an amino acid sequence of seq id no;
7) The heavy chain consisting of SEQ ID NO:128, and the light chain consisting of the amino acid sequence of SEQ ID NO: 117;
8) The heavy chain consisting of SEQ ID NO:129, and the light chain consisting of the amino acid sequence of SEQ ID NO:118, an amino acid sequence of seq id no;
9) The heavy chain consisting of SEQ ID NO:130, and the light chain consisting of the amino acid sequence of SEQ ID NO:119, and a sequence of amino acids;
10A heavy chain consisting of SEQ ID NO:131, and the light chain consisting of the amino acid sequence of SEQ ID NO:120, an amino acid sequence of seq id no; and
11 A heavy chain consisting of SEQ ID NO:132, and the light chain consisting of the amino acid sequence of SEQ ID NO:121, and a fragment thereof.
In some embodiments, the EC50 of the isolated antibody or antigen binding fragment portion thereof is less than 30pM.
In a second aspect, the invention provides an isolated antibody or antigen-binding fragment portion thereof that specifically binds to a CXCL4 protein, wherein said isolated antibody or antigen-binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
1) The heavy chain comprises a heavy chain consisting of SEQ ID NO:140, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:140, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:140, and the amino acid sequence encoded by the nucleotide sequence of 140, and
The light chain comprises a sequence consisting of SEQ ID NO:139 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:139, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:139, and a nucleotide sequence encoded by the nucleotide sequence of 139;
2) The heavy chain comprises a heavy chain consisting of SEQ ID NO:142, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:142, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:142, and the amino acid sequence encoded by the nucleotide sequence of 142, and
The light chain comprises a sequence consisting of SEQ ID NO:141, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:141, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:141, and a nucleotide sequence encoding the same;
3) The heavy chain comprises a heavy chain consisting of SEQ ID NO:144, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:144, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:144, and the amino acid sequence encoded by the nucleotide sequence of 144, and
The light chain comprises a sequence consisting of SEQ ID NO:143 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:143, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:143, a nucleotide sequence encoding an amino acid sequence;
4) The heavy chain comprises a heavy chain consisting of SEQ ID NO:146 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:146, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:146, and the amino acid sequence encoded by the nucleotide sequence of 146, and
The light chain comprises a sequence consisting of SEQ ID NO:145 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:145, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:145, and an amino acid sequence encoded by the nucleotide sequence;
5) The heavy chain comprises a heavy chain consisting of SEQ ID NO:148, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:148, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:148, and an amino acid sequence encoded by the nucleotide sequence of 148, and
The light chain comprises a sequence consisting of SEQ ID NO:147 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:147, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:147, and the amino acid sequence encoded by the nucleotide sequence of 147;
6) The heavy chain comprises a heavy chain consisting of SEQ ID NO:150 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:150, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:150, and the amino acid sequence encoded by the nucleotide sequence of 150, and
The light chain comprises a sequence consisting of SEQ ID NO:149 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:149, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:149, and a nucleotide sequence encoding the same;
7) The heavy chain comprises a heavy chain consisting of SEQ ID NO:152, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:152, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:152, and an amino acid sequence encoded by the nucleotide sequence of 152, and
The light chain comprises a sequence consisting of SEQ ID NO:151 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:151, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:151, and a nucleotide sequence encoding the amino acid sequence;
8) The heavy chain comprises a heavy chain consisting of SEQ ID NO:154 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:154, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:154, and an amino acid sequence encoded by a nucleotide sequence of seq id no
The light chain comprises a sequence consisting of SEQ ID NO:153 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:153, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:153, and an amino acid sequence encoded by the nucleotide sequence of 153;
9) The heavy chain comprises a heavy chain consisting of SEQ ID NO:156, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:156, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:156, and an amino acid sequence encoded by a nucleotide sequence of 156, and
The light chain comprises a sequence consisting of SEQ ID NO:155, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:155, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:155, and an amino acid sequence encoded by the nucleotide sequence of 155;
10 The heavy chain comprises a sequence consisting of SEQ ID NO:158 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:158, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:158, and the amino acid sequence encoded by the nucleotide sequence of 158, and
The light chain comprises a sequence consisting of SEQ ID NO:157 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:157, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:157, and a nucleotide sequence encoding the same;
11 The heavy chain comprises a sequence consisting of SEQ ID NO:160 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:160, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:160, and the amino acid sequence encoded by the nucleotide sequence of 160, and
The light chain comprises a sequence consisting of SEQ ID NO:159 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:159, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:159, and a nucleotide sequence encoding the amino acid sequence of 159.
In some embodiments, the isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
1) The heavy chain comprises a heavy chain consisting of SEQ ID NO:2 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:2, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:2, and the amino acid sequence encoded by the nucleotide sequence of 2, and
The light chain comprises a sequence consisting of SEQ ID NO:1 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1, and the amino acid sequence encoded by the nucleotide sequence of 1;
2) The heavy chain comprises a heavy chain consisting of SEQ ID NO:4, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:4, and the amino acid sequence encoded by the nucleotide sequence of 4, and
The light chain comprises a sequence consisting of SEQ ID NO:3 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3, and the amino acid sequence encoded by the nucleotide sequence of 3;
3) The heavy chain comprises a heavy chain consisting of SEQ ID NO:6 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:6, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:6, and the amino acid sequence encoded by the nucleotide sequence of 6, and
The light chain comprises a sequence consisting of SEQ ID NO:5 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5, and the amino acid sequence encoded by the nucleotide sequence of 5;
4) The heavy chain comprises a heavy chain consisting of SEQ ID NO:8, or an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO8, or an amino acid sequence consisting of SEQ ID NO:8, and the amino acid sequence encoded by the nucleotide sequence of 8, and
The light chain comprises a sequence consisting of SEQ ID NO:7 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7, the amino acid sequence encoded by the nucleotide sequence of 7;
5) The heavy chain comprises a heavy chain consisting of SEQ ID NO:10 or comprises an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:10, and the amino acid sequence encoded by the nucleotide sequence of 10, and
The light chain comprises a sequence consisting of SEQ ID NO:9 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:9, and the amino acid sequence encoded by the nucleotide sequence of 9;
6) The heavy chain comprises a heavy chain consisting of SEQ ID NO:12 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:12, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:12, and the amino acid sequence encoded by the nucleotide sequence of 12, and
The light chain comprises a sequence consisting of SEQ ID NO:11, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:11, and a nucleotide sequence encoding the same;
7) The heavy chain comprises a heavy chain consisting of SEQ ID NO:14 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:14, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:14, and the amino acid sequence encoded by the nucleotide sequence of 14, and
The light chain comprises a sequence consisting of SEQ ID NO:13, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:13, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:13, and a nucleotide sequence encoding the same;
8) The heavy chain comprises a heavy chain consisting of SEQ ID NO:16, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:16, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:16, and the amino acid sequence encoded by the nucleotide sequence of 16, and
The light chain comprises a sequence consisting of SEQ ID NO:15, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:15, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:15, and an amino acid sequence encoded by the nucleotide sequence of seq id no;
9) The heavy chain comprises a heavy chain consisting of SEQ ID NO:18, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:18, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:18, and the amino acid sequence encoded by the nucleotide sequence of 18, and
The light chain comprises a sequence consisting of SEQ ID NO:17, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:17, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:17, and the amino acid sequence encoded by the nucleotide sequence of seq id no;
10 The heavy chain comprises a sequence consisting of SEQ ID NO:20, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:20, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:20, and the amino acid sequence encoded by the nucleotide sequence of 20, and
The light chain comprises a sequence consisting of SEQ ID NO:19 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:19, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:19, and a nucleotide sequence encoding the same;
11 The heavy chain comprises a sequence consisting of SEQ ID NO:22 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:22, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:22, and the amino acid sequence encoded by the nucleotide sequence of 22, and
The light chain comprises a sequence consisting of SEQ ID NO:21 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:21, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:21, and an amino acid sequence encoded by the nucleotide sequence of 21.
In some embodiments, the EC50 of the isolated antibody or antigen binding fragment portion thereof is less than 30pM.
In a third aspect, the invention provides an antibody composition directed against a CXCL4 protein comprising at least two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to the CXCL4 protein,
Wherein the at least one isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
Wherein the at least one further isolated antibody or antigen binding fragment portion thereof is selected from one of the following 5 antibodies:
(1) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:68, (b) HCDR1 of SEQ ID NO:69, and (c) SEQ ID NO:70, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:24, (e) LCDR1 of SEQ ID NO:25, and (f) SEQ ID NO: LCDR3 of 26; or alternatively
The heavy chain variable region comprising SEQ ID NO:67 or an amino acid sequence identical to SEQ ID NO:67, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23 or amino acid sequence corresponding to SEQ ID NO:23, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain consisting of SEQ ID NO:122, and the light chain consisting of the amino acid sequence of SEQ ID NO:111, an amino acid sequence of seq id no;
(2) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42; or alternatively
The heavy chain variable region comprising SEQ ID NO:83 or amino acid sequence identical to SEQ ID NO:83, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence identical to SEQ ID NO:39, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO:115, and a sequence of amino acids;
(3) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:88, (b) HCDR1 of SEQ ID NO:89, and (c) HCDR2 of SEQ ID NO:90, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:44, (e) LCDR1 of SEQ ID NO:45, and (f) LCDR2 of SEQ ID NO:46 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:87 or amino acid sequence corresponding to SEQ ID NO:87, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:43 or amino acid sequence corresponding to SEQ ID NO:43, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:127, and the light chain consisting of the amino acid sequence of SEQ ID NO:116, an amino acid sequence of seq id no;
(4) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:92, (b) HCDR1 of SEQ ID NO:93, and (c) HCDR2 of SEQ ID NO:94, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:48, (e) LCDR1 of SEQ ID NO:49, and (f) LCDR2 of SEQ ID NO:50 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:91 or an amino acid sequence identical to SEQ ID NO:91, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:47 or amino acid sequence corresponding to SEQ ID NO:47, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:128, and the light chain consisting of the amino acid sequence of SEQ ID NO: 117; and
(5) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:108, (b) HCDR1 of SEQ ID NO:109, and (c) HCDR2 of SEQ ID NO:110, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:64, (e) LCDR1 of SEQ ID NO:65, and (f) SEQ ID NO:66 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:107 or amino acid sequence identical to SEQ ID NO:107, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:63 or amino acid sequence identical to SEQ ID NO:63, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:132, and the light chain consisting of the amino acid sequence of SEQ ID NO:121, and a fragment thereof.
In some embodiments, the antibody compositions of the invention directed against CXCL4 proteins comprise two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to CXCL4 proteins,
The first isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
The second isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:68, (b) HCDR1 of SEQ ID NO:69, and (c) SEQ ID NO:70, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:24, (e) LCDR1 of SEQ ID NO:25, and (f) SEQ ID NO: LCDR3 of 26; or alternatively
The heavy chain variable region comprising SEQ ID NO:67 or an amino acid sequence identical to SEQ ID NO:67, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23 or amino acid sequence corresponding to SEQ ID NO:23, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain consisting of SEQ ID NO:122, and the light chain consisting of the amino acid sequence of SEQ ID NO: 111.
In some embodiments, the antibody compositions of the invention directed against CXCL4 proteins comprise two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to CXCL4 proteins,
The first isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
The second isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42; or alternatively
The heavy chain variable region comprising SEQ ID NO:83 or amino acid sequence identical to SEQ ID NO:83, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence identical to SEQ ID NO:39, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO: 115.
In some embodiments, the antibody compositions of the invention directed against CXCL4 proteins comprise two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to CXCL4 proteins,
The first isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
The second isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:88, (b) HCDR1 of SEQ ID NO:89, and (c) HCDR2 of SEQ ID NO:90, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:44, (e) LCDR1 of SEQ ID NO:45, and (f) LCDR2 of SEQ ID NO:46 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:87 or amino acid sequence corresponding to SEQ ID NO:87, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:43 or amino acid sequence corresponding to SEQ ID NO:43, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:127, and the light chain consisting of the amino acid sequence of SEQ ID NO:116, and a polypeptide comprising the amino acid sequence of 116.
In some embodiments, the antibody compositions of the invention directed against CXCL4 proteins comprise two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to CXCL4 proteins,
The first isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
The second isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:92, (b) HCDR1 of SEQ ID NO:93, and (c) HCDR2 of SEQ ID NO:94, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:48, (e) LCDR1 of SEQ ID NO:49, and (f) LCDR2 of SEQ ID NO:50 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:91 or an amino acid sequence identical to SEQ ID NO:91, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:47 or amino acid sequence corresponding to SEQ ID NO:47, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:128, and the light chain consisting of the amino acid sequence of SEQ ID NO: 117.
In some embodiments, the antibody compositions of the invention directed against CXCL4 proteins comprise two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to CXCL4 proteins,
The first isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
The second isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:108, (b) HCDR1 of SEQ ID NO:109, and (c) HCDR2 of SEQ ID NO:110, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:64, (e) LCDR1 of SEQ ID NO:65, and (f) SEQ ID NO:66 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:107 or amino acid sequence identical to SEQ ID NO:107, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:63 or amino acid sequence identical to SEQ ID NO:63, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:132, and the light chain consisting of the amino acid sequence of SEQ ID NO:121, and a fragment thereof.
In some embodiments, the EC50 of the isolated antibody or antigen binding fragment portion thereof is less than 30pM.
In some embodiments, the isolated antibodies or antigen binding fragments thereof can be used as reagents for detection or analysis of CXCL4 protein in biological studies. In some embodiments, the isolated antibody or antigen-binding fragment thereof can be used as a reagent for detection or analysis of CXCL4 protein in medical research.
(IV), nucleic acids, vectors, host cells and methods for producing antibodies
In one aspect, the invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention as described above. In some embodiments, the nucleic acid molecules of the invention encode the heavy and/or light chain variable regions, the individual CDRs in the heavy and/or light chain variable regions, and the heavy and/or light chain constant regions of the antibodies of the invention described previously. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized for the host cell used for expression. In some embodiments, the nucleic acid molecules of the invention are operably linked to expression control sequences.
The invention also provides an isolated nucleic acid molecule that encodes and is capable of inventing an antibody or antigen binding fragment portion thereof.
In some embodiments, the nucleic acid molecules of the invention comprise or consist of a nucleotide sequence selected from any one of the following combinations: SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22.
In some embodiments, the nucleic acid molecule is operably linked to an expression control sequence.
In another aspect, the invention also provides an expression vector comprising a nucleic acid molecule of the invention.
In another aspect, the invention also provides a host cell comprising a nucleic acid molecule of the invention or an expression vector of the invention.
In another aspect, the invention also provides a hybridoma cell expressing an antibody or antigen-binding fragment portion thereof of the invention.
In another aspect, the invention also provides a method of producing an antibody or antigen-binding fragment portion thereof that targets human tumor cells that express CXCL4, comprising:
(i) Expression of a nucleic acid molecule according to the invention, and
(Ii) Isolating and purifying the portion of the antibody or antigen binding fragment thereof expressed by the nucleic acid molecule.
Fifth, diagnostic method
The invention proves that CXCL4 protein can be independently used as a biomarker for specifically diagnosing depressive disorder, and the specific antibody aiming at the CXCL4 protein can be used for detecting the expression level of the CXCL4 protein and can realize the diagnosis of depressive disorder.
In one aspect, the invention provides a method for diagnosing a depressive disorder, comprising determining the expression level of a CXCL4 protein in a sample from a subject and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the antibody or antigen-binding fragment portion thereof of any one of the invention, or the antibody is an antibody composition of the invention against a CXCL4 protein, wherein the subject is suspected of having a psychotic disorder but is difficult to determine as a depressive disorder, wherein an increase in the expression level of the CXCL4 protein compared to the reference value indicates that the subject has a depressive disorder.
In one aspect, the invention provides a method for detecting the expression level of a biomarker in vitro comprising: (a) Collecting a sample from a subject, wherein the subject is suspected of having a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, (b) determining the expression level of a CXCL4 protein in the sample and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the antibody or antigen-binding fragment portion thereof of any one of the invention, or the antibody is an antibody composition of the invention against a CXCL4 protein, wherein an elevated expression level of the CXCL4 protein compared to the reference value is indicative of the subject having a depressive disorder.
In one aspect, the invention provides a method for determining the likelihood of a subject suffering from a depressive disorder, wherein the subject is suspected of suffering from a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, wherein the method comprises determining the expression level of a CXCL4 protein in a sample from the subject and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the antibody or antigen-binding fragment portion thereof of any of the invention, or the antibody is an antibody composition of the invention against a CXCL4 protein, wherein an elevated expression level of the CXCL4 protein compared to the reference value indicates that the subject has a high likelihood of suffering from a depressive disorder.
