CN116023488A - Antibodies against human cardiac troponin I and uses thereof - Google Patents

Abstract

The invention relates to the technical field of biomedicine, in particular to an antibody for resisting human cardiac troponin I and application thereof. The invention provides a high-affinity monoclonal antibody of three different epitopes of anti-human cardiac troponin I22-40, 39-51 and 81-96, which can specifically recognize human cardiac troponin I and has high affinity and high specificity. The invention also provides a preparation method of the antibody and application of the antibody in the fields of development of cardiac troponin I immunodiagnosis reagents and immunodiagnosis kits and clinical diagnosis.

Description

Antibodies against human cardiac troponin I and uses thereof

Cross reference to related applications

The present application claims priority from chinese patent application No. 202111240430.7 filed 10/25/2021, the entire contents of which are incorporated by reference.

Technical Field

The invention relates to the technical field of biomedicine, in particular to an antibody for resisting human cardiac troponin I and application thereof.

Background

Acute Myocardial Infarction (AMI) is myocardial necrosis caused by acute, persistent ischemia and hypoxia of the coronary arteries, and early diagnosis and timely treatment thereof are key to controlling mortality. Creatine kinase, creatine kinase isozymes, glutamic-oxaloacetic transaminase, lactate dehydrogenase, hydroxybutyrate dehydrogenase and the like are early markers traditionally used for diagnosing acute myocardial infarction, but because most of the markers have the problems of low specificity, short half-life and the like, troponin which provides important contribution to the understanding of molecular biology of heart contraction ^[1] Gradually coming into the field of research in the diagnostic field.

Troponin is an important functional protein that maintains myocardial fiber contraction, and is present in filaments of myofibrils, mainly comprising three subunits: troponin I, troponin T and troponin C. Troponin I is the "inhibitory" unit of the filament-associated troponin complex, a key regulator protein representing myocardial contraction and relaxation, activating the cross-bridge response with the filament ^[2] Can be used to determine myocardial damage after or during, for example, myocardial infarction, while troponin I is of increasing interest to clinicians and scholars due to its outstanding advantages in terms of sensitivity, specificity and diagnostic window period in the diagnosis of acute myocardial infarction and other heart diseases. The international union of clinical chemistry (IFCC), national institute of clinical biochemistry (NACB), european cardiology institute (ESC), american cardiology institute/american heart association (ACC/AHA) and the like have issued numerous recommendations for clinical use of troponin in order to emphasize the important value of cardiac troponin I or T in diagnosing myocardial infarction. Currently, troponin detection is often considered as a gold standard biomarker for detecting acute myocardial infarction ^[3] For a pair ofPatients suspected of acute coronary syndrome undergo troponin detection to rule out or determine acute myocardial necrosis ^[4] 。

Numerous studies have been made on the determination of troponin I, such as radioimmunoassay methods developed using polyclonal antibodies ^[5] And utilizing the polyclonal antibody to match with the monoclonal antibody ^[6] Or two monoclonal antibodies are matched to develop a double-antibody sandwich enzyme immunoassay ^[7,8] To detect cardiac troponin I in serum, but these methods either have cross-reactivity or the accuracy still cannot meet the detection requirements, resulting in lower accuracy of the detection results. Therefore, in the detection of troponin I, development of highly specific and highly sensitive immunoassays is required. Because polyclonal antibodies have relatively many binding sites and relatively weak specificity and homogeneity, the development and equipping of high affinity and high specificity anti-troponin I antibodies that meet the clinical diagnostic requirements of cardiovascular disease is critical to the solution of the problem.

The colloidal gold kit, the time-resolved immunochromatography kit, the chemiluminescent kit and the turbidimetric immunoassay kit are four types of immunodiagnosis reagents which are most commonly used for clinically detecting troponin I, wherein an anti-troponin I antibody is one of key raw materials of the diagnosis reagents, and although troponin I antibody raw material suppliers in the market at home and abroad are many at present, a majority of troponin I diagnosis reagent suppliers still feed back troponin I antibody raw materials in the market in terms of detection sensitivity, and can not completely meet the high-sensitivity troponin I detection requirement. A broad range of diagnostic reagent suppliers would like to find on the market high sensitivity troponin I antibody starting materials for developing a minimum detection limit of not more than 10pg/ml on a chemiluminescent technology platform or up to 30pg/ml on a chromatographic technology platform. Furthermore, most of the troponin I diagnostic kits clinically used in China are imported abroad, or foreign raw materials are adopted, so that the high price limits the wide popularization and use of troponin I in clinical diagnosis of heart diseases in China. The development of a completely domestic troponin I diagnostic kit is urgently needed in the domestic market, and the development of high-quality anti-troponin I antibodies is more important. Most of troponin I antibody raw materials in the market cannot completely meet the requirement of high-sensitivity detection, and the requirement of high-affinity and high-specificity anti-troponin I antibody raw materials is urgent.

Disclosure of Invention

A first object of the present invention is to provide an antibody or an antigen-binding fragment thereof capable of specifically recognizing cardiac troponin I (cTnI), comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from the group consisting of the complementarity determining regions shown in a-j:

。

it is a second object of the present invention to provide an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof as described above.

It is a third object of the present invention to provide a vector comprising a nucleic acid as described above.

It is a fourth object of the present invention to provide a host cell comprising a nucleic acid as described above or a vector as described above.

It is a fifth object of the present invention to provide a composition or combination product comprising one or more of the antibodies or antigen binding fragments thereof as described above.

It is a sixth object of the present invention to provide a detection product comprising an antibody or antigen-binding fragment thereof as described above; the detection product comprises a reagent, a test strip or a kit.

A seventh object of the present invention is to provide the use of an antibody or antigen binding fragment thereof as described above for the preparation of a cardiac troponin I assay product; the detection product comprises a reagent, a test strip or a kit.

The invention provides a high-affinity monoclonal antibody of three different epitopes of anti-human cardiac troponin I22-40, 39-51 and 81-96, which can specifically recognize human cardiac troponin I and has high affinity and high specificity. The invention also provides a preparation method of the antibody and application of the antibody in the fields of development of cardiac troponin I immunodiagnosis reagents and immunodiagnosis kits and clinical diagnosis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows SDS-PAGE to detect the purity of clone I50.G11-2 purified antibody according to an embodiment of the invention; lane M, protein Marker (Takara. CAT 3452); lane 1 clone I50.G11-2 under reducing conditions; lane 2, control rabbit IgG under reduced conditions; lane 3 clone I50.G11-2 in non-reducing conditions; lane 4, control rabbit IgG in non-reducing condition;

FIG. 2 is a graph showing the binding of clone I50.F12-3 provided by one embodiment of the present invention;

FIG. 3 is a graph showing the calibration of human cardiac troponin I detection by chemiluminescent detection by an antibody according to one embodiment of the present invention; fitting a four-parameter calibration curve to the concentration of human cardiac troponin I calibrator and the Relative Luminescence Unit (RLU) signal detected by the double antibody sandwich method, the correlation coefficient R of the curve ² ＞0.99；

FIG. 4 is a chemiluminescent normals evaluation of antibodies in one embodiment of the invention.

