CN115353566B - Antibody combination for detecting interleukin 1-beta and application thereof - Google Patents
Antibody combination for detecting interleukin 1-beta and application thereof Download PDFInfo
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Abstract
The present invention provides an antibody combination for detecting interleukin 1-beta, said antibody combination comprising a first antibody and/or a second antibody, the antibodies having unique CDR fragments; specific uses of the antibody combination are also provided. The invention has the advantages that the antibody combination has strong affinity to interleukin 1-beta antigen, high detection sensitivity, simple and convenient use and rapid and stable detection; the concentration of IL-1 beta in normal human blood is about (90.51 +/-5.03) pg/mL, the conventional low-affinity antibody is difficult to realize effective detection in the situation, and the high-affinity antibody combination and the quantitative detection kit containing the same, which adopts a double-antibody sandwich method, can effectively realize the quantitative detection of the interleukin 1-beta with ultralow concentration, and the sensitivity of the kit can reach 0.3pg/mL through verification.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to an antibody combination for detecting interleukin 1-beta and application thereof.
Background
Interleukins are a class of cytokines (also known as lymphocyte stimulators) produced by and acting on a variety of cells, primarily derived from activated monocytes-macrophages. Interleukin 1 exists mainly in the form of IL-1 alpha and IL-1 beta. Interleukins play an important role in the transmission of information, the activation and regulation of immune cells, the mediation of T, B cell activation, proliferation and differentiation, and in inflammatory responses.
The main biological functions are as follows:
(1) Local low concentration-immunomodulation: synergistic stimulation of APC and T cell activation, promotion of B cell proliferation and secretion of antibodies;
(2) Mass production-endocrine effect: inducing liver acute phase protein synthesis; causing fever and cachexia.
IL-1β is an important member of the IL-1 family, and is of great interest due to its important role in inflammation-related diseases. IL-1β has potent pro-inflammatory activity and can induce a variety of pro-inflammatory mediators, such as cytokines and chemokines. IL-1β has a variety of effects on various cells and ultimately leads to a broad range of inflammatory events. In the systemic sense, IL-1. Beta. Signaling can lead to acute phase reactions, hypotension, vasodilation, and fever. Locally, the result of IL-1 signaling leads to upregulation of adhesion molecules, thereby promoting lymphocyte recruitment. Subsequently, immune cells are activated and inflammation is further amplified. In addition to playing an important role in the innate immune response, IL-1β is involved in the adaptive immune response by affecting Th17 and Th 1-induced immune responses. Abnormal IL-1β signaling can cause hereditary auto-inflammatory diseases such as familial mediterranean fever. In addition, it is associated with common diseases such as type 2 diabetes, gout, systemic Lupus Erythematosus (SLE), and cardiovascular disease. IL-1β levels are markedly elevated in degenerated intervertebral discs (IVDs), and IL-1β is involved in many pathological processes of IDD.
However, since the concentration of IL-1β in normal human blood is about (90.51.+ -. 5.03) pg/ml, the existing biological products aiming at IL-1β are mostly low-affinity antibodies, and the detection accuracy and sensitivity of IL-1β are low.
Disclosure of Invention
In view of the limitations presented above, the present invention proposes an antibody combination for detecting interleukin 1-beta and its use; overcoming the deficiencies and drawbacks mentioned in the background art.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides an antibody combination for detecting interleukin 1-beta- (IL-1 beta), which comprises a first antibody and/or a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of a heavy chain variable region of the first antibody are respectively shown as the amino acid sequences of 26-34, 54-63 and 105-116 of SEQ ID No.1, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of a light chain variable region are respectively shown as the amino acid sequences of 24-32, 48-56 and 89-98 of SEQ ID No. 2; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the second antibody are respectively shown as the 26 th-33, 53 th-62 th and 104 th-114 th amino acid sequences of SEQ ID No.3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as the 24 th-35 th, 51 th-60 th and 93 th-102 th amino acid sequences of SEQ ID No. 4.
The first antibody was 11G10 and the second antibody was 7B9.
CDR1 of the first antibody heavy chain variable region is: DYKTVVAFT;
CDR2 of the first antibody heavy chain variable region is: NAVTYGGYPE;
CDR3 of the first antibody heavy chain variable region is: GIYSQVLYDYAM;
CDR1 of the first antibody light chain variable region is: SFTPFGHVA;
CDR2 of the first antibody light chain variable region is: PKFHJLYHS;
CDR3 of the first antibody light chain variable region is: FFYPBYAHKT;
CDR1 of the second antibody heavy chain variable region is: gmaSLINY;
CDR2 of the second antibody heavy chain variable region is: FGTDGTNKPE;
CDR3 of the second antibody heavy chain variable region is: DYGPPTHGDVY;
CDR1 of the second antibody light chain variable region is: QGGLPQWNWLPS;
CDR2 of the second antibody light chain variable region is: NHYTKLETAD;
CDR3 of the second antibody light chain variable region is: LAAGVKNDTT.