In the foregoing aspects, the subject may also be a depressive disorder that has been initially diagnosed by a prior diagnostic criteria, such as a human, for which further diagnosis is required at the molecular level. In the foregoing embodiments, the subject may also be a large random population of subjects, who may include depressive disorder.
In some embodiments, the sample is a blood sample, preferably the sample is a plasma or serum sample.
In some embodiments, the reference value is the expression level of CXCL4 protein in a sample from a healthy subject or from a standard sample.
In some embodiments, the expression level of the CXCL4 protein is determined by a method selected from the group consisting of: immunoassays, including competitive immunoassays, non-competitive immunoassays, enzyme-linked immunosorbent assays (ELISA), immunohistochemical assays, chemiluminescent assays, western assay blots, and Dot blot assays; mass spectrometry; preferably, the expression level of the CXCL4 protein is determined by chemiluminescence.
In some embodiments, the antibody to CXCL4 protein can be further modified to carry a detectable label; preferably, the antibody directed against the CXCL4 protein can be further modified to bear a detectable label, for example a label which can be detected by a chemiluminescent method.
In some embodiments, the subject is a subject who is refractory to diagnosis of a depressive disorder by an existing mental disorder diagnosis and classification system; optionally, the current mental disorder diagnosis and classification system is: a system based on the DSM of the american psychology society (e.g., DSM IV or DSM V), a system based on the ICD of the world health organization (e.g., ICD-10 or ICD-11), and/or a system based on the CCMD of china (e.g., CCMD-3). In some embodiments, the subject is a human.
In some embodiments, the methods of the invention are performed by a full-automatic luminescence detection apparatus or an automated/digital detection platform.
Sixth, kits, test strips and test cards comprising the antibodies/antibody combinations of the invention
In one aspect, the invention provides a kit, test strip or test strip for diagnosing a depressive disorder, comprising an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject suspected of having a psychotic disorder but difficult to determine that the psychotic disorder is a depressive disorder, the antibody being selected from the antibody or antigen-binding fragment portion thereof of any of the invention, or the antibody being an antibody composition against a CXCL4 protein of the invention, the kit, test strip or test strip further comprising instructions or inserts for indicating that up-regulation of the expression level of a CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder.
In one aspect, the invention provides a kit, test strip or test paper card for diagnosing a depressive disorder, wherein the kit, test strip or test paper card comprises a portion for receiving a sample from a subject; and a moiety coated with an antibody capable of specifically binding to a CXCL4 protein, said antibody being selected from the group consisting of an antibody or antigen-binding fragment portion thereof of any one of the present invention, or said antibody being an antibody composition against a CXCL4 protein of the present invention, wherein said subject is suspected to have a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, and an up-regulation of the expression level of the CXCL4 protein compared to a reference value indicates that said subject has a depressive disorder.
In one aspect, the invention provides a kit, strip or card for detecting the expression level of a CXCL4 protein, comprising an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, the antibody being selected from the antibody or antigen-binding fragment portion thereof of any one of the invention, or the antibody being an antibody composition against a CXCL4 protein of the invention, wherein the subject is suspected of having a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, the kit, strip or card further comprising instructions or inserts for indicating that the expression level of a CXCL4 protein is up-regulated compared to a reference value, suggesting that the subject has a depressive disorder.
In one aspect, the invention provides the use of an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, wherein the subject is suspected to have a psychotic disorder but is difficult to determine as a depressive disorder, wherein the antibody is selected from the antibody or antigen-binding fragment portion thereof of any of the invention, or the antibody is an antibody composition of the invention against a CXCL4 protein, wherein an up-regulation of the expression level of the CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder, in the preparation of a test strip, test paper card and/or kit for diagnosing a depressive disorder.
In the foregoing aspects, the subject may also be a depressive disorder that has been initially diagnosed by a prior diagnostic criteria, such as a human, for which further diagnosis is required at the molecular level. In the foregoing embodiments, the subject may also be a large random population of subjects, who may include depressive disorder.
In some embodiments, the sample is a blood sample, preferably the sample is a plasma or serum sample.
In some embodiments, the reference value is the expression level of CXCL4 protein in a sample from a healthy subject or from a standard sample.
In one aspect, the invention provides a kit, strip or card for determining the amount of CXCL4 protein in a biological sample, comprising an antibody for determining the level of expression of CXCL4 protein in a biological sample, said antibody being selected from the group consisting of an antibody or antigen-binding fragment portion thereof of any one of the invention, or said antibody being an antibody composition of the invention directed against CXCL4 protein. In some embodiments, the biological sample may be a sample that requires biological investigation, such as tissue and cells. In some embodiments, the kit, test strip, or test paper card can be used for biological studies of CXCL4 protein function, or for detection or analysis of CXCL4 protein in biological or medical studies.
In some embodiments, the kit, test strip or test paper card may further comprise at least one additional detection reagent for detecting the presence of at least one of the aforementioned monoclonal antibodies of the invention or antigen binding fragments thereof. In some embodiments, the antibody to CXCL4 protein can be further modified to carry a detectable label; preferably, the antibody directed against the CXCL4 protein can be further modified to bear a detectable label, such as a label that can be detected by a chemiluminescent method, such as a label with biotin, fluorescein isothiocyanate, alkaline phosphatase, or acridinium ester. In some embodiments, the marker molecule is selected from one of the following: radiolabels (e.g., isotopes), fluorescent labels (e.g., fluorochromes), chemicals, and enzymes and tags that can detect modifications.
In some embodiments, the kit, test strip or test strip card further comprises a reagent that serves as a control.
Methods for detecting antibody-antigen binding are known in the art, such as ELISA and chemiluminescence, among others. In some embodiments, the expression level of the CXCL4 protein is determined by a method selected from the group consisting of: immunoassays, including competitive immunoassays, non-competitive immunoassays, enzyme-linked immunosorbent assays (ELISA), immunohistochemical assays, chemiluminescent assays, western assay blots, and Dot blot assays; and (5) mass spectrometry.
In some embodiments, the expression level of the CXCL4 protein is determined by chemiluminescence. In some embodiments, chemiluminescent detection is used, using Fluorescein Isothiocyanate (FITC) and alkaline phosphatase as chemiluminescent paired labels conjugated to the antibody, and the magnetic particles are sheep anti-fluorescein isothiocyanate antibody labeled magnetic particles. In some embodiments, chemiluminescent detection is used, conjugation is performed with Fluorescein Isothiocyanate (FITC) and biotin as chemiluminescent paired labels to the antibody, and a conjugate of alkaline phosphatase and streptavidin is used as a third detection reagent, and the magnetic microparticles are sheep anti-fluorescein isothiocyanate antibody labeled magnetic microparticles.
The principle of chemiluminescent detection is known from the prior art and can be seen in particular in, for example, TADESSE HAILE Fereja et al, hindawi Publishing Corporation, ISRN Spectroscopy, volume 2013,Article ID 230858,12pages; yu Wang et al International Journal of Nanomedicine,2019:14 4293-4307; luigi Cinquanta et al, autoimmun Highlights (2017) 8:9.
Without being bound by any theory, the detection principle of the chemiluminescent method for detecting CXCL4 protein of the invention is as follows: fluorescein Isothiocyanate (FITC) -labeled rabbit monoclonal anti-CXCL 4 antibodies bind to CXCL4 in a sample, calibrator, or quality control to form an immune complex. Subsequently, magnetic particles to which goat anti-FITC antibodies were attached were added, and antigen-antibody immune complexes were bound to the magnetic particles by specific binding of the anti-FITC antibodies to FITC. Under the action of an externally applied magnetic field, separating the compound formed by immune reaction from other unbound substances, cleaning the compound, and adding an Alkaline Phosphatase (AP) -labeled rabbit monoclonal anti-CXCL 4 paired antibody to form a sandwich compound with the compound. Under the action of an externally applied magnetic field, the sandwich compound formed by the immune reaction is separated from other unbound substances, and after washing, an enzymatic chemiluminescent substrate is added. The substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, and photons are emitted when the excited state intermediate returns to the ground state to form a luminescence reaction, so that the luminescence intensity of the reaction can be detected by using a chemiluminescent instrument. In the detection wavelength (230-700 nm), the luminous intensity is in proportion to the CXCL4 content in the sample, and the CXCL4 concentration in the sample can be quantitatively calculated through equation fitting.
In some embodiments, the kit is a kit for detecting the expression level of CXCL4 protein by a chemiluminescent method, comprising a magnetic microparticle reagent, an anti-reagent a, an anti-reagent B, and optionally an anti-reagent C, wherein the magnetic microparticle reagent is a detectable label X conjugated magnetic microparticle, the anti-reagent a is a detectable label Y conjugated CXCL4 specific antibody, the anti-reagent B is a detectable label Z conjugated CXCL4 mating antibody, and the anti-reagent C is a detectable label W and V conjugate.
In some embodiments, the detectable label X-conjugated magnetic particle, the detectable label Y-conjugated CXCL4 specific antibody, and the detectable label Z-conjugated CXCL4 mating antibody are selected from the group consisting of:
-anti-fluorescein isothiocyanate antibody conjugated magnetic microparticles, fluorescein isothiocyanate conjugated CXCL4 specific antibody and alkaline phosphatase conjugated CXCL4 pairing antibody;
-avidin-conjugated magnetic microparticles, biotin-conjugated CXCL4 specific antibodies and alkaline phosphatase-conjugated CXCL4 pairing antibodies;
-anti-fluorescein isothiocyanate antibody conjugated magnetic microparticles, fluorescein isothiocyanate conjugated CXCL4 specific antibody and acridinium ester conjugated CXCL4 pairing antibody;
-avidin-conjugated magnetic microparticles, biotin-conjugated CXCL 4-specific antibodies and acridinium ester-conjugated CXCL 4-pairing antibodies.
In some embodiments, the detectable label X-conjugated magnetic particle is a sheep anti-fluorescein isothiocyanate antibody-labeled magnetic particle, the detectable label Y-conjugated CXCL 4-specific antibody is a fluorescein isothiocyanate-labeled CXCL 4-specific antibody, and the detectable label Z-conjugated CXCL 4-pairing antibody is an alkaline phosphatase-labeled CXCL 4-pairing antibody.
In some embodiments, the detectable label X-conjugated magnetic microparticles, the detectable label Y-conjugated CXCL4 specific antibody, the conjugate of the anti-agent C to detectable labels W and V, and the detectable label Z-conjugated CXCL4 mating antibody are anti-fluorescein isothiocyanate antibody-conjugated magnetic microparticles, fluorescein isothiocyanate-conjugated CXCL4 specific antibodies, biotin-conjugated CXCL4 mating antibodies, and conjugates of alkaline phosphatase and streptavidin.
In some embodiments, the kit further comprises a calibrator and a quality control, which are buffers to which different amounts of CXCL4 antigen are added, respectively.
In some embodiments, the kit is a kit for detecting the expression level of CXCL4 protein by a chemiluminescent method, comprising a magnetic particle reagent, an anti-reagent a, and an anti-reagent B, the magnetic particle reagent being a magnetic particle comprising a sheep anti-fluorescein isothiocyanate antibody label, the anti-reagent a being a CXCL4 specific antibody comprising a fluorescein isothiocyanate label, the anti-reagent B being a CXCL4 mating antibody comprising an alkaline phosphatase label. In some embodiments, the kit is a kit for detecting the expression level of CXCL4 protein by a chemiluminescent method, comprising a magnetic microparticle reagent, an anti-reagent a, an anti-reagent B, a calibrator, and a quality control, the magnetic microparticle reagent being a magnetic microparticle comprising a sheep anti-fluorescein isothiocyanate antibody label, the anti-reagent a being a CXCL4 specific antibody comprising a fluorescein isothiocyanate label, the anti-reagent B being a CXCL4 mating antibody comprising an alkaline phosphatase label, and the calibrator and quality control being buffers to which different amounts of CXCL4 antigen are added, respectively. In some embodiments, the magnetic microparticles, CXCL4 specific antibody and CXCL4 mating antibody are each present in Tris buffer. In some embodiments, the CXCL4 specific antibody is an antibody of the previous embodiments of the invention. In some embodiments, the CXCL4 mating antibody is an antibody of the previous embodiments of the invention. In some embodiments, the CXCL4 specific antibody and CXCL4 mating antibody are a combination of the first antibody and a plurality of optional second antibodies of the previous embodiments of the invention.
In some embodiments, the kit is a kit for detecting the expression level of CXCL4 protein by a chemiluminescent method, comprising a magnetic microparticle reagent, an anti-reagent a, an anti-reagent B, and an anti-reagent C, the magnetic microparticle reagent being a magnetic microparticle comprising a sheep anti-fluorescein isothiocyanate antibody label, the anti-reagent a being a CXCL4 specific antibody comprising a fluorescein isothiocyanate label, the anti-reagent B being a CXCL4 mating antibody comprising a biotin label, the anti-reagent C being a conjugate of alkaline phosphatase and streptavidin. In some embodiments, the kit is a kit for detecting the expression level of CXCL4 protein by a chemiluminescent method, comprising a magnetic microparticle reagent, an anti-reagent a, an anti-reagent B, an anti-reagent C, a calibrator, and a quality control, the magnetic microparticle reagent being a magnetic microparticle comprising a sheep anti-fluorescein isothiocyanate antibody label, the anti-reagent a being a CXCL4 specific antibody comprising a fluorescein isothiocyanate label, the anti-reagent B being a CXCL4 pairing antibody comprising a biotin label, the anti-reagent C being a conjugate of alkaline phosphatase and streptavidin, the calibrator and the quality control being buffers to which different amounts of CXCL4 antigen are added, respectively. In some embodiments, the magnetic microparticles, CXCL4 specific antibody, CXCL4 paired antibody, and conjugate of alkaline phosphatase and streptavidin are each present in Tris buffer. In some embodiments, the CXCL4 specific antibody is an antibody of the previous embodiments of the invention. In some embodiments, the CXCL4 mating antibody is an antibody of the previous embodiments of the invention. In some embodiments, the CXCL4 specific antibody and CXCL4 mating antibody are a combination of the first antibody and a plurality of optional second antibodies of the previous embodiments of the invention.
In some embodiments, the kit may further comprise a substrate. In some embodiments, the substrates may be packaged separately in isolated form, e.g., may be placed in additional kits. In some embodiments, the substrate is the luminescent substrate (4-chlorobenzenesulfonyl) (10-methyl-9, 10-acridinium methylene) phosphate disodium salt, APS-5.
In some embodiments, the buffer used to formulate the calibrator and quality control comprises the following components: tris, sodium chloride, bovine serum albumin, tetracycline, neomycin sulfate, and 4M hydrochloric acid. In some embodiments, the method for formulating a calibrator and a quality control buffer comprises: 0.1M Tris, 0.9% sodium chloride, 2% bovine serum albumin, 0.01% tetracycline, 0.01% neomycin sulfate, and an appropriate amount of 4M hydrochloric acid (pH 7.5.+ -. 0.1).
In some embodiments, the anti-agent (Tris) buffer comprises the following components: tris, sodium chloride, bovine serum albumin, tetracycline, neomycin sulfate, and 4M hydrochloric acid. In some embodiments, the anti-agent (Tris) buffer comprises: 0.1M Tris, 0.9% sodium chloride, 1% bovine serum albumin, 0.01% tetracycline, 0.01% neomycin sulfate, and an appropriate amount of 4M hydrochloric acid (pH 8.+ -. 0.1).
In some embodiments, the magnetic particle (Tris) buffer comprises the following components: tris, sodium chloride, bovine serum albumin, methylcellulose, and 4M hydrochloric acid. In some embodiments, the magnetic particle (Tris) buffer comprises: 0.1M Tris, 0.9% sodium chloride, 1% bovine serum albumin, 5% methylcellulose, and an appropriate amount of 4M hydrochloric acid (pH 8.+ -. 0.1).
In some embodiments, a luminescent substrate (Tris) buffer comprises the following components: tris, sodium chloride, luciferin and SDS. In some embodiments, a luminescent substrate (Tris) buffer comprises: 0.1M Tris, 0.9% sodium chloride, 0.3% lucigenin and 1% SDS.
In some embodiments, the (concentrated) cleaning solution comprises the following components: tris, sodium chloride, tween-20 and triton-100. In some embodiments, a luminescent substrate (Tris) buffer comprises: 0.1M Tris, 0.9% sodium chloride, 1% Tween-20 and 1% triton-100.
In some embodiments, the calibrator and quality control are prepared by the following methods: and (3) diluting CXCL4 antigen by using a calibrator buffer, and preparing calibrators and quality control products with different concentrations.
In some embodiments, the substrate is prepared by the following method: the luminescent substrate (4-chlorobenzenesulfide) (10-methyl-9, 10-dihydroacridine methylene) disodium phosphate, APS-5, was diluted with a luminescent buffer.