FIG. 5 is a graph showing the calibration of human cardiac troponin I detection by chemiluminescent detection by an antibody according to another embodiment of the present invention; fitting a four-parameter calibration curve to the concentration of human cardiac troponin I calibrator and the Relative Luminescence Unit (RLU) signal detected by the double antibody sandwich method, the correlation coefficient R of the curve ² ＞0.99；

FIG. 6 is a chemiluminescent normals evaluation of antibodies in another embodiment of the invention.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In the present invention, the combination name and the antibody name are merely for convenience of description, and do not limit the present invention.

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated. Molecular weight is referred to, allowing its meaning to include fluctuations of + -10%.

In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Unless otherwise contradicted by purpose and/or technical solution of the present application, the cited documents related to the present invention are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present invention, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are incorporated into the present application by reference, but are not limited to the embodiments that can be implemented. It should be understood that when a reference is made to the description herein, it is intended to control or adapt the present application in light of the description herein.

The present invention relates to an antibody or antigen binding fragment thereof capable of specifically recognizing cardiac troponin I comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, the amino acid sequences of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 being selected from the group of complementarity determining region combinations represented by a-j:

combination name

HCDR1

HCDR2

HCDR3

LCDR1

LCDR2

LCDR3

a

SEQ ID NO:1

SEQ ID NO:2

SEQ ID NO:3

SEQ ID NO:31

SEQ ID NO:32

SEQ ID NO:33

b

SEQ ID NO:4

SEQ ID NO:5

SEQ ID NO:6

SEQ ID NO:34

SEQ ID NO:35

SEQ ID NO:36

c

SEQ ID NO:7

SEQ ID NO:8

SEQ ID NO:9

SEQ ID NO:37

SEQ ID NO:38

SEQ ID NO:39

d

SEQ ID NO:10

SEQ ID NO:11

SEQ ID NO:12

SEQ ID NO:40

SEQ ID NO:41

SEQ ID NO:42

e

SEQ ID NO:13

SEQ ID NO:14

SEQ ID NO:15

SEQ ID NO:43

SEQ ID NO:44

SEQ ID NO:45

f

SEQ ID NO:16

SEQ ID NO:17

SEQ ID NO:18

SEQ ID NO:46

SEQ ID NO:47

SEQ ID NO:48

g

SEQ ID NO:19

SEQ ID NO:20

SEQ ID NO:21

SEQ ID NO:49

SEQ ID NO:50

SEQ ID NO:51

h

SEQ ID NO:22

SEQ ID NO:23

SEQ ID NO:24

SEQ ID NO:52

SEQ ID NO:53

SEQ ID NO:54

i

SEQ ID NO:25

SEQ ID NO:26

SEQ ID NO:27

SEQ ID NO:55

SEQ ID NO:56

SEQ ID NO:57

j

SEQ ID NO:28

SEQ ID NO:29

SEQ ID NO:30

SEQ ID NO:58

SEQ ID NO:59

SEQ ID NO:60

。

In some embodiments, the antibody or antigen binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), the amino acid sequences of which HCVR and LCVR have any one of the group consisting of the combinations of variable region sequences set forth in A1, A2, and B-J:

/>

。

variants of antibodies or antigen binding fragments thereof are also within the scope of the invention, the antibodies or antigen binding fragments thereof of the invention comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise a mutation of up to 3 amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof) respectively, as compared to any of the sets of complementarity determining regions set forth in a-j; preferably, the mutation is a conservative mutation. In some embodiments, the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the antibody or antigen binding fragment thereof respectively comprise up to 3 amino acid substitutions (e.g., 1, 2 or 3 amino acid substitutions) compared to any one of the sets of complementarity determining regions set forth in a-j.

In some embodiments, an antibody or antigen binding fragment thereof of the invention comprises a HCVR whose amino acid sequence comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the sequences set forth in SEQ ID NOS.61-71. In other embodiments, the amino acid sequence of the HCVR comprises any of the sequences shown in SEQ ID NOS.61-71. In a specific embodiment, the amino acid sequence of the HCVR is as set forth in any one of SEQ ID NOS: 61-71. In some embodiments, the antibody or antigen binding fragment thereof comprises a HCVR whose amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the sequences set forth in SEQ ID NOs 61-71 and/or whose amino acid sequence comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the sequences set forth in SEQ ID NOs 72-82. In other embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR whose amino acid sequence comprises any of the sequences shown in SEQ ID NOs 61-71 and a LCVR; and/or the amino acid sequence of LCVR comprises any of the sequences shown in SEQ ID NOS.72-82. In a specific embodiment, the HCVR has an amino acid sequence as set forth in any one of SEQ ID NOs 61-71; and/or the amino acid sequence of said LCVR is as shown in any one of SEQ ID NOS: 72-82.

In some embodiments, an antibody or antigen binding fragment thereof of the invention comprises a HCVR and a LCVR, wherein the amino acid sequences of the HCVR and LCVR are at least 80% identical to the HCVR and LCVR sequences, respectively, described in any one of the sets of variable region sequence combinations set forth in A1, A2, and B-J. In some embodiments, the amino acid sequence of the HCVR has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the HCVR sequence shown in A1, A2 and B-J; the amino acid sequence of the LCVR has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the LCVR sequence shown in A1, A2, and B-J. In other embodiments, the amino acid sequences of the HCVR and LCVR are selected from any of the group consisting of A1, A2, and the combinations of variable region sequences shown in B-J.

In some cases, the variant of the antibody or antigen binding fragment thereof comprises at least the 6 CDRs described above; in some cases, variants of an antibody include at least one heavy chain and one light chain, while in other cases, variant forms contain two identical light chains and two identical heavy chains (or sub-portions thereof). In some cases, the variants are obtained by conservative mutations (e.g., conservative substitutions or modifications) in the antibody sequences provided herein. "conservative mutations" refer to mutations, preferably conservative substitutions, that normally maintain the function of a protein.

"conservative substitutions" refer to the replacement of an amino acid in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein.

Substitutions generally considered to be conservative substitutions are those in aliphatic amino acids Ala, val, leu and Ile with each other, the exchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the exchange between amide residues Asn and Gln, the exchange of basic residues Lys and Arg, and the exchange between aromatic residues Phe, tyr. Those skilled in The art know that in general, single amino acid substitutions in The non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al (1987) Molecular Biology of The Gene, the Benjamin/Cummings pub. Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.