In a preferred embodiment, the combination of antibodies for detecting interleukin 1-beta, the amino acid sequence of the heavy chain variable region of said first antibody is shown as SEQ ID No.1 or an amino acid sequence having greater than 90% homology with the sequence of SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.2 or an amino acid sequence having greater than 90% homology with the sequence of SEQ ID No. 2; the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID No.3 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.4 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No. 4.
The first antibody heavy chain variable region amino acid sequence SEQ ID No.1 is:
EVQLVESGGGLVQPGGSLRLSCAASDYKTVVAFTGMNWVRQAPGKGLEWMGWINAVTYGGYPEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTA VYYCARGIYSQVLYDYAMWGQGTLVTVSS；
the amino acid sequence of the first antibody light chain variable region SEQ ID No.2 is:
DIQMTQSPSSLSASVGDRVTITCSFTPFGHVAWYQQKPGKAPKALIYPKFHJLYHSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCFFYPBYAHKTFGQGTKVEIKRTVA；
the second antibody heavy chain variable region amino acid sequence SEQ ID No.3 is:
DVQLVESGGGLVQPGRSLKLSCAASGMASLINYYMAWVRQAPTKGLEWVASIFGTDGTNKPETFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTAT YYCTTDYGPPTHGDVYWGQGTLVTVSS；
the second antibody light chain variable region amino acid sequence SEQ ID No.4 is:
DIQMTQSPASLPASPEEIVTITCQGGLPQWNWLPSWYQQKPGKSPQLLIYNHYTKLETADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLAAGVKNDT TFGAGTKLELKRTDA。
in a preferred embodiment, the combination of antibodies for detecting interleukin 1-beta, the heavy chain constant region of said first antibody has an amino acid sequence as shown in SEQ ID No.5 or an amino acid sequence having greater than 90% homology with the sequence of SEQ ID No.5, and the light chain constant region has an amino acid sequence as shown in SEQ ID No.6 or an amino acid sequence having greater than 90% homology with the sequence of SEQ ID No. 6; the amino acid sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.7 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.7, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.8 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No. 8.
The first antibody heavy chain constant region amino acid sequence SEQ ID No.5 is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
the amino acid sequence SEQ ID No.6 of the light chain constant region of the first antibody is:
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS；
the second antibody heavy chain constant region amino acid sequence SEQ ID No.7 is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
the amino acid sequence of the light chain constant region of the second antibody SEQ ID No.8 is:
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS。
the first antibody and the second antibody are independently coated antibodies or detection antibodies.
Further, the above antibody combination for detecting interleukin 1-beta is selected from any one of the following combinations: the first antibody is used as a detection antibody, the second antibody is used as a detection antibody, the first antibody is used as a coating antibody, the second antibody is used as a detection antibody, the second antibody is used as a coating antibody, and the first antibody is used as a detection antibody; preferably, the primary antibody is used as a coating antibody and the secondary antibody is used as a detection antibody.
That is, the first antibody and the second antibody can be used as the detection antibody of interleukin 1-beta separately, at this time, the conventional monoclonal antibody detection method can be adopted to detect interleukin 1-beta by using the first antibody or the second antibody, and meanwhile, the first antibody can be used as the coating antibody and the second antibody can be used as the detection antibody, and the detection of interleukin 1-beta can be carried out by using a double antibody sandwich method; or the second antibody is used as a coating antibody and the first antibody is used as a detection antibody, and the detection of the interleukin 1-beta is carried out by a double-antibody sandwich method.
The second invention provides a polynucleotide for coding the heavy chain and the light chain of the antibody combination for detecting interleukin 1-beta, wherein the polynucleotide sequence for coding the heavy chain variable region of the first antibody is shown as SEQ ID No.9 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.9, and the polynucleotide sequence for coding the light chain variable region of the first antibody is shown as SEQ ID No.10 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No. 10; the polynucleotide sequence of the heavy chain variable region of the second antibody is shown as SEQ ID No.11 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No.11, and the polynucleotide sequence of the light chain variable region of the second antibody is shown as SEQ ID No.12 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No. 12.