In some embodiments, the subject is a subject who is refractory to diagnosis of a depressive disorder by an existing mental disorder diagnosis and classification system; optionally, the current mental disorder diagnosis and classification system is: a system based on the DSM of the american psychology society (e.g., DSM IV or DSM V), a system based on the ICD of the world health organization (e.g., ICD-10 or ICD-11), and/or a system based on the CCMD of china (e.g., CCMD-3).
In some embodiments, the kit, test strip or test paper card can be used for chemiluminescent detection.
In some embodiments, the kit, test strip or test paper card may be used with a fully automated luminescence detection instrument or an automated/digital detection platform. The kit, strip or card generally includes a label that indicates the intended use and/or method of use of its contents. The term label includes any written or recorded material provided on or with or otherwise with the kit. In some embodiments, the kit, test strip or test strip further comprises instructions or inserts for how to use the CXCL4 antibodies of the invention to detect CXCL4 protein expression, and instructions or inserts for use to indicate that the level of CXCL4 protein expression is up-regulated compared to a reference value, suggesting that the subject has a depressive disorder.
The reagent components (such as the concentrated cleaning solution is diluted properly according to the instruction, some other buffers are necessary, etc.), the external package of the reagent kit, the independent packaging containers of the reagent components, etc. which are not mentioned in detail in the chemiluminescent kit can be performed according to the conventional operation in the art, and the reagent components are required to meet the relevant industry regulations. The procedure not mentioned in detail in the method of the invention can also be carried out with reference to the usual procedures in the art, for example, before the detection, the reagents can be brought to room temperature (18-25 ℃) and thoroughly mixed before the sample addition; the use of the detection apparatus used, such as a chemiluminescent type of assay, is carried out according to the instructions; in the present invention, the ratio and the content of the unit are not particularly noted, the solid component is the mass ratio and the content, and the liquid component is the volume ratio and the content.
(Seventh), chemiluminescent detection method for CXCL4 protein, and method for diagnosing depression by detecting CXCL4 protein
In one aspect, the invention provides a chemiluminescent detection method for the content of CXCL4 protein, comprising the steps of:
(1) Adding CXCL4 calibrator, quality control product and sample to be tested, and mixing uniformly;
(2) Adding an anti-reagent A and fully reacting, adding a magnetic particle reagent and fully reacting, then placing the reaction mixture in a magnetic field for standing, and discharging supernatant;
(3) Adding an anti-reagent B (and optionally an anti-reagent C) and fully reacting, then standing the reaction mixture in a magnetic field, and discharging supernatant;
(4) Adding a chemiluminescent substrate and uniformly mixing;
(5) And detecting the luminous intensity, and calculating to obtain the CXCL4 protein content in the sample to be detected.
Optionally, the test sample is from a sample from a subject suspected of having a psychotic disorder but difficult to determine that the psychotic disorder is a depressive disorder.
In one aspect, the invention provides a method for detecting the expression level of CXCL4 protein in vitro, comprising the steps of:
(1) Collecting a sample from a subject, wherein the subject is suspected of having a psychotic disorder but is difficult to determine as a depressive disorder;
(2) Adding CXCL4 calibrator, quality control product and sample to be tested, and mixing uniformly;
(3) Adding an anti-reagent A and fully reacting, adding a magnetic particle reagent and fully reacting, then placing the reaction mixture in a magnetic field for standing, and discharging supernatant;
(4) Adding an anti-reagent B (and optionally an anti-reagent C) and fully reacting, then standing the reaction mixture in a magnetic field, and discharging supernatant;
(5) Adding a chemiluminescent substrate and uniformly mixing;
(6) And detecting the luminous intensity, and calculating to obtain the CXCL4 protein content in the sample to be detected.
In one aspect, the invention provides a method for diagnosing a depressive disorder comprising determining the expression level of a CXCL4 protein in a sample from a subject suspected of having a psychotic disorder but difficult to determine as a depressive disorder, and comparing to a reference value, an increase in the expression level of the CXCL4 protein compared to the reference value is indicative of the subject having a depressive disorder,
Wherein the expression level of the CXCL4 protein is determined by a chemiluminescent method comprising the steps of:
(1) Adding CXCL4 calibrator, quality control product and sample to be tested, and mixing uniformly;
(2) Adding an anti-reagent A and fully reacting, adding a magnetic particle reagent and fully reacting, then placing the reaction mixture in a magnetic field for standing, and discharging supernatant;
(3) Adding an anti-reagent B (and optionally an anti-reagent C) and fully reacting, then standing the reaction mixture in a magnetic field, and discharging supernatant;
(4) Adding a chemiluminescent substrate and uniformly mixing;
(5) And detecting the luminous intensity, and calculating to obtain the CXCL4 protein content in the sample to be detected.
In one aspect, the invention provides a method for determining the likelihood of a subject having a depressive disorder (likelihood), comprising determining the expression level of a CXCL4 protein in a sample from a subject suspected of having a psychotic disorder but difficult to determine the psychotic disorder as a depressive disorder, and comparing to a reference value, an increase in the expression level of the CXCL4 protein compared to the reference value is indicative of the subject having a depressive disorder,
Wherein the expression level of the CXCL4 protein is determined by a chemiluminescent method comprising the steps of:
(1) Adding CXCL4 calibrator, quality control product and sample to be tested, and mixing uniformly;
(2) Adding an anti-reagent A and fully reacting, adding a magnetic particle reagent and fully reacting, then placing the reaction mixture in a magnetic field for standing, and discharging supernatant;
(3) Adding an anti-reagent B (and optionally an anti-reagent C) and fully reacting, then standing the reaction mixture in a magnetic field, and discharging supernatant;
(4) Adding a chemiluminescent substrate and uniformly mixing;
(5) And detecting the luminous intensity, and calculating to obtain the CXCL4 protein content in the sample to be detected.
In the foregoing aspects, the subject may also be a depressive disorder that has been initially diagnosed by a prior diagnostic criteria, such as a human, for which further diagnosis is required at the molecular level. In the foregoing embodiments, the subject may also be a large random population of subjects, who may include depressive disorder.
In some embodiments, the magnetic particle reagent is Tris buffer containing sheep anti-fluorescein isothiocyanate antibody labeled magnetic particles. In some embodiments, the anti-agent a is Tris buffer containing a fluorescein isothiocyanate labeled CXCL4 specific antibody. In some embodiments, the anti-agent B is Tris buffer containing alkaline phosphatase or biotin-labeled CXCL4 paired antibody. In some embodiments, the anti-agent C is Tris buffer containing a conjugate of alkaline phosphatase and streptavidin. In some embodiments, the anti-agent a is Tris buffer containing a fluorescein isothiocyanate labeled CXCL4 specific antibody and the anti-agent B is Tris buffer containing an alkaline phosphatase labeled CXCL4 mating antibody. In some embodiments, the anti-agent a is Tris buffer containing a fluorescein isothiocyanate labeled CXCL4 specific antibody, the anti-agent B is Tris buffer containing a biotin labeled CXCL4 pairing antibody, and the anti-agent C is Tris buffer containing a conjugate of alkaline phosphatase and streptavidin. In some embodiments, the calibrator and the quality control are buffers to which different amounts of CXCL4 antigen are added, respectively. In some embodiments, the CXCL4 specific antibody is an antibody of the previous embodiments of the invention. In some embodiments, the CXCL4 mating antibody is an antibody of the previous embodiments of the invention. In some embodiments, the CXCL4 specific antibody and CXCL4 mating antibody are a combination of the first antibody and a plurality of optional second antibodies of the previous embodiments of the invention.
Examples
A further understanding of the present application may be obtained by reference to the specific examples which are set forth to illustrate, but are not intended to limit the scope of the present application. It will be apparent that various modifications and variations can be made to the present application without departing from the spirit of the application, and therefore, such modifications and variations are also within the scope of the application as claimed.
EXAMPLE 1 identification of CXCL4 protein markers
1. Summary of the experiment
The study enrolled 288 subjects in total, including subjects identified as having MDD according to the DSM-IV standard and Healthy Controls (HC), samples were subjected to a mass spectrometry analysis for Data Independent Acquisition (DIA), and the mass spectrometry data obtained as described above was subjected to a partial least Squares discriminant analysis (PARTIAL LEAST Squares DISCRIMINANT ANALYSIS, PLSDA) (fig. 1), showing the presence of significant clustering between MDD and HC subjects. In addition, the mass spectral signal intensity of CXCL4 showed significant differential expression between MDD and HC (fig. 2), thus CXCL4 was used as a biomarker for MDD diagnosis.
2. Experimental materials and methods
This section describes the experimental methods and materials used for the experiments in the examples.
2.1 Group entry criteria and sample collection
The study was approved by the ethical committee of the hospital at the urban psychosis hospital in certain province of China, and written informed consent was obtained from all participants. The diagnosis of MDD subjects enrolled in this study was performed in a clinical setting by trained psychiatrists, strictly according to manual for diagnosis and statistics of mental diseases, 4 th edition (DSM-IV). The study recruited a total of 135 MDD subjects and 153 diagnostic healthy donor HC who had no mental disorder from the present or past.
For HC and MDD subjects, blood samples were collected in anticoagulation tubes using standard venipuncture protocols. The plasma was extracted by centrifugation at 3000rpm for 10 minutes. Plasma samples were stored at-80 ℃ prior to use.
2.2 Removal of high abundance proteins from plasma samples
The high abundance proteins were removed on an Agilent1290INFINITY II liquid chromatography system equipped with an Agilent multiple affinity removal column-Human-14 (Agilent Multi Affinity Removal Column-Human-14). Protein concentration was measured using BCA protein assay kit (BCA Protein Assay Kit, thermo Scientific). The protein concentration determination was performed according to the instruction manual of the kit.
2.3DIA Mass Spectrometry data analysis
For subsequent mass spectrometry of plasma samples from the discovery cohort, small amounts of peptide fragments were taken from each of these samples, and peptide fragments from each sample were mixed to obtain a peptide fragment mixture. The peptide mixture was subjected to high pH reverse phase fractionation, with liquid collected every 60 seconds, separating the peptide mixture into several fractions. These fractions were pooled pairwise to form new samples. The new sample was analyzed by mass spectrometry to obtain a library of profiles for use in constructing databases. The initial plasma samples were then analyzed sequentially by mass spectrometry and the data obtained (peptide fragment information) was used to search the library of spectra to obtain quantitative information on the protein for data analysis.
Procedures and methods for DIA mass spectrometry data analysis can be found in Y.Chen et al.,Immune Response Pattern Across the Asympthmatic,Symptomatic and Convalescent Periods of COVID-19,BBA-Proteins and Proteomics 1870(2022); and Y.Chen et al.,Proteomic Analysis Identifies Prolonged Disturbances in Pathways Related to Cholesterol Metabolism and Myocardium Function in the COVID-19 Recovery Stage,Journal of Proteome Research,2021.
A) Preparation of peptide samples for Data Independent Acquisition (DIA) mass spectrometry
Plasma proteins were precipitated with trichloroacetic acid (TCA) solution, reduced with 20mM (2-carboxyethyl) phosphine hydrochloride (TCEP) and alkylated with 40mM iodoacetamide. The mixture was digested with trypsin overnight at 37℃in a ratio of trypsin to protein of 1/100 (w/w). The lysate was desalted using MonospinC column (GLSCIENCE, tokyo, japan) and then vacuum centrifuged to dryness. The dried peptides were reconstituted prior to DIA analysis.
B) Preparation of peptide samples for profiling library generation
The peptide fragments from each sample were mixed to obtain 100 μg of peptide fragment mixture, and then vacuum centrifuged to dryness. The dried peptide was reconstituted in Milli-Q water containing 2% Acetonitrile (ACN) for fractionation.
C) High pH reverse phase fractionation
At pH 10, the reaction mixture was purified by chromatography on a chromatography column (BEH C18,1.7 Μm,1 mm. Times.150 mm) was used for fractionation of peptide samples. Approximately 100 μg of purified peptide was separated into several fractions by collecting the liquid every 60 seconds. The fractions were mixed in pairs to give new samples, and then centrifuged in vacuo to dryness. The dried peptide was dissolved in 10 μl Milli-Q water containing 0.1% Formic Acid (FA) and then iRT peptides were added for chromatographic correction. The method of reverse phase separation can be seen Proteomic Analysis Identifies Prolonged Disturbances in Pathways Related to Cholesterol Metabolism and Myocardium Function in the COVID-19 Recovery Stage,J Proteome Res.2021 Jul 2;20(7):3463-3474.doi:10.1021/acs.jproteome.1c00054.Epub 2021Jun 3.
D) Liquid chromatography
The nanoupgradeable reverse phase chromatographic analysis (Nanoflow reversed-phase chromatography) was performed on nanoElute liquid chromatography systems (Bruker Daltonics). Separation of the peptide fragments was achieved by non-linear gradient separation, starting with 2% acetonitrile in 0.1% formic acid, then increasing to 22% acetonitrile in 0.1% formic acid, then increasing to 37% acetonitrile in 8 minutes, then further increasing to 100% acetonitrile in 5 minutes, and finally maintaining acetonitrile at 100% for 7 minutes before re-equilibration. A self-prepared chromatographic column (25 cm. Times.75 μm, 1.5. Mu. m C. Mu.18-AQ particles) was used.
Mobile phases a and B were water and acetonitrile containing 0.1% formic acid, respectively. The separation was carried out at a flow rate of 300 nL/min.
E) Mass spectrometry
Liquid Chromatography (LC) was coupled in-line to timsTOF Pro (Bruker) by CAPTIVESPRAY nm electrospray ion source. Fraction samples were analyzed in a data correlation mode and mass spectra were set in the m/z 100-1700 range in a positive electrospray mode to generate a spectral library. Another analysis employs a data independent model consisting of: MS1 scan from m/z 400 to m/z 1200, and covers 64 MS2 windows in diaPASEF acquisition schemes with mass ranging from m/z 400 to m/z 1200.
Ion mobility was scanned from 0.6Vs/cm to 1.6Vs/cm 2. The collision energy increases linearly as a function of mobility from 59eV at 1/k0=1.6 Vs/cm2 to 20eV at 1/k0=0.6 Vs/cm 2.
F) Generation of profiling libraries and DIA data analysis
A profiling library was generated against UNIPROT human data library (considering only proteins with reviewed identity) using Spectronaut version 14.2 (Biognosys). All parameters are default. The DIA file is processed in default mode except that the correction factor of XIC IM extraction window is set to 0.8.
2.4 Partial Least Squares Discriminant Analysis (PLSDA)
Omicsbean software is used for mass spectrum data interpolation and normalization, and then Partial Least Squares Discriminant Analysis (PLSDA) is performed to analyze and visualize the data of the subjects, each component represents a variable affecting grouping, and the higher the similarity between the subjects, the more the distribution distance tends to be closer, and the farther the similarity between the subjects tends to be.
3. Experimental results
3.1CXCL4 protein has obvious difference in the expression level of MDD and HC subjects' blood plasma
PLSDA analysis (fig. 1) of proteins from large scale MDD (n=135) and HC (n=153) deep DIA plasma protein analysis showed clear separation of MDD from HC, suggesting that protein markers may be used to distinguish MDD from healthy people. Among the numerous differentially altered proteins, CXCL4 had stable expression in all samples (100% identification rate), and the expression level of CXCL4 was significantly elevated for MDD compared to HC group (fig. 2). This result suggests that CXCL4 can be used as a biomarker for MDD diagnosis.
Since there are 17 members of the CXC family of chemokines, there is a high homology, how to obtain antibodies with high specificity and high affinity for CXCL4 would be critical to be able to accurately detect their expression levels by immunological methods.
EXAMPLE 2 preparation of anti-CXCL 4 antibody
1. Vector construction
The CXCL4 antibody sequence design, vector construction, expression, purification, identification and the like of the invention are all entrusted to completion of Nanjing Jinsri Limited company. The construction process mainly comprises the steps of constructing the CXCL4 antibody sequence on a pcDNA3.4 vector, and obtaining plasmids after PCR, enzyme digestion, connection, transformation, identification, sequencing, comparison and extraction.
Vectors comprising the light and heavy chain sequences of the CXCL4 antibodies of the invention are shown in table 5 below.
Table 5:
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the 11 plasmids of the light chain are Kappa type; the heavy chain 11 plasmids are of the IgG type.
It should be noted here that the above 11 antibodies are not all antibodies designed, synthesized and tested during the course of the experiment. Some antibodies that do not perform well in subsequent experiments and are therefore not intended to be included within the scope of the invention will not be described in this example.