The modification may be a derivative obtained by natural processes (such as processing and other post-translational modifications), or by chemical modification techniques, for example by the addition of one or more polyethylene glycol molecules, sugars, phosphates and/or other such molecules, wherein one or more molecules are not naturally attached to the protein. Derivatives include salts. Such chemical modifications are described in detail in basic textbooks and in more detailed monographs, as well as in a number of research literature, and are well known to those skilled in the art. It will be appreciated that the same type of modification may be present to the same or different extent at several sites in a given antibody or antigen binding fragment thereof. In addition, a given antibody or antigen binding fragment thereof may contain many types of modifications. Modifications may occur anywhere in the antibody or antigen binding fragment thereof, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking formation, cysteine formation, pyroglutamic acid formation, methylation, gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydrocarbylation and ADP-ribosylation, selenization, sulfation, transfer RNA-mediated addition of amino acids of proteins (such as arginylation), and ubiquitination. They may also bind to vitamins, such as biotin, folic acid, or vitamin B12. See, e.g., proteins-Structure And Molecular Properties,2nd Ed., T.E.Creighton, W.H.Freeman and Company, new York (1993) and Wold, f., "Posttranslational Protein Modifications: perspectives and Prospects, "pgs.1-12in Posttranslational Covalent Modification Of Proteins,B.C.Johnson,Ed," Academic Press, new York (1983); seifter et al, meth. Enzymol.182:626-646 (1990) and Rattan et al, "Protein Synthesis: posttranslational Modifications and Aging, "ann.n.y.acad.sci.663:48-62 1992).

The variants retain the ability to specifically bind cTnI. One of ordinary skill in the art will be able to determine suitable variants of the antigen binding molecules as set forth herein using well known techniques. In certain embodiments, one of skill in the art can identify suitable regions in an antibody or antigen binding fragment thereof that are not important for activity to specifically bind cTnI to alter without disrupting activity. For nucleotide and amino acid sequences, the term "identity" indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

All the above types of antibodies have good binding ability to human cTnI.

In the present invention, the term "antibody" is a protein that binds to a specific antigen, which refers broadly to all proteins and protein fragments, particularly full length antibodies, that contain CDR regions. The term "full length antibody" includes polyclonal antibodies as well as monoclonal antibodies, and the term "antigen binding fragment" is a substance comprising a portion or all of the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. In some embodiments, the antibody functional fragment has the effect of specifically recognizing and binding cTnI. In one aspect, such fragments will comprise a single heavy chain and a single light chain, or portions thereof. The fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

The term "complementarity determining regions" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al (Kabat et al Sequences of proteins of immunological interest,5th Ed"US Department of Health and Human Services,NIH,1991, and later versions) are employed in the present invention. There are three heavy chain CDRs (HCDR) and three light chain CDRs (LCDR). Here, the terms "CDR" and "CDRs" are used to refer to regions comprising one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope, depending on the circumstances.

The term "antigen binding fragment" includes antigen compound binding fragments of these antibodies, including Fab, F (ab') ₂ Fd, fv, scFv, antibody minimal recognition units, and single chain derivatives of these antibodies and fragments, e.g., scFv-Fc and the like, preferably F (ab') ₂ 、Fab、scFv。

The term "F (ab') ₂ "is obtained after pepsin digestion of the entire full length antibody to remove most of the Fc region while leaving some of the hinge region intact. F (ab') ₂ Fragments have two antigen-binding Fab portions linked together by a disulfide bond, thus F (ab') ₂ The fragment is a bivalent antibody, and F (ab') prepared from an IgG antibody ₂ For example, the molecular weight is about 110kDa.

The term "Fab" is an antibody structure that is monovalent and does not contain an Fc portion, yet can bind to an antigen. Papain digested the full length antibody gave two Fab fragments, each of about 50kDa, and one Fc fragment.

The term "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior artThe operative linker is a flexible or rigid linker, e.g. consisting of a repeated GGGGS amino acid sequence or variant thereof, e.g. using 1-4 repeated variants (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), cancer Immunol.

The terms "specifically bind" and "specifically bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 ^-7 M, e.g. less than about 10 ^-8 M、10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M or greater affinity (K _D ) And (5) combining.

In some embodiments, the antibody or antigen binding fragment thereof further comprises an antibody constant region sequence.

In some embodiments, the heavy chain constant region sequence is selected from the constant region sequences of any of IgG, igA, igM, igE, igD. In some embodiments, the heavy chain constant region sequence may be selected from a human heavy chain constant region, wherein IgG may be further divided into subclasses, such as IgG1, igG2, igG3, or IgG4.

In some embodiments, the light chain constant region is a kappa or lambda chain.

In some embodiments, the heavy chain constant region of the antibody is IgG; the light chain constant region is a kappa chain.

In some embodiments, the heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO. 83, or a sequence that is at least 80%, 85%, 90%, 93%, 95%, 97% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 83.

In some embodiments, the amino acid sequence of the light chain constant region is as set forth in SEQ ID NO. 84, or a sequence that is at least 80%, 85%, 90%, 93%, 95%, 97% or 99% identical to the amino acid sequence set forth in SEQ ID NO. 84.

In some embodiments, the constant region is of a species source selected from the group consisting of rabbit, cow, horse, pig, sheep, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, or human.

In some embodiments, the antibody or antigen binding fragment thereof is a rabbit antibody, chimeric antibody, or humanized antibody.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a first animal-derived antibody is fused to a constant region of a second animal-derived antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting a first animal-derived specific monoclonal antibody, cloning a variable region gene from hybridoma cells, cloning a constant region gene of a second animal-derived antibody according to requirements, connecting the first animal-derived variable region gene and the second animal-derived constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the invention, the first animal source is rabbit and the second animal source is preferably human, so that the immune response elicited by the first animal source antibody is reduced. The antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of a human IgG1, igG2, igG3, igG4 or variant thereof, preferably comprises a human IgG1, igG2 or IgG4 heavy chain constant region, or an IgG1, igG2 or IgG4 variant using amino acid mutations (such as YTE mutations).

The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting CDR sequences of a first animal origin into the framework of human antibody variable regions, i.e., into framework sequences of different types of human germline antibodies. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes are available, for example, in the "VBase" human germline sequence database (www.mrccpe.com.ac.uk/VBase) and are found in Kabat, E.A. et al, 1991,Sequences of Proteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the invention also include humanized antibodies that are further affinity matured for the CDRs by phage display. In a preferred embodiment of the invention, the first animal source is a rabbit source. The framework of the human antibody variable region is chosen, for example, wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from the combined sequence of human germline heavy chains IGHV1-18 x 01 and hjh 6.1.1, or the combined sequence of human germline heavy chains IGHV1-3 x 01 and hjh 6.1.1; wherein the light chain FR region sequence on the light chain variable region of said antibody is derived from the combined sequence of human germline heavy chain IGKV1-39 x 01 and hjk 4.1. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region may be subjected to minimal reverse mutation to maintain activity.

Grafting of CDRs may result in reduced affinity of the generated cTnI antibody or antigen binding fragment thereof to the antigen due to framework residues that contact the antigen. Such interactions may be the result of somatic hypermutations. Thus, it may still be desirable to graft such donor framework amino acids to the framework of a humanized antibody. Amino acid residues involved in antigen binding from a non-human cTnI antibody or antigen binding fragment thereof can be identified by examining the sequence and structure of the variable region of a first animal-derived (e.g., rabbit) monoclonal antibody. Residues in the CDR donor framework that differ from the germline can be considered relevant. If the closest germ line cannot be determined, the sequence can be compared to a subtype consensus sequence or a consensus sequence of a first animal origin (e.g., rabbit) sequence with a high percentage of similarity. Rare framework residues are thought to be the result of highly mutated somatic cells, thereby playing an important role in binding.

In some embodiments, the antibody or antigen binding fragment thereof is a monoclonal antibody.