The polynucleotide sequence SEQ ID No.9 encoding the heavy chain variable region of the first antibody is:
gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcg gcgagcgattataaaaccgtggtggcgtttaccggcatgaactgggtgcgccaggcgccgggcaaaggcctggaatggatgggctggattaacgcggtgacctatggcggctatccggaaccgatttatgcggatagcgtgaaaggccgctttacctttagcctggataccagcaaaagcaccgcgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcggcatttatagccaggtgctgtatgattatgcgatgtggggccagggcaccctggtgaccgtgagcagc；
the polynucleotide sequence SEQ ID No.10 encoding the light chain variable region of the first antibody is:
gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcag ctttaccccgtttggccatgtggcgtggtatcagcagaaaccgggcaaagcgccgaaagcgctgatttatccgaaatttcatctgtatcatagcggcgtgccgtatcgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagattttgcgacctattattgctttttttatccgtatgcgcataaaacctttggccagggcaccaaagtggaaattaaacgcaccgtggcg；
the polynucleotide sequence SEQ ID No.11 encoding the heavy chain variable region of the second antibody is:
gatgtgcagctggtggaaagcggcggcggcctggtgcagccgggccgcagcctgaaactgagctgcgcg gcgagcggcatggcgagcctgattaactattatatggcgtgggtgcgccaggcgccgaccaaaggcctggaatgggtggcgagcatttttggcaccgatggcaccaacaaaccggaaaccttttatcgcgatagcgtgaaaggccgctttaccgtgag ccgcgataacgcgcgcagcagcctgtatctgcagatggatagcctgcgcagcgaagataccgcgacctattattgcaccaccgattatggcccgccgacccatggcgatgtgtattggggccagggcaccctggtgaccgtgagcagc；
the polynucleotide sequence SEQ ID No.12 encoding the light chain variable region of the second antibody is:
gatattcagatgacccagagcccggcgagcctgccggcgagcccggaagaaattgtgaccattacctgcca gggcggcctgccgcagtggaactggctgccgagctggtatcagcagaaaccgggcaaaagcccgcagctgctgatttataaccattataccaaactggaaaccgcggatggcgtgccgagccgctttagcgcgagccgcagcggcacccagtatagcctgaaaattagccgcctgcaggtggaagattttggcattttttattgcctggcggcgggcgtgaaaaacgataccacctttggcgcgggcaccaaactggaactgaaacgcaccgatgcg。
in a preferred embodiment, the polynucleotide encoding the heavy chain constant region of the first antibody is as shown in SEQ ID No.13 or a nucleotide sequence having greater than 90% sequence homology to SEQ ID No.13, and the polynucleotide encoding the light chain constant region of the first antibody is as shown in SEQ ID No.14 or a nucleotide sequence having greater than 90% sequence homology to SEQ ID No. 14; the polynucleotide sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.15 or the nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.15, and the polynucleotide sequence of the light chain constant region of the second antibody is shown as SEQ ID No.16 or the nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No. 16.
The polynucleotide sequence SEQ ID No.13 encoding the heavy chain constant region of the first antibody is:
gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgc ggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa；
the polynucleotide sequence SEQ ID No.14 encoding the light chain constant region of the first antibody is:
ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaa gcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtggaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc；
the polynucleotide sequence SEQ ID No.15 encoding the heavy chain constant region of the second antibody is:
gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgc ggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa；
the polynucleotide sequence SEQ ID No.16 encoding the light chain constant region of the second antibody is:
ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaa gcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtggaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc。
a third aspect of the present invention provides an expression system of the polynucleotide for detecting a heavy chain and a light chain of an antibody combination of interleukin 1-beta described above, which is a mammalian expression vector.
In a preferred embodiment, the above-described expression system, the antigen expression system for interleukin 1-beta is the Expi293 expression system.
In a preferred embodiment, the expression system described above, the antibody expression system is a CHO expression system.
A fourth aspect of the invention provides a host cell comprising the expression system described above.
The fifth invention provides the application of the antibody combination in preparing biological products for quantitatively detecting the interleukin 1-beta content in vitro.
In a preferred embodiment, the use as described above, the in vitro quantitative assay is for the detection of the amount of interleukin 1-beta in serum, plasma, whole blood and/or peripheral blood.
In a preferred embodiment, the above-described use, the biologic is a kit.
In a preferred embodiment, the use as described above, the kit is a double antibody sandwich assay kit.
Compared with the prior art, the invention has the following advantages:
the antibody combination for detecting the interleukin 1-beta and the application thereof provided by the invention have the advantages of strong affinity for the interleukin 1-beta antigen, high detection sensitivity, simple and convenient use, and rapid and stable detection; the concentration of IL-1 beta in normal human blood is about (90.51 +/-5.03) pg/mL, and the conventional low-affinity antibody is difficult to realize effective detection in the situation, while the high-affinity antibody combination and the interleukin 1-beta quantitative detection kit containing the antibody combination and adopting a double-antibody sandwich method can effectively realize the quantitative detection of the interleukin 1-beta with ultralow concentration, and the sensitivity of the kit can reach 0.3pg/mL through verification.