The coding sequences for the light and heavy chains in each plasmid vector in the above table are as follows (underlined sections correspond to the constant region sequences)
Rabbit monoclonal antibody FS003-4C7 light chain coding sequence (SEQ ID NO: 1) for expression of rabbit light chain variable region + rabbit light chain constant region
CAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAACTGCCAGGCCAGTCAGAGTCTTTATGATGAGAAAAATTTGGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGATGCATCCGATCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCATTCTCACCATCAGCGGTGTGCAGTGTGACGATGCTGCCAGTTACTACTGTCAAGGTGAATTTGACTGTAGTAGTGGTGATTGTAGTGTTTTCGGCGGAGGGACCGAGGTGGTGGTCGAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-4C7 heavy chain coding sequence (SEQ ID NO: 2) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTAACGCCTGGAGGACCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGGAGTGAGCTGGGTCCGCCAGGCTCCAGGGGAGGGGCTGGAATGGATCGGAACCATTACTGTTTCAAGAAATACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAGGATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGGAGTCTTAGTAGTTATTATTACGCCTTTAACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
Rabbit monoclonal antibody FS003-11A1 light chain coding sequence (SEQ ID NO: 3) for expression of rabbit light chain variable region + rabbit light chain constant region
GCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGGTGACAACCGCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATCTGGCATCCAAACTGGCAACTGGGGTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGTGGCGTGCAGTGTGCCGATGCTGCCACTTATTATTGTGTAGGCGCTTATTTTGGTGAGGCCACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-11A1 heavy chain coding sequence (SEQ ID NO: 4) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTAACGCCTGGAGGATCCCTGACACTCACCTGCACCGTCTCTGGAATCGACCTCAGTAGCTTTGCAATAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGGATGGATCGGGTGGATTGGCACTGATGGTGACACATACTACGCGAGCTGGACAAAAGGCCGATTCACCATGTCCAAAACCTCGTCGACCACGGTAGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGATTCCCACAGTGGGGGGGGTGCCTATGACATCTGGGGCCCAGGCACCCTTGTCACCGTCTCGTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
A rabbit monoclonal antibody FS003-16E1 light chain coding sequence (SEQ ID NO: 5) for expressing a rabbit light chain variable region + a rabbit light chain constant region
ATTGATATGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATAATAACAACCTCTTAGCCTGGTATCAGCAGAAAGCAGGGCAGCCTCCCAACCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTCCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTATGATGATGATAGTGATACTGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-16E1 heavy chain coding sequence (SEQ ID NO: 6) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTGTCTATGCAATGGGCTGGGTCCGCCAGGCTCCAGGGGAGGGGCTGGAATACATCGGAATCATTGATCCTAGTGGTAGCGCATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAACCCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGGGGTGGGGTTTGCTGATGGTAGTGACATTTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
Rabbit monoclonal antibody FS003-21F12 light chain coding sequence (SEQ ID NO: 7) for expressing rabbit light chain variable region+rabbit light chain constant region
GCCATTGAGATGACCCAGTCTCCACCCTCCCTGTCTGCATCTGTGGGAGAAAGTGTCAGGATTAGGTGCCTGGCCAGTGAGAACGTTTACAGTGATATATACTGGTATCAGCAGAAGCCAGGGAAGCCTCCTACACTCCTGCTCTATGGTGCATCCAATTTAGAATCTGGGGTCCCACCACGGTTCAGTGGCAGTGGATCCGGGACAGATTACACCCTCACCATTGGCGGCGTGCAGGCTGAAGATGCTGCCACCTACTACTGTCTAGGCGGTTATAGTTACACTACTATAGGTTTGACTTTTGGAGCTGGCACCAAGGTGGAAATCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-21F12 heavy chain coding sequence (SEQ ID NO: 8) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGCTGGAGGAGTCCGGGGGAGACCTGGTCACGCCTGCAGGAACCCTGACACTCACCTGCACAGCCTCTGGATCCGACCTCAGTAACTACGCAATAAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATACATTAGTTATGGTGGGAGCGCACACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCTGGAGATTTGGGCCATGATGAATATGGCGATTATATAATATTGACCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
A rabbit monoclonal antibody FS003-28H12 light chain coding sequence (SEQ ID NO: 9) for expressing a rabbit light chain variable region + a rabbit light chain constant region
GCCATTGAGATGACCCAGTCTCCACCCTCCCTGTCTGCATCTGTGGGAGAAACTGTCAGGATTAGGTGCCTGGCCAGTGAGGACATTTACAGTGGTATATCCTGGTATCAACAGAAGCCAGGGAAACCTCCTACACTCCTGATCTATGGTGGATCCCATTTAGAATCTGGGGTCCCACCACGGTTCAGTGGCAGTGGATCCGGGACAGATTACACCCTCACCATCGGCGGCGTGCAGGCTGAAGATGTTGCCACCTACTACTGTCTAGGCGGTTGGGCTTACAGTAGTAGCGGATATGCTTTCGGTGCGGGGACCAAGGTGGAGATCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-28H12 heavy chain coding sequence (SEQ ID NO: 10) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGTTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAATTATATAGTGATGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGTTCCGTTAGTAGTCCTGGTACCACATACTACACGAGCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAATAGAGGACACGGCCACCTATTTCTGTGCCAGAGATGATGCCTACGATGAGTATGCTGATTACGAGATTATTAATTTCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
A rabbit monoclonal antibody FS003-30B10 light chain coding sequence (SEQ ID NO: 11) for expressing a rabbit light chain variable region + a rabbit light chain constant region
CAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGTTCCTTTACAAATCTCTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCCGATCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAAGCTATGTTTATCGCAGTATTGTTTACACTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-30B10 heavy chain coding sequence (SEQ ID NO: 12) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTGACTATGGAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAATTATTAGTAGTGGTAATAGCGCATCCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGACCACGGTGGAGCTGAAAGTCACCAGTCCGACAACCGAGGACACGGCCACGTATTTCTGTGCCAGAGAGGTCTATGATGACTATGGTGATTCCCTGGGGGCTTTTGATCCCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
Rabbit monoclonal antibody FS003-47E3 light chain coding sequence (SEQ ID NO: 13) for expression of rabbit light chain variable region + rabbit light chain constant region
GCATTCGAATTGACCCAGACTCCATCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTGAGTTCCTTTACAAATCTCTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCCGATCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAAGCTATGTTTATCGCAGTATTGTTTACACTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-47E3 heavy chain coding sequence (SEQ ID NO: 14) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTGACTATGGAATGGGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAATTATTAGTAGTGGTAATAGCGCATCCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGACCACGGTGGAGCTGAAAGTCACCAGTCCGACAACCGAGGACACGGCCACGTATTTCTGTGCCAGAGAGGTCTATGATGACTATGGTGATTCCCTGGGGGCTTTTGATCCCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
Rabbit monoclonal antibody FS003-47F2 light chain coding sequence (SEQ ID NO: 15) for expression of rabbit light chain variable region + rabbit light chain constant region
GACATCGTGTTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTAGGAACCTCTTATCCTGGTATCAGCAGAAACCAGGACAGCCTCCCAAGCTCCTGATTTATGGTGCATCCAATCTGGAATCTGGGGTCCCATCGCGTTTCCGTGGCAGTGGATCTGGGACAGAGTACACTCTCACCATTAGTGGCATGAAGGCTGAAGATGCTGCCACTTATTACTGTCACGGTGGTGCCTATGATACTGGTGTAACTTTTGGAGCTGGCACCAAGGTGGAAATCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-47F2 heavy chain coding sequence (SEQ ID NO: 16) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATCCCTGACACTCACCTGTACAGTCTCTGGAATCGACCTCAGCAGCTATTCAGTGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATCAGTGTTAGTACTAGGACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATGACCCGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCTGGAGAGGGGGACGATGACTATGGTGATTACTTTTTTTCCGCTTTTGATTCCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
Rabbit monoclonal antibody FS003-56D1 light chain coding sequence (SEQ ID NO: 17) for expressing rabbit light chain variable region+rabbit light chain constant region
CAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTGAGTTCCTTTACAAATCTCTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGATGCATCCGATCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAAGCTATCTTTATCGCAGTTATGTTTACACTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-56D1 heavy chain coding sequence (SEQ ID NO: 18) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTGTCTGGAATCGACCTCAGTGACTATGGAATGGGCTGGGTCCGCCAGGCTCCAGGGGAGGGGCTGGAATGGATCGGAACCATTAGTACTGGTGGTAGCATATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACGTATTTCTGTGCCAGAGAGGTCTACGATGACTATGGTGATTCCCTGGGGGCTTTTGATCCCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
Rabbit monoclonal antibody FS003-57D6 light chain coding sequence (SEQ ID NO: 19) for expression of rabbit light chain variable region + rabbit light chain constant region
GCCGATGTCGTGATGACCCAGACTCCAGCCTCCGTGGAGGCAGCTGTGGGAGGCACAGTCACCATAAAGTGCCAGGCCAGTGAGAGCATTGGCAAGCCATTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAATCTCTTGATCTACAGGACATCCAATCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTAGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAGCTATTCTTTAATTAATATTAATACTGATACATTTAATACTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-57D6 heavy chain coding sequence (SEQ ID NO: 20) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAGCTACCACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATATCTAATACTGATAACACATATTATGCGAGCTGGACGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTACTTCTGTGCCAGAGTTTATGATGGTTTTAGTTGTGGAGACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
Rabbit monoclonal antibody FS003-72A12 light chain coding sequence (SEQ ID NO: 21) for expression of rabbit light chain variable region + rabbit light chain constant region
CAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCGTCAACTGCCAGGCCAGTCAGAGTCTTTATAATAAGAAGAATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGAATCAGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTATTGTCAAGGCGAATTTGATTGTAGTAGTGGTGATTGTAATGTTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
Rabbit monoclonal antibody FS003-72A12 heavy chain coding sequence (SEQ ID NO: 22) for expression of rabbit heavy chain variable region + rabbit heavy chain constant region
CAGTCGGTGGAGGAATCCGGGGGTCGCCTAGTCACGCCTGGGACACCCCTCACACTCACCTGCATACTCTCTGGAATCGACCTCAGTAGCGATGCAATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTTATGCTGGTAATGATGGCACATGGTACGCGAGCTGGGTGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGCACATTACGATGATTATGATTATTCTTATGGTGCATTTAACATCTGGGGCCCAGGCACCCTGGTCACCATCTCTTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCATCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
The amino acid sequences of the light chain variable region and the heavy chain variable region in the antibody expressed by the above vector are as follows:
light chain variable region (amino acid sequence):
FS003-4C7 light chain variable region (SEQ ID NO: 23)
The underlined italics are respectively labeled in turn
FS003-11A1 light chain variable region (SEQ ID NO: 27)
The underlined italics are respectively labeled in turn
FS003-16E1 light chain variable region (SEQ ID NO: 31)
The underlined italics are respectively labeled in turn
FS003-21F12 light chain variable region (SEQ ID NO: 35)
The underlined italics are respectively labeled in turn
FS003-28H12 light chain variable region (SEQ ID NO: 39)
The underlined italics are respectively labeled in turn
FS003-30B10 light chain variable region (SEQ ID NO: 43)
The underlined italics are respectively labeled in turn/>
FS003-47E3 light chain variable region (SEQ ID NO: 47)
The underlined italics are respectively labeled in turn
FS003-47F2 light chain variable region (SEQ ID NO: 51)
The underlined italics are respectively labeled in turn
FS003-56D1 light chain variable region (SEQ ID NO: 55)
The underlined italics are respectively labeled in turn
FS003-57D6 light chain variable region (SEQ ID NO: 59)
The underlined italics are respectively labeled in turn
FS003-72A12 light chain variable region (SEQ ID NO: 63)
The underlined italics are respectively labeled in turn
Heavy chain variable region (amino acid sequence):
FS003-4C7 heavy chain variable region (SEQ ID NO: 67)
The underlined italics are respectively labeled in turn />
FS003-11A1 heavy chain variable region (SEQ ID NO: 71)
The underlined italics are respectively labeled in turn
FS003-16E1 heavy chain variable region (SEQ ID NO: 75)
The underlined italics are respectively labeled in turn
FS003-21F12 heavy chain variable region (SEQ ID NO: 79)
The underlined italics are respectively labeled in turn
FS003-28H12 heavy chain variable region (SEQ ID NO: 83)
The underlined italics are respectively labeled in turn
FS003-30B10 heavy chain variable region (SEQ ID NO: 87)
The underlined italics are respectively labeled in turn
FS003-47E3 heavy chain variable region (SEQ ID NO: 91)
The underlined italics are respectively labeled in turn
FS003-47F2 heavy chain variable region (SEQ ID NO: 95)
The underlined italics are respectively labeled in turn
FS003-56D1 heavy chain variable region (SEQ ID NO: 99)
The underlined italics are respectively labeled in turn
FS003-57D6 heavy chain variable region (SEQ ID NO: 103)
The underlined italics are respectively labeled in turn
FS003-72A12 heavy chain variable region (SEQ ID NO: 107)
The underlined italics are respectively labeled in turn
The full-length amino acid sequence of the light chain of the antibody is as follows (underlined corresponds to the constant region sequence):
FS003-4C7 light chain (SEQ ID NO: 111)
QVLTQTPSSVSAAVGGTVTINCQASQSLYDEKNLAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFILTISGVQCDDAASYYCQGEFDCSSGDCSVFGGGTEVVVEGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-11A1 light chain (SEQ ID NO: 112)
AVLTQTPSPVSAAVGGTVTINCQASQSVYGDNRLSWYQQKPGQPPKLLIYLASKLATGVPSRFSGSGSGTQFTLTISGVQCADAATYYCVGAYFGEATFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-16E1 light chain (SEQ ID NO: 113)
IDMTQTPSSVSAAVGGTVTIKCQSSQSVYNNNLLAWYQQKAGQPPNLLIYYASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDSDTVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-21F12 light chain (SEQ ID NO: 114)
AIEMTQSPPSLSASVGESVRIRCLASENVYSDIYWYQQKPGKPPTLLLYGASNLESGVPPRFSGSGSGTDYTLTIGGVQAEDAATYYCLGGYSYTTIGLTFGAGTKVEIKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-28H12 light chain (SEQ ID NO: 115)
AIEMTQSPPSLSASVGETVRIRCLASEDIYSGISWYQQKPGKPPTLLIYGGSHLESGVPPRFSGSGSGTDYTLTIGGVQAEDVATYYCLGGWAYSSSGYAFGAGTKVEIKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-30B10 light chain (SEQ ID NO: 116)
QVLTQTPSSVSEPVGGTVTINCQASEFLYKSLAWYQQKPGQPPKLLIYDASDLASGVPSRFSGSGSGTEFTLTISGVQCDDAATYYCQSYVYRSIVYTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-47E3 light chain (SEQ ID NO: 117)
AFELTQTPSSVSEPVGGTVTIKCQASEFLYKSLAWYQQKPGQPPKLLIYDASDLASGVPSRFSGSGSGTEFTLTISGVQCDDAATYYCQSYVYRSIVYTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-47F2 light chain (SEQ ID NO: 118)
DIVLTQTPSSVSAAVGGTVTINCQASQSVRNLLSWYQQKPGQPPKLLIYGASNLESGVPSRFRGSGSGTEYTLTISGMKAEDAATYYCHGGAYDTGVTFGAGTKVEIKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-56D1 light chain (SEQ ID NO: 119)
QVLTQTPSSVSEPVGGTVTINCQASEFLYKSLAWYQQKPGQPPKLLIYDASDLASGVPSRFSGSGSGTEFTLTISGVQCDDAATYYCQSYLYRSYVYTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-57D6 light chain (SEQ ID NO: 120)
ADVVMTQTPASVEAAVGGTVTIKCQASESIGKPLAWYQQKPGQPPNLLIYRTSNLASGVSSRFKGSRSGTEYTLTISDLECADAATYYCQSYSLININTDTFNTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
FS003-72A12 light chain (SEQ ID NO: 121)
QVLTQTPSSVSAAVGGTVTVNCQASQSLYNKKNLAWYQQKPGQPPKLLIYRASTLESGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCQGEFDCSSGDCNVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
The full-length amino acid sequence of the heavy chain of the antibody is as follows (underlined corresponds to the constant region sequence):
FS003-4C7 heavy chain (SEQ ID NO: 122)
QSLEESGGRLVTPGGPLTLTCTVSGFSLSSYGVSWVRQAPGEGLEWIGTITVSRNTYYASWAKGRFTISKTSTTVDLRITSPTTEDTATYFCARSLSSYYYAFNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-11A1 heavy chain (SEQ ID NO: 123)
QSLEESGGRLVTPGGSLTLTCTVSGIDLSSFAINWVRQAPGKGLGWIGWIGTDGDTYYASWTKGRFTMSKTSSTTVDLKITSPTTEDTATYFCARDSHSGGGAYDIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-16E1 heavy chain (SEQ ID NO: 124)
QSVEESGGRLVTPGTPLTLTCTVSGIDLSVYAMGWVRQAPGEGLEYIGIIDPSGSAYYANWAKGRFTISKPSSTTVDLKMTSPTTEDTATYFCARGVGFADGSDIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-21F12 heavy chain (SEQ ID NO: 125)
QSLEESGGDLVTPAGTLTLTCTASGSDLSNYAISWVRQAPGKGLEYIGYISYGGSAHYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCAGDLGHDEYGDYIILTIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-28H12 heavy chain (SEQ ID NO: 126)
QSLEESGGRLVTPGTPLTLTCTVSGIDLSNYIVMWVRQAPGKGLEWIGSVSSPGTTYYTSWAKGRFTISKTSSTTVDLKITSPTIEDTATYFCARDDAYDEYADYEIINFWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-30B10 heavy chain (SEQ ID NO: 127)
QSVEESGGRLVTPGTPLTLTCTVSGIDLSDYGMGWVRQAPGKGLEWIGIISSGNSASYASWAKGRFTISKTSTTVELKVTSPTTEDTATYFCAREVYDDYGDSLGAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-47E3 heavy chain (SEQ ID NO: 128)
QSVEESGGRLVTPGTPLTLTCTVSGIDLSDYGMGWVRQAPGKGLEWIGIISSGNSASYASWAKGRFTISKTSTTVELKVTSPTTEDTATYFCAREVYDDYGDSLGAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-47F2 heavy chain (SEQ ID NO: 129)
QSLEESGGRLVTPGGSLTLTCTVSGIDLSSYSVAWVRQAPGKGLEYIGIISVSTRTYYASWAKGRFTISKTSSTTVDLKMTRLTTEDTATYFCAGEGDDDYGDYFFSAFDSWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-56D1 heavy chain (SEQ ID NO: 130)
QSVEESGGRLVTPGTPLTLTCTVSGIDLSDYGMGWVRQAPGEGLEWIGTISTGGSIYYANWAKGRFTISKTSTTVDLKITSPTTEDTATYFCAREVYDDYGDSLGAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-57D6 heavy chain (SEQ ID NO: 131)
QSVEESGGRLVTPGTPLTLTCTASGFSLSSYHMSWVRQAPGKGLEWIGVISNTDNTYYASWTKGRFTISKTSTTVDLKMTSLTTEDTATYFCARVYDGFSCGDIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
FS003-72A12 heavy chain (SEQ ID NO: 132)
QSVEESGGRLVTPGTPLTLTCILSGIDLSSDAMTWVRQAPGKGLEWIGIIYAGNDGTWYASWVKGRFTISKTSTTVDLKITSPTTEDTATYFCARAHYDDYDYSYGAFNIWGPGTLVTISSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
The amino acid sequences of the constant regions of the light and heavy chains of the aforementioned antibodies are as follows:
light chain constant region (SEQ ID NO: 133)
GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
Heavy chain constant region (SEQ ID NO: 134)
GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSIRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
The sequence of the signal peptide used in the preparation of the antibody is as follows:
light chain signal peptide coding sequence (SEQ ID NO: 135)
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCC
Heavy chain signal peptide coding sequence (SEQ ID NO: 136)
ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGT
Light chain signal peptide amino acid sequence (SEQ ID NO: 137)
MDTRAPTQLLGLLLLWLPGATFA
Heavy chain signal peptide amino acid sequence (SEQ ID NO: 138)
METGLRWLLLVAVLKGVQC
The signal peptide sequence in the antibody is excised during transfection of the plasmid expressing the antibody into cells and subsequent secretion into the supernatant.