In some embodiments, the antibody or antigen binding fragment thereof has a detectable label.

The detectable label may be selected from any one or more of chromophores, digoxin-labeled probes, electron dense substances, colloidal gold or enzymes. These labels are listed in the following non-limiting section:

Enzymes that produce detectable signals, such as by colorimetry, fluorescence and luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.

Chromophores such as fluorescence, quantum dots, fluorescent microspheres, luminescent compounds and dyes.

Groups having electron densities that can be detected by electron microscopy or by their electrical properties, such as conductivity, amperometry, voltage measurement, and resistance.

A detectable group, such as a molecule of sufficient size to induce a modification detectable in its physical and/or chemical properties; such detection may be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variation and angle of contact variation) or physical methods (e.g., atomic force spectroscopy and tunneling).

Electronically dense substances, e.g. radioactive molecules (e.g ³² P， ³⁵ S or ¹²⁵ I)。

In some embodiments, the detectable label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, ruthenium terpyridyl, isoluminol, or a rare earth element, preferably alkaline phosphatase, acridinium ester, or horseradish peroxidase.

The invention also relates to a composition or combination product comprising one or more of the antibodies or antigen binding fragments thereof as described above.

When selected from a-j, it means that it is a composition of antibodies (in a mixed state, e.g., polyclonal antibodies) or a combination product (at least one antibody or antigen binding fragment thereof is a separately packaged product).

The antigen epitopes aimed by different antibodies in the invention are respectively:

in some embodiments, the HCVR and LCVR of the antibody or antigen binding fragment thereof have at least one group selected from the group consisting of the variable region amino acid sequence combinations of A1, A2, and B-J:

。

in a preferred embodiment, the composition or combination product contains antibodies from 2 or 3 different epitopes thereof for combined use, e.g. ac, ad, ae, af, ag, ah, ai, aj, ach, ade, aij, bc, bd, be, bf, bg, bh, bi, bj, ch, ci, cj, dh, di, dj, eh, ei, ej, fh, fi, fj, gh, gi, gj, etc. The combination of subordinate antibodies in the composition or combination product is not particularly limited, and for example, a may be A1 and/or A2 (e.g., A2 and I, A2 and J, A1 and I, A1 and J may be combined). In some embodiments, the composition or combination has at least one antibody selected from i. In some embodiments, the composition or combination has at least one antibody selected from j. In some embodiments, the composition or combination has at least one antibody selected from a and at least one antibody selected from j.

The invention also relates to nucleic acids encoding an antibody or antigen binding fragment thereof as described above.

Herein, nucleic acids include conservatively substituted variants thereof (e.g., substitution of degenerate codons) and complementary sequences, as well as variants that are optimized by codons for more efficient expression in the desired host cell. Nucleic acids are typically RNA or DNA, including genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acids are preferably used when they are incorporated into vectors.

Furthermore, since antibodies are membrane proteins, nucleic acids typically carry signal peptide sequences.

The invention also relates to a vector comprising a nucleic acid as described above.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

In the present invention, the vector may be a composition, for example, a mixture of plasmids, different plasmids carrying a portion of an antibody or antigen binding fragment thereof.

The invention also provides a host cell comprising a nucleic acid as described above or a vector as described above.

Host cells or cell lines suitable for expressing the antigen binding proteins of the invention include: mammalian cells such as NS0, sp2/0, CHO, COS, HEK, fibroblasts and myeloma cells. Human cells can be used, thus allowing the molecule to be modified with a human glycosylation pattern. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening, and product generation and purification, are known in the art.

It can be demonstrated that bacterial cells can be used as host cells suitable for expressing the recombinant Fab or other embodiments of the invention. However, since proteins expressed in bacterial cells tend to be in an unfolded or incorrectly folded form or in a non-glycosylated form, any recombinant Fab produced in bacterial cells must be screened to preserve antigen binding capacity. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell will be the desired host, or, in alternative embodiments, the molecule may be expressed in a bacterial host, followed by refolding. For example, various E.coli strains for expression are well known host cells in the biotechnology field. Various strains of Bacillus subtilis, streptomyces, other Bacillus, and the like may also be used in the method.

Yeast cell strains known to those skilled in the art, as well as insect cells, such as Drosophila and lepidopteran insects and viral expression systems, can also be used as host cells, if desired.

In some embodiments, the nucleic acid is inserted into the genome of the cell and is capable of stable expression.

The manner of insertion may be by way of a vector as described above, or by direct transfer of the nucleic acid into the cell without attachment of the vector (e.g., liposome-mediated transfection techniques).

The invention also relates to a method of producing an antibody or antigen-binding fragment thereof, comprising:

culturing a host cell as described above under suitable culture conditions; and

recovering the antibody or antigen-binding fragment thereof so produced from the culture medium or from the cultured cells.

The culture method of the present invention is usually a serum-free culture method, and cells are usually cultured by serum-free suspension. Similarly, once the antibodies of the invention are produced, they can be purified from the cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Such techniques are within the skill of the art and do not limit the present invention. Another method of expressing antibodies may utilize expression in animals, particularly transgenic animals or nude mice. This involves an expression system that utilizes animal casein promoters, which when transgenically incorporated into a mammal, allow a female to produce the desired recombinant protein in its milk. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The invention also provides a detection product comprising an antibody or antigen-binding fragment thereof as described above.

The invention also relates to the use of an antibody or antigen binding fragment thereof as described above for the preparation of a cardiac troponin I assay product.

The detection product comprises a reagent, a test strip or a kit.

The detection product can be used for diagnosing cTnI related diseases. Thus, in particular, the present invention also relates to a method for diagnosing cTnI-related diseases in a subject in need thereof, the method comprising:

a) Contacting cTnI in the test sample with an antibody or antigen binding fragment thereof as described above under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) Detecting the presence of the immune complex, the presence of the complex being indicative of the presence of cTnI in the test sample.

The cTnI-related disease is selected from, for example, diagnosing acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, and myocardial injury.

In some embodiments, in step a), two antibodies are included in the immune complex, both of which bind to the cTnI;

in this embodiment, the antibody or antigen binding fragment thereof forms a partner antibody in the form of a first antibody with the second antibody for binding to a different epitope of cTnI.

Any cTnI-containing sample is suitable for use in the present invention and may be derived from a tissue or organ, tissue lysate of an animal (preferably including at least a mammal, such as a primate, including a human); cells (cells in a subject, taken directly from a subject, or maintained in culture or from a cultured cell line), cell lysates (or lysate fractions), or cell extracts; or a solution containing a naturally or non-naturally occurring (e.g., artificially expressed cTnI) cTnI, which is or can be assayed as described herein. In particular, the biological samples of the present invention are preferably derived from body fluids, including liquids from animals, semi-solids, aerated liquids, liquid-gas mixtures, and the like. Such body fluids may include, but are not limited to, saliva, sputum, serum, plasma, blood, urine, mucus, amniotic fluid or lymph.