Table 6: the heavy chain, light chain, constant region, and variable region of the aforementioned antibody, and the sequence numbers of the respective CDRs are summarized as follows (SEQ ID NO: SEQ ID NO.)
2. Expression and purification of antibodies, and SDS-PAGE identification
The plasmids were expressed in CHO cells as required for 7 days using ExpiCHO-s expression system and purified using Protein A affinity chromatography on day 6 to give antibodies.
Subsequently, purity of the resulting antibody was checked by SDS-PAGE. In SDS-PAGE, reducing band purity or reducing heavy chain plus light chain sum purity is calculated by imageJ according to a peak area normalization method; the molecular weight of the non-reducing band of the reference IPI is about 150kDa, and the purity is more than 90 percent; the molecular weight of the reduced heavy chain is about 55kDa, the molecular weight of the light chain is about 25kDa, and the purity of the heavy chain and the light chain is more than 90 percent.
Example 3-evaluation of binding specificity of anti-CXCL 4 antibodies to CXCL4 at the protein level and affinity to CXCL4 protein by ELISA method
The following methods were used to determine the specificity and affinity of CXCL4 antibodies at the protein level to CXCL4 protein, including coating ELISA plates with 1ug/mL CXCL4-hFc, human total IgG (Total Human IgG), human IgG1 (Human IgG 1), human IgG4 (Human IgG 4), CXCL4-His, or His-tagged unrelated proteins. Plates were washed 3 times with PBST and blocked with 5% PBS-Mill for 2 hours at room temperature. Plates were washed 3 times with PBST, then diluted antibodies (1 ug/ml) were added to ELISA plates, 30. Mu.L per well, and left at room temperature for 60 minutes. Plates were washed 3 times with PBST, then anti-rabbit IgG-Fc-HRP (Boster; BA 1061) was added to ELISA plates and left at room temperature for 60 minutes. TMB was added and the reaction stopped with 2M stop solution and OD450 was read. The binding specificity of the antibodies to CXCL4 was finally determined by OD450 reading to compare the binding capacity of each antibody to CXCL 4. The measured result data are shown in table 7 below.
Table 7: binding ability of anti-CXCL 4 antibodies to different proteins at the same concentration (ELISA OD450 reading)
As a result of the above table, 11 antibodies to be detected had a high binding capacity to both CXCL4-hFc protein and CXCL4-His, but none of them bound to their tag control Human IgG Fc or His. The results show that the binding of the antibody to be detected to CXCL4 is specific.
The following table shows the results of the affinity assay.
Table 8: affinity assay of anti-CXCL 4 antibodies for CXCL4
| Assayed anti-CXCL 4 antibodies | CXCL4-hFc affinity, EC50 (pM) determined by ELISA |
| FS003-4C7 | 20.420 |
| FS003-11A1 | 19.107 |
| FS003-16E1 | 19.740 |
| FS003-21F12 | 13.973 |
| FS003-28H12 | 22.327 |
| FS003-30B10 | 28.267 |
| FS003-47E3 | 27.367 |
| FS003-47F2 | 11.293 |
| FS003-56D1 | 20.787 |
| FS003-57D6 | 27.973 |
| FS003-72A12 | 27.093 |
As a result of the above table, 11 antibodies to be detected all have a good affinity with CXCL4-hFc protein (EC 50 < 30 pM).
Example 4-double antibody Sandwich ELISA test for different anti-CXCL 4 antibody Pair detection of CXCL4 antigen of the invention
The binding linearity of CXCL4 antibodies to CXCL4 proteins at the protein level was determined using the following method, which included coating ELISA plates with unlabeled anti-CXCL 4 antibodies. Plates were washed 3 times with PBST and blocked with 5% PBS-Mill for 2 hours at room temperature. Plates were washed 3 times with PBST, then diluted CXCL4-hFc antigen at different concentrations was added to ELISA plates and left at room temperature for 60 minutes. Plates were washed 3 times with PBST, then anti-CXCL 4 detection antibody with biotin label was added to ELISA plates and left at room temperature for 60 minutes. Plates were washed 3 times with PBST, and finally NeutrAvidin-HRP (Thermo, 31001) was added to ELISA plates and left at room temperature for 60 minutes. TMB was added and the reaction stopped with 2M stop solution and OD450 values were read. Finally, a linear correlation between the different anti-CXCL 4 antibody pairs and CXCL4 antigen concentrations was determined by reading. The measured result data are shown in table 9 below.
Table 9: preliminary screening of anti-CXCL 4 antibody pairs suitable for ELISA detection Using 4 antigen concentration gradients
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From the above results, further pairs of antibodies with a better linear range were selected 5 from them for further ELISA validation. Specific antibody pairings and ELISA data are shown in table 10 below, with OD450 values determined. In embodiments of the present invention, the term "coated antibody" in antibody pairing may also be referred to as "capture antibody".
Table 10: further validation of 5-pair anti-CXCL 4 antibody pairing suitable for ELISA detection using 12 antigen concentration gradients
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Table 11: information on 5 anti-CXCL 4 antibody pairings in the final validated ELISA assay and their sensitivity
| Antibody pairs | Coated antibodies | Detection antibodies | Sensitivity of |
| First antibody pairing | FS003-11A1 | FS003-47E 3-Biotin | 1-50pg/ml |
| Second antibody pairing | FS003-11A1 | FS003-30B 10-Biotin | 1-150pg/ml |
| Third antibody pairing | FS003-11A1 | FS003-72A 12-Biotin | 1-150pg/ml |
| Fourth antibody pairing | FS003-11A1 | FS003-28H 12-Biotin | 1-150pg/ml |
| Fifth antibody pairing | FS003-4C7 | FS003-11A 1-Biotin | 1-300pg/ml |
Example 5 preparation of chemiluminescent kit and detection of CXCL4 antigen
The chemiluminescent detection kit was further prepared using the 5 anti-CXCL 4 antibody pairs obtained in the above examples. Kits for detecting expression levels of CXCL4 proteins by chemiluminescence include magnetic microparticle reagents, anti-reagent a, anti-reagent B, calibrator, and quality control.
The anti-agent A is prepared by the following method:
(1) Coupling of fluorescein isothiocyanate with CXCL4 specific antibodies: the concentration of the fluorescein isothiocyanate prepared by Tris buffer solution is 1.0-5.0 mg/mL, and the volume ratio is 1: 100-1: 10000, diluting fluorescein isothiocyanate to obtain a diluent, adding CXCL4 specific antibody into the diluent according to the molar ratio of 1:2-1:10, and uniformly mixing at 20+/-5 ℃ for 1-2 h;
(2) Purification of fluorescein isothiocyanate and CXCL4 specific antibody conjugate: eluting with bicarbonate buffer solution with pH=8-9, collecting eluent, and adding the obtained fluorescein isothiocyanate marked CXCL4 specific antibody conjugate into an anti-reagent buffer solution to prepare the anti-reagent A.
The anti-agent B is prepared by the following method:
(1) Coupling of alkaline phosphatase with CXCL4 partner antibody: alkaline phosphatase is prepared by phosphate buffer solution with pH of 7-8 and concentration of 1.0-5.0 mg/mL to obtain alkaline phosphatase solution, CXCL4 paired antibody is prepared according to the mol ratio of 1: 2-1: 10 adding alkaline phosphatase solution, and uniformly mixing for 4-5 h at 20+/-5 ℃; obtaining a conjugate of alkaline phosphatase and CXCL4 paired antibody;
(2) Purifying the alkaline phosphatase and CXCL4 paired antibody conjugate, eluting with bicarbonate buffer with pH=8-9, collecting the eluent, and adding the obtained alkaline phosphatase marked CXCL4 paired antibody conjugate into an anti-reagent buffer to prepare the anti-reagent B.
The magnetic particle reagent is prepared by the following method:
(1) Fully and uniformly mixing the carboxyl magnetic bead concentrated solution; taking out the carboxyl magnetic beads, placing the carboxyl magnetic beads in a magnetic field with the magnetic field strength of 3800mT for 15min, and sucking the supernatant after the carboxyl magnetic beads are totally settled; adding phosphate buffer solution with the volume 5 times of that of the carboxyl magnetic beads, and uniformly mixing for 20-30 min; placing the mixture in a magnetic field with the magnetic field strength of 3800mT, and sucking the supernatant after the magnetic beads are totally settled for 15 min; repeating the step 3 to clean the carboxyl magnetic beads fully;
(2) The carboxylic magnetic bead solution is fixed to 10-50mg/mL; according to the carboxyl magnetic beads: the mass ratio of the sheep anti-fluorescein isothiocyanate antibody is 100:1 adding sheep anti-fluorescein isothiocyanate antibody, and keeping the mixture to react for 18 hours at the temperature of 2-8 ℃ under the uniform mixing state;
(3) Closing: adding 0.5-2M Tris solution, 5-12% bovine serum albumin, 7-15% casein solution and 1-10 g mixed sealing solution of fish skin gel, uniformly mixing for 3-4 h at 20+/-5 ℃, and cleaning for 3 times by using phosphate buffer solution; the concentrated magnetic particle reagent is fixed to a volume of 10-20 mg/mL by using a magnetic particle buffer solution; preserving at 2-8 ℃ for standby;
(4) When in use, the magnetic particle buffer solution is used for preparing the magnetic particle reagent with the concentration of 1-2 mg/mL.
The calibrator and the quality control product are buffers to which different amounts of CXCL4 antigen are added respectively.
In the above method, different 5 antibody pairs are used, respectively, wherein the anti-reagent A corresponds to the capture antibody and the anti-reagent B corresponds to the detection antibody. The specific antibody pairs employed are shown in table 12 below:
| antibody pairing mode | Coated antibodies | Detection antibodies |
| First antibody pairing | FS003-11A1-FITC | FS003-47E 3-alkaline phosphatase |
| Second antibody pairing | FS003-11A1-FITC | FS003-30B 10-alkaline phosphatase |
| Third antibody pairing | FS003-11A1-FITC | FS003-72A12 alkaline phosphatase |
| Fourth antibody pairing | FS003-11A1-FITC | FS003-28H12 alkaline phosphatase |
| Fifth antibody pairing | FS003-4C7-FITC | FS003-11A1 alkaline phosphatase |
The buffers used in the above method are as follows:
Table 13-calibrator, quality control buffer:
| Reagent name | Manufacturer (S) | Concentration of | 1L buffer dosage |
| Tris | Sigma | 0.1M | 12.12g |
| Sodium chloride | Sigma | 0.9％ | 9g |
| Bovine serum albumin | Proliant | 2％ | 20g |
| Tetracycline | Sigma | 0.01％ | 10mg |
| Neomycin sulfate | Sigma | 0.01％ | 10mg |
| 4M hydrochloric acid | Sigma | Proper amount of | Adjusting pH to 7.5+ -0.1 |
Table 14-anti-agent (Tris) buffer:
| Reagent name | Manufacturer (S) | Concentration of | 1L buffer dosage |
| Tris | Sigma | 0.1M | 12.12g |
| Sodium chloride | Sigma | 0.9％ | 9g |
| Bovine serum albumin | Proliant | 1％ | 10g |
| Tetracycline | Sigma | 0.01％ | 10mg |
| Neomycin sulfate | Sigma | 0.01％ | 10mg |
| 4M hydrochloric acid | Sigma | Proper amount of | Adjusting pH to 8+ -0.1 |
Table 15-magnetic particle (Tris) buffer:
| Reagent name | Manufacturer (S) | Concentration of | 1L buffer dosage |
| Tris | Sigma | 0.1M | 12.12g |
| Sodium chloride | Sigma | 0.9％ | 9g |
| Bovine serum albumin | Proliant | 1％ | 10g |
| Methylcellulose and process for producing the same | Sigma | 5％ | 50g |
| 4M hydrochloric acid | Sigma | Proper amount of | Adjusting pH to 8+ -0.1 |
TABLE 16 luminescent substrate (Tris) buffer
Table 17- (formulation of concentrated) cleaning solution, the following formulation:
| Reagent name | Manufacturer (S) | Specification of specification | 1L buffer dosage |
| Tris | Sigma | 0.1M | 12.12g |
| Sodium chloride | Sigma | 0.9％ | 9g |
| Tween-20 | Sigma | 1％ | 50mL |
| Triton-100 | Sigma | 1％ | 50mL |
Further, by using the kit prepared as described above, plasma samples (98 cases in total) of healthy persons and patients suffering from depression who were collected from psychosis control hospitals in certain provinces of China were measured. The results showed that the average concentration of CXCL4 in the healthy population was about 1300ng/ml, whereas the average concentration of CXCL4 in the depressed population was about 2400ng/ml, and that there was a significant difference in the concentrations (p < 0.0001). Further calculations showed that when using a concentration of 1800ng/ml CXCL4 plasma protein as the dividing line for diagnosing health and depressive disorders, the specificity of this kit was as high as 92.5%, the sensitivity was as high as 93.3% and the accuracy was as high as 92.8%.
The technical scheme is as follows:
technical solution group A-antibodies of the invention and antibody combinations
A1. an isolated antibody or antigen-binding fragment portion thereof that specifically binds to a CXCL4 protein, wherein the isolated antibody or antigen-binding fragment portion thereof comprises a heavy chain variable region comprising an HCDR1, HCDR2, HCDR3 sequence; and a light chain variable region comprising LCDR1, LCDR2, LCDR3 sequences wherein the antibody or antigen binding fragment portion thereof has a heavy chain variable region and a light chain variable region selected from at least one of the group consisting of:
1) The heavy chain variable region comprises (a) SEQ ID NO:68, (b) HCDR1 of SEQ ID NO:69, and (c) SEQ ID NO:70, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:24, (e) LCDR1 of SEQ ID NO:25, and (f) SEQ ID NO: LCDR3 of 26;
2) The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30;
3) The heavy chain variable region comprises (a) SEQ ID NO:76, (b) HCDR1 of SEQ ID NO:77, and (c) HCDR2 of SEQ ID NO:78, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:32, (e) LCDR1 of SEQ ID NO:33, and (f) SEQ ID NO:34 LCDR3;
4) The heavy chain variable region comprises (a) SEQ ID NO:80, (b) HCDR1 of SEQ ID NO:81, and (c) HCDR2 of SEQ ID NO:82, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:36, (e) LCDR1 of SEQ ID NO:37, and (f) LCDR2 of SEQ ID NO: LCDR3 of 38;
5) The heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42;
6) The heavy chain variable region comprises (a) SEQ ID NO:88, (b) HCDR1 of SEQ ID NO:89, and (c) HCDR2 of SEQ ID NO:90, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:44, (e) LCDR1 of SEQ ID NO:45, and (f) LCDR2 of SEQ ID NO:46 LCDR3;
7) The heavy chain variable region comprises (a) SEQ ID NO:92, (b) HCDR1 of SEQ ID NO:93, and (c) HCDR2 of SEQ ID NO:94, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:48, (e) LCDR1 of SEQ ID NO:49, and (f) LCDR2 of SEQ ID NO:50 LCDR3;
8) The heavy chain variable region comprises (a) SEQ ID NO:96 HCDR1, (b) SEQ ID NO:97, and (c) HCDR2 of SEQ ID NO:98, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:52, (e) LCDR1 of SEQ ID NO:53, and (f) SEQ ID NO:54 LCDR3;
9) The heavy chain variable region comprises (a) SEQ ID NO:100, (b) HCDR1 of SEQ ID NO:101, and (c) HCDR2 of SEQ ID NO:102, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:56, (e) LCDR1 of SEQ ID NO:57, and (f) SEQ ID NO: LCDR3 of 58;
10 The heavy chain variable region comprises (a) SEQ ID NO:104, (b) HCDR1 of SEQ ID NO:105, and (c) HCDR2 of SEQ ID NO:106, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:60, (e) LCDR1 of SEQ ID NO:61, and (f) SEQ ID NO: LCDR3 of 62; and
11 The heavy chain variable region comprises (a) SEQ ID NO:108, (b) HCDR1 of SEQ ID NO:109, and (c) HCDR2 of SEQ ID NO:110, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:64, (e) LCDR1 of SEQ ID NO:65, and (f) SEQ ID NO: LCDR3 of 66.
A2. The isolated antibody or antigen-binding fragment portion thereof of claim A1, wherein the isolated antibody or antigen-binding fragment portion thereof comprises a light chain variable region and a heavy chain variable region having at least one selected from the group consisting of:
1) The heavy chain variable region comprising SEQ ID NO:67 or an amino acid sequence identical to SEQ ID NO:67, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23 or amino acid sequence corresponding to SEQ ID NO:23, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical;
2) The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical;
3) The heavy chain variable region comprising SEQ ID NO:75 or amino acid sequence corresponding to SEQ ID NO:75, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:31 or amino acid sequence corresponding to SEQ ID NO:31, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
4) The heavy chain variable region comprising SEQ ID NO:79 or amino acid sequence identical to SEQ ID NO:79, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:35 or an amino acid sequence identical to SEQ ID NO:35, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
5) The heavy chain variable region comprising SEQ ID NO:83 or amino acid sequence identical to SEQ ID NO:83, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence identical to SEQ ID NO:39, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
6) The heavy chain variable region comprising SEQ ID NO:87 or amino acid sequence corresponding to SEQ ID NO:87, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:43 or amino acid sequence corresponding to SEQ ID NO:43, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
7) The heavy chain variable region comprising SEQ ID NO:91 or an amino acid sequence identical to SEQ ID NO:91, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:47 or amino acid sequence corresponding to SEQ ID NO:47, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
8) The heavy chain variable region comprising SEQ ID NO:95 or an amino acid sequence identical to SEQ ID NO:95, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:51 or amino acid sequence corresponding to SEQ ID NO:51, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
9) The heavy chain variable region comprising SEQ ID NO:99 or amino acid sequence corresponding to SEQ ID NO:99, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:55 or amino acid sequence identical to SEQ ID NO:55, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
10 A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:103 or amino acid sequence identical to SEQ ID NO:103, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:59 or amino acid sequence identical to SEQ ID NO:59, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; and
11 A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:107 or amino acid sequence identical to SEQ ID NO:107, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:63 or amino acid sequence identical to SEQ ID NO:63, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
A3. The isolated antibody or antigen-binding fragment portion thereof according to claim A1 or A2, wherein the isolated antibody or antigen-binding fragment portion thereof further comprises a heavy chain constant region and/or a light chain constant region,
The heavy chain constant region comprises SEQ ID NO:134 or an amino acid sequence identical to SEQ ID NO:134, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and
The light chain constant region comprises SEQ ID NO:133 or amino acid sequence identical to SEQ ID NO:133, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
A4. The isolated antibody or antigen-binding fragment portion thereof of any one of claims A1-A3, wherein the isolated antibody or antigen-binding fragment portion thereof comprises a heavy chain and a light chain having at least one selected from the group consisting of:
1) The heavy chain comprising the amino acid sequence of SEQ ID NO:122 or amino acid sequence corresponding to SEQ ID NO:122, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:111 or amino acid sequence corresponding to SEQ ID NO:111, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
2) The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
3) The heavy chain comprising the amino acid sequence of SEQ ID NO:124 or amino acid sequence identical to SEQ ID NO:124, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain comprising the amino acid sequence of SEQ ID NO:113 or an amino acid sequence identical to SEQ ID NO:113, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
4) The heavy chain comprising the amino acid sequence of SEQ ID NO:125 or amino acid sequence identical to SEQ ID NO:125, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:114 or amino acid sequence corresponding to SEQ ID NO:114, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
5) The heavy chain comprising the amino acid sequence of SEQ ID NO:126 or amino acid sequence identical to SEQ ID NO:126, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:115 or amino acid sequence identical to SEQ ID NO:115, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
6) The heavy chain comprising the amino acid sequence of SEQ ID NO:127 or amino acid sequence identical to SEQ ID NO:127, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:116 or amino acid sequence corresponding to SEQ ID NO:116, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
7) The heavy chain comprising the amino acid sequence of SEQ ID NO:128 or an amino acid sequence identical to SEQ ID NO:128, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain comprising the amino acid sequence of SEQ ID NO:117 or an amino acid sequence identical to SEQ ID NO:117, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
8) The heavy chain comprising the amino acid sequence of SEQ ID NO:129 or amino acid sequence corresponding to SEQ ID NO:129, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain comprising the amino acid sequence of SEQ ID NO:118 or an amino acid sequence identical to SEQ ID NO:118, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
9) The heavy chain comprising the amino acid sequence of SEQ ID NO:130 or amino acid sequence corresponding to SEQ ID NO:130, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain comprising the amino acid sequence of SEQ ID NO:119 or an amino acid sequence identical to SEQ ID NO:119, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
10 A) the heavy chain comprising SEQ ID NO:131 or amino acid sequence identical to SEQ ID NO:131, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:120 or an amino acid sequence identical to SEQ ID NO:120, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
11 A) the heavy chain comprising SEQ ID NO:132 or amino acid sequence corresponding to SEQ ID NO:132, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:121 or an amino acid sequence identical to SEQ ID NO:121, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
A5. The isolated antibody or antigen-binding fragment portion thereof of any one of claims A1-A4, wherein the isolated antibody or antigen-binding fragment portion thereof consists of a heavy chain and a light chain selected from at least one of the group consisting of:
1) The heavy chain consisting of SEQ ID NO:122, and the light chain consisting of the amino acid sequence of SEQ ID NO:111, an amino acid sequence of seq id no;
2) The heavy chain consisting of SEQ ID NO:123, and said light chain consisting of the amino acid sequence of SEQ ID NO:112, an amino acid sequence of seq id no;
3) The heavy chain consisting of SEQ ID NO:124, and the light chain consisting of the amino acid sequence of SEQ ID NO:113, and a fragment thereof;
4) The heavy chain consisting of SEQ ID NO:125, and the light chain consisting of the amino acid sequence of SEQ ID NO:114, and a sequence of amino acids;
5) The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO:115, and a sequence of amino acids;
6) The heavy chain consisting of SEQ ID NO:127, and the light chain consisting of the amino acid sequence of SEQ ID NO:116, an amino acid sequence of seq id no;
7) The heavy chain consisting of SEQ ID NO:128, and the light chain consisting of the amino acid sequence of SEQ ID NO: 117;
8) The heavy chain consisting of SEQ ID NO:129, and the light chain consisting of the amino acid sequence of SEQ ID NO:118, an amino acid sequence of seq id no;
9) The heavy chain consisting of SEQ ID NO:130, and the light chain consisting of the amino acid sequence of SEQ ID NO:119, and a sequence of amino acids;
10A heavy chain consisting of SEQ ID NO:131, and the light chain consisting of the amino acid sequence of SEQ ID NO:120, an amino acid sequence of seq id no; and
11 A heavy chain consisting of SEQ ID NO:132, and the light chain consisting of the amino acid sequence of SEQ ID NO:121, and a fragment thereof.
A6. an isolated antibody or antigen-binding fragment portion thereof that specifically binds to CXCL4 protein, wherein said isolated antibody or antigen-binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
1) The heavy chain comprises a heavy chain consisting of SEQ ID NO:140, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:140, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:140, and the amino acid sequence encoded by the nucleotide sequence of 140, and
The light chain comprises a sequence consisting of SEQ ID NO:139 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:139, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:139, and a nucleotide sequence encoded by the nucleotide sequence of 139;
2) The heavy chain comprises a heavy chain consisting of SEQ ID NO:142, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:142, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:142, and the amino acid sequence encoded by the nucleotide sequence of 142, and
The light chain comprises a sequence consisting of SEQ ID NO:141, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:141, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:141, and a nucleotide sequence encoding the same;
3) The heavy chain comprises a heavy chain consisting of SEQ ID NO:144, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:144, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:144, and the amino acid sequence encoded by the nucleotide sequence of 144, and
The light chain comprises a sequence consisting of SEQ ID NO:143 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:143, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:143, a nucleotide sequence encoding an amino acid sequence;
4) The heavy chain comprises a heavy chain consisting of SEQ ID NO:146 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:146, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:146, and the amino acid sequence encoded by the nucleotide sequence of 146, and
The light chain comprises a sequence consisting of SEQ ID NO:145 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:145, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:145, and an amino acid sequence encoded by the nucleotide sequence;
5) The heavy chain comprises a heavy chain consisting of SEQ ID NO:148, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:148, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:148, and an amino acid sequence encoded by the nucleotide sequence of 148, and
The light chain comprises a sequence consisting of SEQ ID NO:147 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:147, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:147, and the amino acid sequence encoded by the nucleotide sequence of 147;
6) The heavy chain comprises a heavy chain consisting of SEQ ID NO:150 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:150, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:150, and the amino acid sequence encoded by the nucleotide sequence of 150, and
The light chain comprises a sequence consisting of SEQ ID NO:149 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:149, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:149, and a nucleotide sequence encoding the same;
7) The heavy chain comprises a heavy chain consisting of SEQ ID NO:152, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:152, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:152, and an amino acid sequence encoded by the nucleotide sequence of 152, and
The light chain comprises a sequence consisting of SEQ ID NO:151 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:151, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:151, and a nucleotide sequence encoding the amino acid sequence;
8) The heavy chain comprises a heavy chain consisting of SEQ ID NO:154 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:154, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:154, and an amino acid sequence encoded by a nucleotide sequence of seq id no
The light chain comprises a sequence consisting of SEQ ID NO:153 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:153, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:153, and an amino acid sequence encoded by the nucleotide sequence of 153;
9) The heavy chain comprises a heavy chain consisting of SEQ ID NO:156, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:156, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:156, and an amino acid sequence encoded by a nucleotide sequence of 156, and
The light chain comprises a sequence consisting of SEQ ID NO:155, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:155, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:155, and an amino acid sequence encoded by the nucleotide sequence of 155;
10 The heavy chain comprises a sequence consisting of SEQ ID NO:158 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:158, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:158, and the amino acid sequence encoded by the nucleotide sequence of 158, and
The light chain comprises a sequence consisting of SEQ ID NO:157 or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:157, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:157, and a nucleotide sequence encoding the same;
11 The heavy chain comprises a sequence consisting of SEQ ID NO:160 or an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:160, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:160, and the amino acid sequence encoded by the nucleotide sequence of 160, and
The light chain comprises a sequence consisting of SEQ ID NO:159 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:159, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:159, and a nucleotide sequence encoding the amino acid sequence of 159.
A7. the isolated antibody or antigen-binding fragment portion thereof of claim A6, wherein the isolated antibody or antigen-binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
1) The heavy chain comprises a heavy chain consisting of SEQ ID NO:2 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:2, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:2, and the amino acid sequence encoded by the nucleotide sequence of 2, and
The light chain comprises a sequence consisting of SEQ ID NO:1 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1, and the amino acid sequence encoded by the nucleotide sequence of 1;
2) The heavy chain comprises a heavy chain consisting of SEQ ID NO:4, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:4, and the amino acid sequence encoded by the nucleotide sequence of 4, and
The light chain comprises a sequence consisting of SEQ ID NO:3 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3, and the amino acid sequence encoded by the nucleotide sequence of 3;
3) The heavy chain comprises a heavy chain consisting of SEQ ID NO:6 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:6, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:6, and the amino acid sequence encoded by the nucleotide sequence of 6, and
The light chain comprises a sequence consisting of SEQ ID NO:5 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5, and the amino acid sequence encoded by the nucleotide sequence of 5;
4) The heavy chain comprises a heavy chain consisting of SEQ ID NO:8, or an amino acid sequence comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO8, or an amino acid sequence consisting of SEQ ID NO:8, and the amino acid sequence encoded by the nucleotide sequence of 8, and
The light chain comprises a sequence consisting of SEQ ID NO:7 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7, the amino acid sequence encoded by the nucleotide sequence of 7;
5) The heavy chain comprises a heavy chain consisting of SEQ ID NO:10 or comprises an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:10, and the amino acid sequence encoded by the nucleotide sequence of 10, and
The light chain comprises a sequence consisting of SEQ ID NO:9 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:9, and the amino acid sequence encoded by the nucleotide sequence of 9;
6) The heavy chain comprises a heavy chain consisting of SEQ ID NO:12 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:12, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:12, and the amino acid sequence encoded by the nucleotide sequence of 12, and
The light chain comprises a sequence consisting of SEQ ID NO:11, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:11, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:11, and a nucleotide sequence encoding the same;
7) The heavy chain comprises a heavy chain consisting of SEQ ID NO:14 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:14, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:14, and the amino acid sequence encoded by the nucleotide sequence of 14, and
The light chain comprises a sequence consisting of SEQ ID NO:13, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:13, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:13, and a nucleotide sequence encoding the same;
8) The heavy chain comprises a heavy chain consisting of SEQ ID NO:16, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:16, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:16, and the amino acid sequence encoded by the nucleotide sequence of 16, and
The light chain comprises a sequence consisting of SEQ ID NO:15, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:15, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:15, and an amino acid sequence encoded by the nucleotide sequence of seq id no;
9) The heavy chain comprises a heavy chain consisting of SEQ ID NO:18, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:18, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:18, and the amino acid sequence encoded by the nucleotide sequence of 18, and
The light chain comprises a sequence consisting of SEQ ID NO:17, or comprises an amino acid sequence that is identical to the amino acid sequence encoded by SEQ ID NO:17, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:17, and the amino acid sequence encoded by the nucleotide sequence of seq id no;
10 The heavy chain comprises a sequence consisting of SEQ ID NO:20, or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:20, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:20, and the amino acid sequence encoded by the nucleotide sequence of 20, and
The light chain comprises a sequence consisting of SEQ ID NO:19 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:19, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:19, and a nucleotide sequence encoding the same;
11 The heavy chain comprises a sequence consisting of SEQ ID NO:22 or comprises an amino acid sequence that is identical to the nucleotide sequence encoded by SEQ ID NO:22, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:22, and the amino acid sequence encoded by the nucleotide sequence of 22, and
The light chain comprises a sequence consisting of SEQ ID NO:21 or comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:21, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:21, and an amino acid sequence encoded by the nucleotide sequence of 21.