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

The invention obtains the high-affinity monoclonal antibodies aiming at three different epitopes of human cardiac troponin I22-40, 39-51 and 81-96 through rabbit monoclonal antibody technology, and can be used for detecting the human cardiac troponin I through chemiluminescence technology and immunochromatography technology. The invention develops a chemiluminescence detection method for detecting troponin I antigen in a test sample by using the provided antibody, and the detection method has good linearity and high sensitivity.

EXAMPLE 1 hybridoma cell line acquisition of Rabbit monoclonal antibody

1. New Zealand rabbits were immunized with three different sequences of polypeptides, human cardiac troponin 22-40 epitope, 39-51 epitope and 81-96 epitope (sequences shown in Table 1).

TABLE 1

Epitope(s)	Sequence(s)
		22-40	CSNYRAYATEPHAKKKSKI(SEQ ID NO:85)
39-51	CISASRKLQLKTL(SEQ ID NO:86)
		81-96	PLELAGLGFAELQDLC(SEQ ID NO:87)

1.1 preparation of a macromolecular immunogen by coupling three epitope polypeptide fragments to a carrier protein KLH (Sigma-Aldrich, CAT#: 374805);

1.2 mixing 200. Mu.g of immunogen with 200. Mu.L of Freund's complete adjuvant (Sigma-Aldrich, CAT#: F5881) in a volume ratio of 1:1 to form an emulsion, subcutaneously immunizing 2 New Zealand rabbits each with each immunogen, and immunizing 6 New Zealand rabbits with the three immunogens;

1.3 subcutaneous injections of a 1:1 emulsion containing 200 μg of immunogen and Freund's incomplete adjuvant (Sigma-Aldrich, CAT#: F5506) 4 times every 2 weeks to boost the New Zealand rabbits;

1.4 serum of animals is diluted according to an initial proportion of 1:1,000 4 days before myeloma fusion, then is diluted in a gradient way of 2 times proportion, and the serum antibody titer is detected by an indirect ELISA method;

indirect ELISA detection method:

(1) ELISA plates were coated overnight at 4deg.C with 1. Mu.g/mL of three epitope polypeptides of human cardiac troponin I and recombinant human cardiac troponin I (GenScript, CAT#: Z03320) in 100. Mu.L/well PBS, respectively;

(2) Plates were washed with PBS-T (0.05% Tween 20) and blocked with 200. Mu.L/well of 1% BSA in PBST for 0.5 hours at 37 ℃;

(3) The blocking solution was aspirated, 100. Mu.L of the above-mentioned animal serum and its dilutions were added to each well, and then incubated at room temperature for 1 hour;

(4) After the incubation, the supernatant was discarded, the plate was washed three times with PBST and incubated with 100. Mu.L/well of mouse anti-rabbit IgG Fc (GenScript, CAT#: A01856) conjugated with horseradish peroxidase for 0.5 hours at 37 ℃;

(5) Plates were washed five times with PBST, then TMB color development was added and incubated in the dark for 15 minutes at room temperature. The reaction was stopped by adding 50. Mu.L of 1M HCl stop solution, reading the plate at 450nm using a microplate reader, passing through the OD ₄₅₀ The readings were used to determine the serum titers of the animals to be fused (as shown in table 2).

TABLE 2

1.5 results showed that animals #6645 for 39-51 epitope, #6575 for 22-40 epitope and #6597 for 81-96 epitope reacted well with human cardiac troponin polypeptide and recombinant human cardiac troponin I, and that the animals were immunized intraperitoneally and intravenously with 400. Mu.g immunogen (without adjuvant).

2. Hybridoma fusion and screening

2.1 spleens of the above-mentioned post-boost rabbits were extracted and homogenized to produce single cell suspensions, while myeloma cell single cell suspensions were prepared. Fusing spleen cells with myeloma cells using electrofusion;

2.2 the fused cells were resuspended in 100mL DMEM/10% FBS medium containing the hybridoma cell selection agents thymine nucleoside pyrimidine, hypoxanthine and aminopterin, and pipetted into 96-well plates in a volume of 100. Mu.L using a pipette;

2.3 plates were incubated at 37℃with 6% CO ₂ And (3) incubating. After 9 days incubation, the binding capacity of the antibodies in the cell supernatant to recombinant human cardiac troponin I was assessed by reference to the indirect ELISA assay of 1.4, indicating the presence of antibodies recognizing recombinant human cardiac troponin I.

3. Hybridoma subcloning was performed by limiting dilution.

3.1 serial dilutions of cells in DMEM/10% fbs medium containing hybridoma cell selective agents thymine nucleoside pyrimidine, hypoxanthine and aminopterin were used to determine cell number using a hemocytometer until cell density reached 5-15/mL;

3.2 for each hybridoma, 200. Mu.L of the cell solution was pipetted into 96 wells with a density of 1-3 cells/well;

3.3 cultures were incubated at 37℃at 5% CO ₂ After 1 week of culture, the cell supernatants were subjected to binding assays by referring to the indirect ELISA assay method of 1.4, and the cell supernatant stock of each clone was subjected to 3-fold dilution 6 times by using human cardiac troponin I epitope polypeptide and recombinant human cardiac troponin I-coated ELISA assay plates, respectively, and titers were calculated from the assay values. As shown in Table 3, the supernatants of the 11 subclones were positive for both human cardiac troponin I epitope polypeptide and recombinant human cardiac troponin I binding.

TABLE 3 Table 3

EXAMPLE 2 variable region sequencing of antibodies and recombinant production of antibodies

From 3X 10 using TRIzol (Ambion, CAT#: 15596-026) ⁶ –5×10 ⁶ Total RNA was extracted from each hybridoma cell and reverse transcribed into cDNA using antibody subtype specific primers and universal primers (Takara, CAT#: 6110A). Rabbit immunoglobulin heavy and light chain variable region fragments were then amplified by RACE PCR and the resulting PCR fragments subcloned into the pMD18-T vector system (Takara, CAT#: 6011) and the insert fragments were sequenced using vector-specific primers. Finally obtaining the variable region nucleotide/protein sequences of the heavy chain and the light chain of 11 strains of rabbit monoclonal antibodies, comprising 5 strains of rabbit monoclonal antibodies aiming at 22-40 epitopes, namely clone I40.F8-1, clone I40.C5-3, clone I40.H7-2, clone I40.F12-1 and clone I40.F10-1; 3 strains of rabbit monoclonal antibodies aiming at 39-51 epitope are clone I50.F12-1, clone I50.F12-3 and clone I50.G11-2 respectively; 3 strains of rabbit monoclonal antibodies directed against the 81-96 epitope were clone I80.C1-1, clone I80.E3-1 and clone I80.C10-1, respectively. The antibodies are produced by recombinant expression, taking one clone I50.G11-2 as an example:

1. Respectively synthesizing DNA fragments comprising a light chain variable region and a constant region (the light chain variable region has an amino acid sequence shown as SEQ ID NO:74, the constant region has an amino acid sequence shown as SEQ ID NO: 84) and a heavy chain variable region and a constant region (the heavy chain variable region has an amino acid sequence shown as SEQ ID NO:63 and the constant region has an amino acid sequence shown as SEQ ID NO: 83), respectively inserting the DNA fragments into pTT5 expression vectors to form expression plasmids;

2. the plasmid is co-transfected with CHO-3E7 cells, and after 6 days of culture in shake flask at 37 ℃, the supernatant is collected for antibody purification;

3. the tubing and protein A column were depyrogenated with 0.2M NaOH, and the column was then re-equilibrated with buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0);

4. the harvested cell culture supernatant was diluted 1:1 with 2 Xthe above buffer and sterilized by filtration. The filtered supernatant and protein a column were incubated for 2 hours at room temperature,

5. after washing the column with 1 Xof the above buffer, igG was eluted using sterile 0.1M sodium citrate (pH 3.5), the eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH 9.0);

6. under sterile conditions, the product buffer was exchanged to PBS (pH 7.4) to remove any elution buffer and concentrate the sample. After concentration, the extinction coefficient Ec of 1.43 (0.1%) was used to pass through the OD ₂₈₀ Quantifying the antibodies at nm;

7. purified antibodies were analyzed for purity by SDS-PAGE. When analyzed by the reduction and non-reduction SDS-PAGE method, 10% of the pre-prepared gel (GenScript, CAT#: M42012) was stained with eStain2.0 (GenScript), analyzed by the BioRad electrophoresis system, the molecular size and purity were estimated by comparing the stained band with the Protein Ladder, and the sample treatment required addition of the reducing agent during the reduction test (FIG. 1, lane M was Protein Marker (TaKaRa, CAT#: 3452), lane 1 was clone I50.G11-2 under reduction, lane 2 was control rabbit IgG under reduction, lane 3 was clone I50.G11-2 under non-reduction, and lane 2 was control rabbit IgG under non-reduction).

8. The other 10 purified antibodies were analyzed for purity by SDS-PAGE or SEC-HPLC methods.

When the SDS-PAGE method is used for analysis, the purity and the molecular weight of the target antibody can be determined by non-reducing SDS-PAGE, the purity is not lower than 90%, the reducing SDS-PAGE can degrade the antibody into a heavy chain and a light chain, the structural purity of the subunit of the detected antibody is not lower than 90% as shown in steps 1-7 in the embodiment;

when analyzed by SEC-HPLC method, the purity was not lower than 90% by analysis on instrument Agilent 1260 (Agilent) at a flow rate of 0.7mL/min at room temperature using a chromatographic column TSKgel G3000SWxl (Tosohbioscience, CAT: 08541). The purity identification results of the 11 antibodies are shown in Table 4, and the results show that the purity of the 11 antibodies is more than 90%, meets the standards of antibody production and purification, and can be used for subsequent experimental study.

TABLE 4 Table 4

Clone numbering	Detection mode	Purity (%)
			I50.F12-1	Reducing condition SDS-PAGE	99
I50.F12-1	Non-reducing condition SDS-PAGE	99
			I50.F12-3	Reducing condition SDS-PAGE	99
I50.F12-3	Non-reducing condition SDS-PAGE	99
			I50.G11-2	Reducing condition SDS-PAGE	99
I50.G11-2	Non-reducing condition SDS-PAGE	98
			I40.C5-3	Reducing condition SDS-PAGE	99
I40.C5-3	Non-reducing condition SDS-PAGE	94
			I40.H7-2	Reducing condition SDS-PAGE	99
I40.H7-2	Non-reducing condition SDS-PAGE	94
			I40.F12-1	Reducing condition SDS-PAGE	99
I40.F12-1	Non-reducing condition SDS-PAGE	96
			I40.F10-1	Reducing condition SDS-PAGE	99
I40.F10-1	Non-reducing condition SDS-PAGE	96
			I40.F8-1	SEC-HPLC	99
I80.C10-1	SEC-HPLC	91
			I80.C1-1	SEC-HPLC	96
I80.E3-1	SEC-HPLC	96

Example 3 determination of antibody affinity

1. The probe with the surface coupled Protein A Protein is soaked in 250 mu L buffer K (PBS+0.002% Tween 20+0.02% BSA) for 10 minutes;

2. the 11 antibodies were prepared as 5. Mu.g/mL working solution using buffer K;

3. preparing three working solutions with the concentration of 5 mug/mL, 2.5 mug/mL and 0 mug/mL by using buffer K for recombinant human cardiac troponin I;

4. according to the Gator non-labeled analyzer (Star child medical technology, CAT#: gator) indicated the addition of reagent, the affinity data are shown in Table 5, the partial binding curves are shown in FIG. 2, and the results show that the 11 cloned antibodies have higher affinity levels with human cardiac troponin I, wherein the K of 9 antibodies in the 11 antibodies _D In the low nanomolar range (10 ^-9 ) K belonging to high affinity antibody, 2-strain antibody _D In the picomolar range (10) ^-12 ) Belonging to antibodies with very high affinity.

TABLE 5

Clone numbering	koff(1/s)	kon(1/Ms)	K D (M)
				I50.F12-1	6.55E-04	1.60E+05	4.10E-09
I50.F12-3	6.42E-04	1.85E+05	3.48E-09
				I50.G11-2	9.33E-04	1.87E+05	5.00E-09
I40.F8-1	3.63E-04	5.32E+04	6.81E-09
				I40.C5-3	3.10E-04	7.60E+04	4.07E-09
I40.H7-2	2.10E-04	6.45E+04	3.25E-09
				I40.F12-1	2.36E-04	7.58E+04	3.11E-09
I40.F10-1	1.61E-04	3.81E+04	4.24E-09
				I80.C1-1	1.24E-04	1.36E+05	9.13E-09
I80.C10-1	<10E-6	6.87E+04	<10E-12
				I80.E3-1	<10E-6	4.20E+04	<10E-12

EXAMPLE 4 application of antibody I50.F12-3 to detection of human cardiac troponin I by chemiluminescence

1. Preparation of detection antibodies conjugated to Alkaline Phosphatase (AP)

1.1 1mg (4 mg/mL,0.25 mL) of detection antibody clone 24E40HC (GenScript, CAT#: V00308) was mixed with 6.7. Mu.L of Traut's Reagent (Thermo, CAT#: 26101) solution at a concentration of 13.76mg/mL, and after mixing, the mixture was allowed to stand at room temperature for 20 minutes to activate the detection antibody;

1.2 mixing 1mg (50. Mu.L, 20 mg/mL) of alkaline phosphatase solution (Roche, CAT#: 03535452103) with 3. Mu.L of (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Thermo, CAT#: 22322) solution at a concentration of 17.5mg/mL, standing at room temperature for 20 minutes after mixing uniformly, and activating the alkaline phosphatase solution;

1.3 adding 6-14 mu L of 1M glycine solution into the activated detection antibody solution, uniformly mixing, standing at room temperature for 10 minutes, adding 1-2 mu L of 1M glycine solution into the activated alkaline phosphatase solution, uniformly mixing, and standing at room temperature for 10 minutes;

1.4 Zeba ^TM The rotary desalting column (Thermo, CAT#: 89891) was used to replace the buffer (0.1M triethanolamine; 5mM Mg) with the two solutions respectively ²⁺ ；1mM Zn ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the pH 7.2-7.4), and collecting the activated detection antibody solution and alkaline phosphatase solution;

1.5 adding 0.8mg of alkaline phosphatase solution into 1mg of detection antibody solution, mixing uniformly, standing at 2-8 ℃ for reaction for 18-24 hours;

1.6 adding 8-10 mu L of 12.5mg/mL N-ethyl maleimide solution into the reactant of alkaline phosphatase coupled detection antibody, and standing at room temperature for 30 minutes;

1.7 Zeba ^TM Rotary desalting column (Thermo, CAT#: 89891) displacement buffer (0.1M triethanolamine; 5mM Mg) ²⁺ ；1mM Zn ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the 0.05% ProClin300 (v/v); pH 7.2-7.6), and obtaining alkaline phosphatase coupled detection antibody concentrate.