A8. An antibody composition directed against CXCL4 protein comprising at least two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to CXCL4 protein,
Wherein the at least one isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
Wherein the at least one further isolated antibody or antigen binding fragment portion thereof is selected from one of the following 5 antibodies:
(1) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:68, (b) HCDR1 of SEQ ID NO:69, and (c) SEQ ID NO:70, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:24, (e) LCDR1 of SEQ ID NO:25, and (f) SEQ ID NO: LCDR3 of 26; or alternatively
The heavy chain variable region comprising SEQ ID NO:67 or an amino acid sequence identical to SEQ ID NO:67, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23 or amino acid sequence corresponding to SEQ ID NO:23, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain consisting of SEQ ID NO:122, and the light chain consisting of the amino acid sequence of SEQ ID NO:111, an amino acid sequence of seq id no;
(2) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42; or alternatively
The heavy chain variable region comprising SEQ ID NO:83 or amino acid sequence identical to SEQ ID NO:83, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence identical to SEQ ID NO:39, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO:115, and a sequence of amino acids;
(3) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:88, (b) HCDR1 of SEQ ID NO:89, and (c) HCDR2 of SEQ ID NO:90, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:44, (e) LCDR1 of SEQ ID NO:45, and (f) LCDR2 of SEQ ID NO:46 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:87 or amino acid sequence corresponding to SEQ ID NO:87, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:43 or amino acid sequence corresponding to SEQ ID NO:43, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:127, and the light chain consisting of the amino acid sequence of SEQ ID NO:116, an amino acid sequence of seq id no;
(4) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:92, (b) HCDR1 of SEQ ID NO:93, and (c) HCDR2 of SEQ ID NO:94, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:48, (e) LCDR1 of SEQ ID NO:49, and (f) LCDR2 of SEQ ID NO:50 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:91 or an amino acid sequence identical to SEQ ID NO:91, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:47 or amino acid sequence corresponding to SEQ ID NO:47, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:128, and the light chain consisting of the amino acid sequence of SEQ ID NO: 117; and
(5) The isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:108, (b) HCDR1 of SEQ ID NO:109, and (c) HCDR2 of SEQ ID NO:110, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:64, (e) LCDR1 of SEQ ID NO:65, and (f) SEQ ID NO:66 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:107 or amino acid sequence identical to SEQ ID NO:107, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:63 or amino acid sequence identical to SEQ ID NO:63, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:132, and the light chain consisting of the amino acid sequence of SEQ ID NO:121, and a fragment thereof.
A9. the isolated antibody or antigen-binding fragment portion thereof of any one of claims A1-A7,
Wherein the antibody is capable of binding CXCL4 protein in vitro with an EC50 of less than 30 pM;
and/or wherein the antibody is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a humanized antibody.
A10. an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment portion thereof of any one of claims A1-A7; or alternatively
The nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of the following combinations: SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22.
A11. The isolated nucleic acid molecule of claim a10, said nucleic acid molecule being operably linked to an expression control sequence.
A12. An expression vector comprising the nucleic acid molecule of claim a10 or a 11.
A13. a host cell comprising the nucleic acid molecule of claim a10 or a11 or the expression vector of claim a 12.
A14. A hybridoma cell expressing the antibody or antigen binding fragment portion thereof of any one of claims A1-A7.
A15. A method of producing an antibody or antigen-binding fragment portion thereof that targets human tumor cells that express CXCL4, comprising:
(i) Expression of the nucleic acid molecule according to the A10 or A11 of the technical scheme, and
(Ii) Isolating and purifying the portion of the antibody or antigen binding fragment thereof expressed by the nucleic acid molecule.
Technical scheme group B-diagnostic kit, test strip and test paper card containing the antibody and antibody combination of the invention
B1. A kit, test strip or test strip for diagnosing a depressive disorder comprising an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, wherein the subject is suspected to have a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, the antibody is selected from the antibodies or antigen-binding fragment portions thereof of any of claims A1-A7 in claim set a, or the antibody is an antibody composition for a CXCL4 protein of claim A8, the kit, test strip or test strip further comprising instructions or inserts indicating that up-regulation of the expression level of a CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder.
B2. a kit, test strip or test paper card for diagnosing a depressive disorder, wherein the kit, test strip or test paper card comprises a portion for receiving a sample from a subject; and a moiety coated with an antibody capable of specifically binding to a CXCL4 protein, said antibody being selected from the group consisting of the antibodies of any one of claims A1-A7 of claim group a or antigen binding fragment thereof, or the antibody being an antibody composition against a CXCL4 protein of claim A8, wherein the subject is suspected to have a psychotic disorder but is difficult to determine as a depressive disorder, and an up-regulation of the expression level of the CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder.
B3. A kit, strip or card for detecting the expression level of a CXCL4 protein, comprising an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, said antibody being selected from the group consisting of an antibody or antigen-binding fragment portion thereof of any one of claims A1-A7 of technical group a, or said antibody being an antibody composition for a CXCL4 protein of technical group A8, wherein said subject is suspected of having a psychotic disorder but it is difficult to determine that the psychotic disorder is a depressive disorder, said kit, strip or card further comprising instructions or inserts indicating that up-regulation of the expression level of a CXCL4 protein compared to a reference value indicates that said subject has a depressive disorder.
B3. The kit, test strip or test card of any one of claims B1-B2, wherein the subject is a depressive disorder that has been initially diagnosed by a preceding diagnostic criteria, a subject, e.g., a human, in need of further diagnosis at the molecular level; or alternatively
Wherein the subject is a large random population of people, who may include people with depressive disorders.
B4. the kit, test strip or test card of any one of claims B1-B3, wherein the sample is a blood sample, preferably the sample is a plasma or serum sample.
B5. The kit, test strip or test paper card of any one of claims B1-B4, wherein the reference value is the expression level of CXCL4 protein in a sample from a healthy subject or from a standard sample.
B6. The kit, test strip or test card of any one of claims B1-B5, wherein the expression level of the CXCL4 protein is determined by a method selected from the group consisting of: immunoassays, including competitive immunoassays, non-competitive immunoassays, enzyme-linked immunosorbent assays (ELISA), immunohistochemical assays, chemiluminescent assays, western assay blots, and Dot blot assays; and (5) mass spectrometry.
B7. The kit, test strip, or test card of any one of claims B1-B6, wherein the antibody to CXCL4 protein can be further modified to carry a detectable label; preferably, the antibody directed against the CXCL4 protein can be further modified to bear a detectable label, for example a label which can be detected by a chemiluminescent method.
B8. The kit, test strip or test card of any one of claims B1-B7, wherein the subject is a subject who is refractory to diagnosis of a depressive disorder by an existing mental disorder diagnosis and classification system; optionally, the current mental disorder diagnosis and classification system is: a system based on the DSM of the american psychology society (e.g., DSM IV or DSM V), a system based on the ICD of the world health organization (e.g., ICD-10 or ICD-11), and/or a system based on the CCMD of china (e.g., CCMD-3).
B9. The kit, test strip or test paper card of any one of claims B1-B8, wherein the kit, test strip or test paper card is useful for chemiluminescent detection.
B10. the kit, test strip or test paper card of any one of claims B1-B9, wherein the kit, test strip or test paper card can be used in combination with a full-automatic luminescence detection instrument or an automated/digital detection platform.
Technical solution group C-diagnostic methods of antibodies and antibody combinations of the invention
C1. A method for diagnosing a depressive disorder comprising determining the expression level of a CXCL4 protein in a sample from a subject and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the group consisting of the antibodies or antigen-binding fragment portions thereof of any one of claims A1-A7 of claim group a, or the antibody is the antibody composition of claim A8 against a CXCL4 protein, wherein the subject is suspected of having a psychotic disorder but is refractory to determining that the psychotic disorder is a depressive disorder, wherein an increase in the expression level of the CXCL4 protein compared to the reference value is indicative of the subject suffering from a depressive disorder.
C2. A method of detecting in vitro the expression level of a biomarker comprising: (a) Collecting a sample from a subject, wherein the subject is suspected of having a psychotic disorder but it is difficult to determine that the psychotic disorder is a depressive disorder, (b) determining the expression level of a CXCL4 protein in the sample and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the group consisting of an antibody or antigen-binding fragment portion thereof of any one of claims A1-A7 in claim set a, or an antibody composition for a CXCL4 protein of claim A8, wherein an elevated expression level of the CXCL4 protein compared to the reference value indicates that the subject has a depressive disorder.
C3. A method for determining the likelihood of a subject having a depressive disorder, wherein the subject is suspected of having a psychotic disorder but is difficult to determine that the psychotic disorder is a depressive disorder, wherein the method comprises determining the expression level of a CXCL4 protein in a sample from the subject and comparing it to a reference value, wherein the expression level of the CXCL4 protein is determined by an antibody selected from the antibody or antigen-binding fragment portion thereof of any one of claims A1-A7 of claim group a, or the antibody is an antibody composition of claim A8 against a CXCL4 protein, wherein an elevated expression level of the CXCL4 protein compared to the reference value indicates a high likelihood of the subject having a depressive disorder.
C4. the method of any one of claims C1-C3, wherein the subject is a depressive disorder that has been initially diagnosed by advanced diagnostic criteria, a subject in need of further diagnosis at the molecular level, e.g., a human; or alternatively
Wherein the subject is a large random population of people, who may include people with depressive disorders.
C5. The method of any one of claims C1-C4, wherein the sample is a blood sample, preferably the sample is a plasma or serum sample.
C6. The method of any one of claims C1-C5, wherein the reference value is the expression level of CXCL4 protein in a sample from a healthy subject or from a standard sample.
C7. The method of any one of claims C1-C6, wherein the expression level of the CXCL4 protein is determined by a method selected from the group consisting of: immunoassays, including competitive immunoassays, non-competitive immunoassays, enzyme-linked immunosorbent assays (ELISA), immunohistochemical assays, chemiluminescent assays, western assay blots, and Dot blot assays; mass spectrometry; preferably, the expression level of the CXCL4 protein is determined by chemiluminescence.
C8. The method of any one of claims C1-C7, wherein the antibody to CXCL4 protein can be further modified to bear a detectable label; preferably, the antibody directed against the CXCL4 protein can be further modified to bear a detectable label, for example a label which can be detected by a chemiluminescent method.
C9. the method of any one of claims C1-C8, wherein the subject is a subject who is refractory to diagnosis of a depressive disorder by an existing mental disorder diagnosis and classification system; optionally, the current mental disorder diagnosis and classification system is: a system based on the DSM of the american psychology society (e.g., DSM IV or DSM V), a system based on the ICD of the world health organization (e.g., ICD-10 or ICD-11), and/or a system based on the CCMD of china (e.g., CCMD-3).
C10. The method of any one of claims C1-C9, wherein the method is performed by a full-automatic luminescence detection apparatus or an automated/digital detection platform.
Technical group D-diagnostic uses of the antibodies and antibody combinations of the invention
D1. Use of an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, wherein the subject is suspected to have a psychotic disorder but is difficult to determine as a depressive disorder, wherein the antibody is selected from the antibody or antigen-binding fragment portion thereof of any of claims A1-A7 in technical group a, or the antibody is an antibody composition of claim A8 against a CXCL4 protein, wherein an up-regulation of the expression level of the CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder, in the preparation of a test strip, test paper card and/or kit for diagnosing a depressive disorder.
D2. The use of claim D1, wherein the subject is a depressive disorder that has been initially diagnosed by a preceding diagnostic criteria, a subject, e.g., a human, in need of further diagnosis at the molecular level; or alternatively
Wherein the subject is a large random population of people, who may include people with depressive disorders.
D3. The use according to any one of claims D1-D2, wherein the sample is a blood sample, preferably the sample is a plasma or serum sample.
D4. the use of any one of claims D1-D3, wherein the reference value is the expression level of CXCL4 protein in a sample from a healthy subject or from a standard sample.
D5. The use of any one of claims D1-D4, wherein the expression level of the CXCL4 protein is determined by a method selected from the group consisting of: immunoassays, including competitive immunoassays, non-competitive immunoassays, enzyme-linked immunosorbent assays (ELISA), immunohistochemical assays, chemiluminescent assays, western assay blots, and Dot blot assays; and (5) mass spectrometry.
D6. The use of any one of claims D1-D5, wherein the antibody to CXCL4 protein can be further modified to bear a detectable label; preferably, the antibody directed against the CXCL4 protein can be further modified to bear a detectable label, for example a label which can be detected by a chemiluminescent method.
D7. The use of any one of claims D1-D6, wherein the subject is a subject who is refractory to diagnosis of a depressive disorder by an existing mental disorder diagnosis and classification system; optionally, the current mental disorder diagnosis and classification system is: a system based on the DSM of the american psychology society (e.g., DSM IV or DSM V), a system based on the ICD of the world health organization (e.g., ICD-10 or ICD-11), and/or a system based on the CCMD of china (e.g., CCMD-3).
D8. The use according to any one of claims D1 to D7, wherein the kit, test strip or test card is for chemiluminescent detection.
D9. The use according to any one of claims D1-D8, wherein the kit, strip or card can be used in combination with a full-automatic luminescence detection instrument or an automated/digital detection platform.
Technical group E-immunoassay compositions
E1. An immunoassay composition comprising a CXCL4 protein in a sample from a subject and an effective amount of an antibody (preferably a binding partner) for detecting the expression level of the CXCL4 protein, and/or comprising a conjugate formed from a CXCL4 protein in a sample from a subject and an effective amount of an antibody (preferably a binding partner) for detecting the expression level of the CXCL4 protein, wherein the subject is suspected of having a psychotic disorder but is difficult to determine as a depressive disorder, wherein the antibody is selected from the antibody or antigen-binding fragment portion thereof of any one of claims A1-A7 of claim a, or the antibody is an antibody composition for a CXCL4 protein of claim A8, wherein an increase in the expression level of the CXCL4 protein in the immunoassay composition compared to a reference value is detected to indicate that the subject has a depressive disorder.
E2. A container comprising an immunoassay composition comprising a CXCL4 protein in a sample from a subject and an effective amount of an antibody (preferably a binding partner) for detecting the expression level of the CXCL4 protein, and/or comprising a conjugate formed from a CXCL4 protein in a sample from a subject and an effective amount of an antibody (preferably a binding partner) for detecting the expression level of the CXCL4 protein, wherein the subject is suspected of having a psychotic disorder but is difficult to determine as a depressive disorder, wherein the antibody is selected from the antibody or antigen-binding fragment portion thereof of any one of claims A1-A7 of claim a, or the antibody is an antibody composition for a CXCL4 protein of claim A8, wherein detection of an elevated expression level of the biomarker in the immunoassay composition in the container compared to a reference value indicates that the subject has a depressive disorder.
E3. The composition of claim E1 or the container of claim E2, wherein the subject is a depressive disorder that has been initially diagnosed by advanced diagnostic criteria, a subject, e.g., a human, in need of further diagnosis at the molecular level; or alternatively
Wherein the subject is a large random population of people, who may include people with depressive disorders.
E4. The composition according to claim E1 or the container according to claim E2 or the composition or container according to claim E3, wherein the sample is a blood sample, preferably the sample is a plasma or serum sample.
E5. The composition of claim E1 or E3-E4 or the container of claim E2 or E3-E4, wherein the reference value is the expression level of CXCL4 protein in a sample from a healthy subject or from a standard sample.
E6. The composition of any one of claims E1, E3-E5 or the container of any one of claims E2-E5, wherein the expression level of the biomarker is determined by a method selected from the group consisting of: immunoassays, including competitive immunoassays, non-competitive immunoassays, enzyme-linked immunosorbent assays (ELISA), immunohistochemical assays, chemiluminescent assays, western assay blots, and Dot blot assays; and (5) mass spectrometry.
E7. the composition of any one of claims E1, E3-E6 or the container of any one of claims E2-E6, wherein the antibody or binding partner is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an antibody fragment, a humanized antibody, a camelid antibody, a chimeric antibody.
E8. The composition of any one of claims E1, E3-E7 or the container of any one of claims E2-E7, wherein the antibody or binding partner can be further modified to bear a detectable label; optionally, the antibody or binding partner may be further modified to bear a detectable label, such as a label that can be detected by chemiluminescent means.
E9. The composition of any one of claims E1, E3-E8 or the container of any one of claims E2-E8, wherein the subject is a subject who is refractory to diagnosis of a depressive disorder by an existing mental disorder diagnosis and classification system; optionally, the current mental disorder diagnosis and classification system is: a system based on the DSM of the american psychology society (e.g., DSM IV or DSM V), a system based on the ICD of the world health organization (e.g., ICD-10 or ICD-11), and/or a system based on the CCMD of china (e.g., CCMD-3).