2. Preparation of magnetic particle coupling coated antibody I50.F12-3

2.1 the supernatant was separated magnetically by placing 500. Mu.L of magnetic particles (JSR, MS 160/Carboxyl) at a concentration of 100mg/mL into a 10mL centrifuge tube. The method comprises the steps of carrying out a first treatment on the surface of the

2.2 magnetic particles were resuspended in 5mL of MES buffer (0.1 mol/L4-morpholinoethanesulfonic acid solution; pH 5.0), and the supernatant was magnetically separated by mixing for 1-5 min. Repeating the washing step 1 time;

2.3 adding 3.2mL MES buffer (0.1 mol/L; pH 5.0) to the centrifuge tube, re-suspending the magnetic particles, and mixing for 1-5min;

2.4 adding 10mg/mL NHS (Thermo, CAT#: 24500) solution 1.15mL and 10mg/mL EDC (Thermo, CAT#: 22980) solution 0.5mL into the centrifuge tube, and uniformly mixing at room temperature for reaction for 30min;

2.5 after the reaction, magnetically separating the supernatant, adding 4.35mL MES buffer (0.1 mol/L; pH 5.0) to resuspend the magnetic particles, and uniformly mixing for 1-5min;

2.6 adding 1mg of coated antibody I50.F12-3 into the centrifuge tube, and uniformly mixing at room temperature for reaction for 3-20h;

2.7 adding 250 mu L of blocking solution (JSR, CE 210) into the centrifuge tube, and uniformly mixing at room temperature for reaction for 18-24h;

2.8 magnetic separation of supernatant, re-suspending the magnetic particles with 5mL of washing solution (25mM Tris;150mM NaCl;0.1%BSA;0.05%Tween-20 (v/v); 0.1%ProClin300;pH 7.4-7.5), mixing for 1-5min, and magnetic separation of supernatant. Repeating the washing step for 2-3 times;

2.9 adding 10mL of the diluent prepared in the step 3, shaking and uniformly mixing for 1-5min, re-suspending the magnetic particles to 5mg/mL, and dispersing the aggregated magnetic beads in the suspension into a single particle state by using an ultrasonic shaking instrument to obtain a magnetic separation reagent concentrated solution;

2.10 diluting the above liquid with magnetic separation reagent diluent to obtain magnetic particles with concentration of 0.1-0.5mg/mL, coating antibody I50.F12-3 and coupling magnetic particles with concentration of 2-10 μg/mL to I50.F12-3.

3. Dilution liquid preparation

Adding 800mL of purified water, 3.5g of disodium hydrogen phosphate dodecahydrate and 0.26g of sodium dihydrogen phosphate dihydrate into a container, stirring and mixing uniformly, adding 8.5g of sodium chloride, stirring until the mixture is completely dissolved, adjusting the pH value to 7.0-7.5, adding 20g of sucrose, stirring until the mixture is completely dissolved, adding 0.5mL of Tween 20 and 10mL of bovine serum into the container, stirring until the mixture is completely dissolved, fixing the volume to 1L by using purified water, filtering by using a 0.22 mu m filter, and storing at 2-8 ℃ for standby.

4. Calibrator and detection antibody working solution dilution

4.1 human cardiac troponin I antigen (Genscript, CAT#: Z03320) was diluted with dilution to serial calibrator concentration spots: 50000pg/mL,10000pg/mL,2000pg/mL,500pg/mL,100pg/mL,20pg/mL,5pg/mL and 0 pg/mL.

4.2 the detection antibody concentrated solution of the coupled AP prepared in the step 1 of the embodiment is diluted to 1 mug/mL by using a diluent, and the detection antibody working solution is obtained.

5. Double antibody sandwich detection procedure

5.1 adding 25. Mu.L of calibrator or 25. Mu.L of sample, 50. Mu.L of detection antibody working solution and 50. Mu.L of magnetic particle coupled I50.F12-3 to the corresponding well in the detection tube, and incubating at 42℃for 10 min. After the completion, magnetically separating, and discarding the supernatant;

5.2 Add 300. Mu.L of wash (10 mM Tris,0.15M sodium chloride, 0.05% Tween 20) to the tube, mix well and magnetically separate the supernatant. Repeating the washing step for 3-4 times;

5.3 adding 100 μl of chemiluminescent substrate (CAT#: APSUB-1) into each well, mixing, and detecting the luminous intensity with chemiluminescent instrument;

5.4, calculating a result, namely fitting the concentration of the calibrator and the signal into a calibration curve by four parameters, wherein the signal of the calibrator is shown in a table 6, the standard curve is shown in fig. 3, and the sample signal value can be substituted into the corresponding curve to calculate the concentration;

TABLE 6

Standard substance concentration (pg/ml)	Relative luminescence unit mean (RLU)
		50000	30580000
10000	10850000
		2000	2076667
500	495067
		100	84387
20	18373
		5	6995
0	3154

5.5 establishment of the condition: correlation coefficient of calibration curve R ² Not less than 0.99, the result of the curve is true, which indicates that the detection of human cardiac troponin I antigen can be performed under the condition.

6. Performance evaluation

6.1 limit of detection: according to step 5 of this example, a low value sample having a concentration of about 5pg/ml was tested 10 times, the mean recovery rate tested should be in the range of 80%,120%, and the coefficient of variation CV should be no greater than 20%. The results are shown in Table 7, which demonstrate that the minimum detection limit of the detection method is not higher than 5pg/ml.

TABLE 7

6.2 linearities: the linear correlation coefficient r is calculated by using a least square method for testing 5 human cardiac troponin I antigen samples with the concentration of 10000pg/mL,2000pg/mL,500pg/mL,100pg/mL and 20pg/mL, and the test concentration and the theoretical concentration of the 5 samples, and the linear correlation coefficient r should not be lower than 0.99, and the results are shown in Table 8 and FIG. 4, which illustrate that the linear range of the detection method is not less than 20-10000pg/mL.

TABLE 8

Theoretical concentration (pg/ml)	Measuring value (pg/ml)
		10000	9436.8
2000	1788.3
		500	511.3
100	114.7
		20	19.4
Linear coefficient r	0.9999

6.3 accuracy: the human cardiac troponin I national standard was prepared into 10000, 1000 and 100pg/ml concentrations by using the dilution obtained in step 3 of this example, and tested as samples according to step 5 of this example, and the deviation of each concentration point measurement value from the theoretical value was within + -15%, and the results are shown in Table 9.

TABLE 9

EXAMPLE 5 application of antibody I80.C10-1 to detection of human cardiac troponin I by chemiluminescence

1. Preparation of detection antibodies conjugated to Alkaline Phosphatase (AP)

Detection antibody clone 24E40HC (GenScript, CAT#: V00308) was conjugated to Alkaline Phosphatase (AP) according to the method of example 4, step 1.

2. Preparation of magnetic particle coupling coated antibody I80.C10-1

The coated antibody I80.C10-1 was coupled to magnetic microparticles as described in example 4, step 2.

3. Dilution liquid preparation

The procedure of example 4, step 3, was followed.

4. Calibrator and detection antibody working solution dilution

Step 4 is the same as in example 4.

5. Double antibody sandwich detection procedure

The procedure is as in example 4, step 5, replaced with the corresponding calibrator, sample, magnetic particle conjugated antibody I80.C10-1 and AP conjugated detection antibody 24E40HC of this example.

Calculating a result, namely fitting the concentration of the calibrator and the signal into a calibration curve by four parameters, wherein the signal of the calibrator is shown in a table 10, the standard curve is shown in fig. 5, and the signal value of a sample can be substituted into the corresponding curve to calculate the concentration;

table 10

Standard substance concentration (pg/ml)	Relative luminescence unit mean (RLU)
		50000	18740000
10000	4434333
		2000	539600
500	127400
		100	26000
20	7002
		5	4157
0	2161

The curve establishment condition: correlation coefficient of calibration curve R ² Not less than 0.99, the result of the curve is true, which indicates that the detection of human cardiac troponin I antigen can be performed under the condition.

6. Performance evaluation

6.1 limit of detection: according to step 5 of this example, a low value sample having a concentration of about 5pg/ml was tested 10 times, the mean recovery rate tested should be in the range of 80%,120%, and the coefficient of variation CV should be no greater than 20%. The results are shown in Table 11, which shows that the lowest detection limit of the detection method is not higher than 5pg/ml.

TABLE 11

6.2 linearities: the linear correlation coefficient r is calculated by using a least square method for testing 5 human cardiac troponin I antigen samples with the concentration of 10000pg/mL,2000pg/mL,500pg/mL,100pg/mL and 20pg/mL, and the test concentration and the theoretical concentration of the 5 samples, and the linear correlation coefficient r should not be lower than 0.99, and the results are shown in Table 12 and FIG. 6, which illustrate that the linear range of the detection method is not less than 20-10000pg/mL.

Table 12

Theoretical concentration (pg/ml)	Measuring value (pg/ml)
		10000	9842.4
2000	1657.3
		500	508.2
100	107.9
		20	17.5
Linear coefficient r	0.9994

6.3 accuracy: the human cardiac troponin I national standard was prepared into 10000, 1000 and 100pg/ml concentrations by using the dilution obtained in step 3 of this example, and tested as samples according to step 5 of this example, and the deviation of each concentration point measurement value from the theoretical value was within + -15%, and the results are shown in Table 13.

TABLE 13

Theoretical concentration (pg/ml)	Measuring value (pg/ml)	Deviation (%)
			10000	8943.0	-11
1000	1030.5	3
			100	111.7	12

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.

Reference to the literature

[1].Greaser ML,Gergely J.Purification and properties of the components from troponin.JBiol Chem1973；248:2125–2133.

[2].Joanne Layland,R.John Solaro,Ajay M.Shah,Regulation of cardiac contractile function by troponin I phosphorylation,Cardiovascular Research,Volume 66,Issue 1,April 2005,Pages 12–21.

[3].Park KC,Gaze DC,Collinson PO,Marber MS.Cardiac troponins:from myocardial infarction to chronic disease.Cardiovasc Res.2017Dec 1；113(14):1708-1718.

[4].Thygesen K,Alpert J S,Jaffe A S.Third universal definition of myocardial infarction[J].European Heart Journal,2012,50(20):2173-2195.

[5].Cummins B,Auckland ML,Cummins P.Cardiac-specific troponin-I radioimmunoassay in the diagnosis of acute myocardial infarction.Am Heart J.1987Jun；113(6):1333-44.

[6].Davies E,Gawad Y,Takahashi M,Shi Q,Lam P,Styba G,Lau A,Heeschen C,Usategui M,Jackowski G.Analytical performance and clinical utility of a sensitive immunoassay for determination of human cardiac troponin I.Clin Biochem.1997Aug；30(6):479-90.

[7].Bodor GS,Porter S,Landt Y,Ladenson JH.Development of monoclonal antibodies for an assay of cardiac troponin-I and preliminary results in suspected cases of myocardial infarction.Clin Chem.1992 Nov；38(11):2203-14.

[8].Suetomi K,Takahama K.A sandwich enzyme immunoassay for cardiac troponin I.NihonHoigaku Zasshi.1995 Feb；49(1):26-32.

Claims (15)

1. An antibody or antigen binding fragment thereof capable of specifically recognizing cardiac troponin I comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 being selected from the group of complementarity determining region combinations represented by a-j:

combination name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 a SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33 b SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 SEQ ID NO:34 SEQ ID NO:35 SEQ ID NO:36 c SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39 d SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:40 SEQ ID NO:41 SEQ ID NO:42 e SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:43 SEQ ID NO:44 SEQ ID NO:45 f SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:46 SEQ ID NO:47 SEQ ID NO:48 g SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:49 SEQ ID NO:50 SEQ ID NO:51 h SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 SEQ ID NO:52 SEQ ID NO:53 SEQ ID NO:54 i SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27 SEQ ID NO:55 SEQ ID NO:56 SEQ ID NO:57 j SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:60

。

2. The antibody or antigen binding fragment thereof of claim 1, comprising a heavy chain variable region HCVR and a light chain variable region LCVR, the amino acid sequences of said HCVR and LCVR having a variable region sequence selected from the group consisting of A1, A2, and B-J:

。

3. the antibody or antigen-binding fragment thereof of claim 1 or 2, which is F (ab') ₂ Fab, scFv, and bispecific antibody.

4. The antibody or antigen binding fragment thereof of claim 1 or 2, which has a constant region, a heavy chain constant region sequence selected from the constant region sequences of any one of IgG, igA, igM, igE, igD; the light chain constant region is a kappa or lambda chain.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the heavy chain constant region of the antibody is IgG; the light chain constant region is a kappa chain.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO. 83 and the light chain constant region has an amino acid sequence as shown in SEQ ID NO. 84.

7. The antibody or antigen binding fragment thereof of claim 1, which is a rabbit antibody, chimeric antibody, or humanized antibody.

8. The antibody or antigen-binding fragment thereof of claim 1, which is a monoclonal antibody.

9. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, and 5-8, which has a detectable label.

10. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9.

11. A vector comprising the nucleic acid of claim 10.

12. A host cell comprising the nucleic acid of claim 10 or the vector of claim 11.

13. A composition or combination comprising one or more of the antibodies or antigen-binding fragments thereof of any one of claims 1-9.

14. A test product comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9; the detection product comprises a reagent, a test strip or a kit.

15. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for the preparation of a cardiac troponin I assay product; the detection product comprises a reagent, a test strip or a kit.

Images