E10. the composition of any one of claims E1, E3-E9 or the container of any one of claims E2-E9, wherein the container may be in the form of a tube, well, vial, or the like.
Technical scheme group F-CXCL4 protein chemiluminescence detection kit
F1. A chemiluminescent detection kit for detecting the amount of CXCL4 protein, or a chemiluminescent detection kit for diagnosing a depressive disorder, or a chemiluminescent detection kit for detecting in vitro the expression level of CXCL4 protein, or a chemiluminescent detection kit for determining the likelihood of a subject suffering from a depressive disorder, the kit comprising a magnetic particle reagent, an anti-reagent A, an anti-reagent B and optionally a reagent C,
Wherein the magnetic particle reagent is a detectable label X conjugated magnetic particle, the anti-reagent A is a detectable label Y conjugated CXCL4 specific antibody, the anti-reagent B is a detectable label Z conjugated CXCL4 paired antibody, the anti-reagent C is a detectable label W and V conjugate,
Wherein the CXCL4 specific antibody has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
Wherein the CXCL4 mating antibody has one of the following 4 antibodies:
(1) The CXCL4 mating antibody has a heavy chain and a light chain selected from at least one of the following groups:
the heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42; or alternatively
The heavy chain variable region comprising SEQ ID NO:83 or amino acid sequence identical to SEQ ID NO:83, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence identical to SEQ ID NO:39, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO:115, and a sequence of amino acids;
(2) The CXCL4 mating antibody has a heavy chain and a light chain selected from at least one of the following groups:
The heavy chain variable region comprises (a) SEQ ID NO:88, (b) HCDR1 of SEQ ID NO:89, and (c) HCDR2 of SEQ ID NO:90, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:44, (e) LCDR1 of SEQ ID NO:45, and (f) LCDR2 of SEQ ID NO:46 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:87 or amino acid sequence corresponding to SEQ ID NO:87, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:43 or amino acid sequence corresponding to SEQ ID NO:43, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:127, and the light chain consisting of the amino acid sequence of SEQ ID NO:116, an amino acid sequence of seq id no;
(3) The CXCL4 mating antibody has a heavy chain and a light chain selected from at least one of the following groups:
The heavy chain variable region comprises (a) SEQ ID NO:92, (b) HCDR1 of SEQ ID NO:93, and (c) HCDR2 of SEQ ID NO:94, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:48, (e) LCDR1 of SEQ ID NO:49, and (f) LCDR2 of SEQ ID NO:50 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:91 or an amino acid sequence identical to SEQ ID NO:91, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:47 or amino acid sequence corresponding to SEQ ID NO:47, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:128, and the light chain consisting of the amino acid sequence of SEQ ID NO: 117; and
(4) The CXCL4 mating antibody has a heavy chain and a light chain selected from at least one of the following groups:
The heavy chain variable region comprises (a) SEQ ID NO:108, (b) HCDR1 of SEQ ID NO:109, and (c) HCDR2 of SEQ ID NO:110, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:64, (e) LCDR1 of SEQ ID NO:65, and (f) SEQ ID NO:66 LCDR3; or alternatively
The heavy chain variable region comprising SEQ ID NO:107 or amino acid sequence identical to SEQ ID NO:107, and an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:63 or amino acid sequence identical to SEQ ID NO:63, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Or alternatively
The heavy chain consisting of SEQ ID NO:132, and the light chain consisting of the amino acid sequence of SEQ ID NO:121, an amino acid sequence of seq id no;
Or alternatively
Wherein the CXCL4 specific antibody has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:68, (b) HCDR1 of SEQ ID NO:69, and (c) SEQ ID NO:70, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:24, (e) LCDR1 of SEQ ID NO:25, and (f) SEQ ID NO: LCDR3 of 26; or alternatively
The heavy chain variable region comprising SEQ ID NO:67 or an amino acid sequence identical to SEQ ID NO:67, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23 or amino acid sequence corresponding to SEQ ID NO:23, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain consisting of SEQ ID NO:122, and the light chain consisting of the amino acid sequence of SEQ ID NO:111, an amino acid sequence of seq id no;
Wherein the CXCL4 mating antibody has a heavy chain and a light chain selected from at least one of the following groups:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region comprising SEQ ID NO:71 or amino acid sequence corresponding to SEQ ID NO:71, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:27 or amino acid sequence corresponding to SEQ ID NO:27, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical; or alternatively
The heavy chain comprising the amino acid sequence of SEQ ID NO:123 or amino acid sequence identical to SEQ ID NO:123, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, and the light chain comprising the amino acid sequence of SEQ ID NO:112 or amino acid sequence corresponding to SEQ ID NO:112, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
F2. The kit of claim F1, wherein the detectable label X-conjugated magnetic microparticles, the detectable label Y-conjugated CXCL4 specific antibody, and the detectable label Z-conjugated CXCL4 mating antibody are selected from the group consisting of:
-anti-fluorescein isothiocyanate antibody conjugated magnetic microparticles, fluorescein isothiocyanate conjugated CXCL4 specific antibody and alkaline phosphatase conjugated CXCL4 pairing antibody;
-avidin-conjugated magnetic microparticles, biotin-conjugated CXCL4 specific antibodies and alkaline phosphatase-conjugated CXCL4 pairing antibodies;
-anti-fluorescein isothiocyanate antibody conjugated magnetic microparticles, fluorescein isothiocyanate conjugated CXCL4 specific antibody and acridinium ester conjugated CXCL4 pairing antibody; and
-Avidin-conjugated magnetic microparticles, biotin-conjugated CXCL 4-specific antibodies and acridinium ester-conjugated CXCL 4-pairing antibodies.
F3. The kit of claim F1 or F2, wherein the detectable label X-conjugated magnetic particle is a sheep anti-fluorescein isothiocyanate antibody-labeled magnetic particle, the detectable label Y-conjugated CXCL 4-specific antibody is a fluorescein isothiocyanate-labeled CXCL 4-specific antibody, and the detectable label Z-conjugated CXCL 4-pairing antibody is an alkaline phosphatase-labeled CXCL 4-pairing antibody.
F4. The kit of claim F1, wherein the detectable label X-conjugated magnetic particle is a sheep anti-fluorescein isothiocyanate antibody-labeled magnetic particle, the detectable label Y-conjugated CXCL 4-specific antibody is a fluorescein isothiocyanate-labeled CXCL 4-specific antibody, the detectable label Z-conjugated CXCL 4-pairing antibody is a biotin-labeled CXCL 4-pairing antibody, and the conjugate of detectable labels W and V is a conjugate of alkaline phosphatase and streptavidin.
F5. The kit of any one of claims F1-F4, wherein the magnetic particle reagent, the anti-reagent a, the anti-reagent B, and the anti-reagent C are each present in Tris buffer.
F6. The kit of claim F5, wherein
The Tris buffer of the magnetic particle reagent comprises: 0.1M Tris, 0.9% sodium chloride, 1% bovine serum albumin, 5% methylcellulose, and an appropriate amount of 4M hydrochloric acid (pH 8.+ -. 0.1); and is also provided with
The Tris buffer for anti-agent a, anti-agent B and anti-agent C comprises: 0.1M Tris, 0.9% sodium chloride, 1% bovine serum albumin, 0.01% tetracycline, 0.01% neomycin sulfate, and an appropriate amount of 4M hydrochloric acid (pH 8.+ -. 0.1).
F7. The kit of any one of claims F1-F6, further comprising a calibrator and/or a quality control; or the calibrator and/or the quality control product can be independently placed in another kit in an isolated form, wherein the calibrator and the quality control product are buffers to which different amounts of CXCL4 antigen are added respectively.
F8. The kit of any one of claims F1-F7, further comprising a substrate; alternatively, the substrate may be separately placed in an additional kit in an isolated form, wherein the substrate is the luminescent substrate (4-chlorobenzenesulfonyl) (10-methyl-9, 10-dihydroacridine methylene) disodium phosphate, APS-5.
F9. The kit of claim F8, wherein the calibrator and the quality control are present in Tris buffer, respectively, the calibrator and the quality control buffer comprising: 0.1M Tris, 0.9% sodium chloride, 2% bovine serum albumin, 0.01% tetracycline, 0.01% neomycin sulfate, and an appropriate amount of 4M hydrochloric acid (pH 7.5.+ -. 0.1).
F10. The kit of any one of claims F1-F9, which can be used in combination with a full-automatic chemiluminescence detection instrument or an automated/digital detection platform.
F11. The kit of any one of claims F1-F10, wherein the subject to which the kit is directed is a subject who is refractory to diagnosis of a depressive disorder by an existing psychotic disorder diagnosis and classification system; optionally, the current mental disorder diagnosis and classification system is: a system based on the DSM of the american psychology society (e.g., DSM IV or DSM V), a system based on the ICD of the world health organization (e.g., ICD-10 or ICD-11), and/or a system based on the CCMD of china (e.g., CCMD-3).
Claims (13)
1. An isolated antibody or antigen-binding fragment portion thereof that specifically binds to a CXCL4 protein, wherein the isolated antibody or antigen-binding fragment portion thereof comprises a heavy chain variable region comprising an HCDR1, HCDR2, HCDR3 sequence; and a light chain variable region comprising LCDR1, LCDR2, LCDR3 sequences wherein the antibody or antigen binding fragment portion thereof has a heavy chain variable region and a light chain variable region selected from the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42.
2. The isolated antibody or antigen-binding fragment portion thereof of claim 1, wherein the isolated antibody or antigen-binding fragment portion thereof comprises a heavy chain variable region and a light chain variable region having:
The heavy chain variable region consisting of SEQ ID NO:83, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:39, and a sequence of amino acids.
3. The isolated antibody or antigen-binding fragment portion thereof of claim 2, wherein the isolated antibody or antigen-binding fragment portion thereof further comprises a heavy chain constant region and/or a light chain constant region,
The heavy chain constant region consists of SEQ ID NO:134, and
The light chain constant region consists of SEQ ID NO:133, and a sequence of amino acids.
4. The isolated antibody or antigen-binding fragment portion thereof of claim 3, wherein the isolated antibody or antigen-binding fragment portion thereof consists of heavy and light chains of:
the heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO: 115.
5. An isolated antibody or antigen-binding fragment portion thereof that specifically binds to CXCL4 protein, wherein said isolated antibody or antigen-binding fragment portion thereof has the following heavy and light chain variable regions:
the heavy chain variable region consists of SEQ ID NO:148, and an amino acid sequence encoded by the nucleotide sequence of 148, and
The light chain variable region consists of SEQ ID NO:147, and the amino acid sequence encoded by the nucleotide sequence of 147.
6. The isolated antibody or antigen-binding fragment portion thereof of claim 5, wherein the isolated antibody or antigen-binding fragment portion thereof has heavy and light chains of:
the heavy chain consists of SEQ ID NO:10, and the amino acid sequence encoded by the nucleotide sequence of 10, and
The light chain consists of SEQ ID NO:9, and an amino acid sequence encoded by the nucleotide sequence of 9.
7. An antibody composition directed against CXCL4 protein comprising two isolated antibodies or antigen-binding fragment portions thereof that specifically bind to CXCL4 protein,
Wherein said one isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region consisting of SEQ ID NO:71, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:27, an amino acid sequence of seq id no; or alternatively
The heavy chain consisting of SEQ ID NO:123, and said light chain consisting of the amino acid sequence of SEQ ID NO:112, an amino acid sequence of seq id no;
wherein the further isolated antibody or antigen binding fragment portion thereof has a heavy chain and a light chain selected from at least one of the group consisting of:
the heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42; or alternatively
The heavy chain variable region consisting of SEQ ID NO:83, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:39, and a sequence of amino acids; or alternatively
The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO: 115.
8. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment portion thereof of any one of claims 1-6; or alternatively
The nucleic acid molecule consists of the following nucleotide sequence: SEQ ID NO. 10 and SEQ ID NO. 9.
9. An expression vector or host cell comprising the nucleic acid molecule of claim 8.
10. Use of an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, wherein the subject is suspected to have a psychotic disorder but is difficult to determine as a depressive disorder, wherein the antibody is selected from the antibody or antigen-binding fragment portion thereof of any one of claims 1-6, or the antibody is an antibody composition of claim 7 against a CXCL4 protein, wherein an up-regulation of the expression level of the CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder, in the manufacture of a test strip, test paper card and/or kit for diagnosing a depressive disorder.
11. A kit, test strip or test strip for diagnosing a depressive disorder comprising an antibody for detecting the expression level of a CXCL4 protein in a sample from a subject, wherein the subject is suspected of having a psychotic disorder but is difficult to determine as a depressive disorder, the antibody is selected from the antibody or antigen-binding fragment portion thereof of any one of claims 1-6, or the antibody is an antibody composition for a CXCL4 protein of claim 7, the kit, test strip or test strip further comprising instructions or inserts indicating that up-regulation of the expression level of a CXCL4 protein compared to a reference value indicates that the subject has a depressive disorder.
12. A chemiluminescent detection kit for detecting the amount of CXCL4 protein, or a chemiluminescent detection kit for diagnosing a depressive disorder, or a chemiluminescent detection kit for detecting the expression level of CXCL4 protein in vitro, or a chemiluminescent detection kit for determining the likelihood of a subject suffering from a depressive disorder, the kit comprising a magnetic particle reagent, an anti-reagent A, an anti-reagent B and an anti-reagent C,
Wherein the magnetic particle reagent is a sheep anti-fluorescein isothiocyanate antibody labeled magnetic particle, the anti-reagent a is a fluorescein isothiocyanate labeled CXCL4 specific antibody, the anti-reagent B is a biotin labeled CXCL4 pairing antibody, and the anti-reagent C is a conjugate of alkaline phosphatase and streptavidin, wherein the CXCL4 specific antibody has a heavy chain and a light chain selected from at least one of the group consisting of:
The heavy chain variable region comprises (a) SEQ ID NO:72, (b) HCDR1 of SEQ ID NO:73, and (c) HCDR2 of SEQ ID NO:74, and said light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:28, (e) LCDR1 of SEQ ID NO:29, and (f) SEQ ID NO: LCDR3 of 30; or alternatively
The heavy chain variable region consisting of SEQ ID NO:71, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:27, an amino acid sequence of seq id no; or alternatively
The heavy chain consisting of SEQ ID NO:123, and said light chain consisting of the amino acid sequence of SEQ ID NO:112, an amino acid sequence of seq id no;
Wherein the CXCL4 mating antibody has a heavy chain and a light chain selected from at least one of the following groups:
the heavy chain variable region comprises (a) SEQ ID NO:84, (b) HCDR1 of SEQ ID NO:85, and (c) HCDR2 of SEQ ID NO:86, and the light chain variable region comprises (d) the amino acid sequence of SEQ ID NO:40, (e) LCDR1 of SEQ ID NO:41, and (f) SEQ ID NO: LCDR3 of 42; or alternatively
The heavy chain variable region consisting of SEQ ID NO:83, and said light chain variable region consisting of the amino acid sequence of SEQ ID NO:39, and a sequence of amino acids; or alternatively
The heavy chain consisting of SEQ ID NO:126, and the light chain consisting of the amino acid sequence of SEQ ID NO: 115.
13. The kit of claim 12, wherein the magnetic particle reagent, the anti-reagent a, the anti-reagent B, and the anti-reagent C are each present in Tris buffer; and is also provided with
Wherein,
The Tris buffer of the magnetic particle reagent comprises: 0.1M Tris, 0.9% sodium chloride, 1% bovine serum albumin, 5% methylcellulose, and an appropriate amount of 4M hydrochloric acid, pH 8.+ -. 0.1; and is also provided with
The Tris buffer for anti-agent a, anti-agent B and anti-agent C comprises: 0.1M Tris, 0.9% sodium chloride, 1% bovine serum albumin, 0.01% tetracycline, 0.01% neomycin sulfate, and an appropriate amount of 4M hydrochloric acid, pH 8.+ -. 0.1;
Wherein the kit further comprises a calibrator and/or a quality control; or the calibrator and/or the quality control product can be independently placed in another kit in an isolated form, wherein the calibrator and the quality control product are buffers to which different amounts of CXCL4 antigen are added respectively;
Wherein the kit further comprises a substrate; or the substrate can be separately placed in an additional kit in an isolated form, wherein the substrate is the luminescent substrate (4-chlorobenzenesulfonyl) (10-methyl-9, 10-acridinium methylene) phosphate disodium salt; wherein, the calibrator with the quality control article exists in Tris buffer respectively, and the calibrator with the buffer of quality control article includes: 0.1M Tris, 0.9% sodium chloride, 2% bovine serum albumin, 0.01% tetracycline, 0.01% neomycin sulphate, and an appropriate amount of 4M hydrochloric acid, pH 7.5.+ -. 0.1.
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