The double antibody sandwich method adopted by the invention is used for measuring the IL-1 beta level in a sample, namely, the sandwich compound structure of the coated antibody-antigen-detection antibody is formed by the antibody coated on the ELISA plate, the detection antibody and the IL-1 beta of the detected sample, thereby being more beneficial to the detection of the IL-1 beta. In addition, the invention effectively adopts the avidin-biotin-enzyme complex to realize high sensitivity of detection.
The detection kit of the invention not only can rapidly and accurately measure the content of IL-1 beta in serum, but also provides reliable clinical reference value for early diagnosis and early treatment of inflammation; and because the combination of two specific monoclonal antibodies is used, the IL-1 beta detection sensitivity is greatly improved.
Drawings
FIG. 1 is a standard curve of ELISA kit containing an antibody combination for detecting interleukin 1-beta according to an embodiment of the invention.
Detailed Description
The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent. It is to be understood that the description is only intended to illustrate the invention and is not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. Reagents and instruments used herein are commercially available, and reference to characterization means is made to the relevant description of the prior art and will not be repeated herein.
For a further understanding of the present invention, the present invention will be described in further detail with reference to the following preferred embodiments.
Example 1
The antibody combination for detecting interleukin 1-beta (IL-1 beta) comprises a first antibody and/or a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of a heavy chain variable region of the first antibody are respectively shown as 26-34, 54-63 and 105-116 amino acid sequences of SEQ ID No.1, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of a light chain variable region are respectively shown as 24-32, 48-56 and 89-98 amino acid sequences of SEQ ID No. 2; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the second antibody are respectively shown as the 26 th-33 th, 53 th-62 th and 104 th-114 th amino acid sequences of SEQ ID No.3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as the 24 th-35 th, 51 th-60 th and 93 th-102 th amino acid sequences of SEQ ID No. 4.
The first antibody was 11G10 and the second antibody was 7B9.
CDR1 of the first antibody heavy chain variable region is: DYKTVVAFT;
CDR2 of the first antibody heavy chain variable region is: NAVTYGGYPE;
CDR3 of the first antibody heavy chain variable region is: GIYSQVLYDYAM;
CDR1 of the first antibody light chain variable region is: SFTPFGHVA;
CDR2 of the first antibody light chain variable region is: PKFHJLYHS;
CDR3 of the first antibody light chain variable region is: FFYPBYAHKT;
CDR1 of the second antibody heavy chain variable region is: gmaSLINY;
CDR2 of the second antibody heavy chain variable region is: FGTDGTNKPE;
CDR3 of the second antibody heavy chain variable region is: DYGPPTHGDVY;
CDR1 of the second antibody light chain variable region is: QGGLPQWNWLPS;
CDR2 of the second antibody light chain variable region is: NHYTKLETAD;
CDR3 of the second antibody light chain variable region is: LAAGVKNDTT.
In the antibody combination, the amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID No.1 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.2 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No. 2; the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID No.3 or the amino acid sequence with the sequence homology of more than 90 percent with SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.4 or the amino acid sequence with the sequence homology of more than 90 percent with SEQ ID No. 4.
The first antibody heavy chain variable region amino acid sequence SEQ ID No.1 is:
EVQLVESGGGLVQPGGSLRLSCAASDYKTVVAFTGMNWVRQAPGKGLEWMGWINAVTYGGYPEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTA VYYCARGIYSQVLYDYAMWGQGTLVTVSS；
the amino acid sequence of the first antibody light chain variable region SEQ ID No.2 is:
DIQMTQSPSSLSASVGDRVTITCSFTPFGHVAWYQQKPGKAPKALIYPKFHJLYHSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCFFYPBYAHKTFGQGTKVEIKRTVA；
the second antibody heavy chain variable region amino acid sequence SEQ ID No.3 is:
DVQLVESGGGLVQPGRSLKLSCAASGMASLINYYMAWVRQAPTKGLEWVASIFGTDGTNKPETFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTDYGPPTHGDVYWGQGTLVTVSS；
the second antibody light chain variable region amino acid sequence SEQ ID No.4 is:
DIQMTQSPASLPASPEEIVTITCQGGLPQWNWLPSWYQQKPGKSPQLLIYNHYTKLETADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLAAGVKNDT TFGAGTKLELKRTDA。
in the antibody combination, the amino acid sequence of the heavy chain constant region of the first antibody is shown as SEQ ID No.5 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.5, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.6 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No. 6; the amino acid sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.7 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No.7, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.8 or the amino acid sequence with the homology of more than 90 percent with the sequence of SEQ ID No. 8.
The first antibody heavy chain constant region amino acid sequence SEQ ID No.5 is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
the amino acid sequence SEQ ID No.6 of the light chain constant region of the first antibody is:
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS；
the second antibody heavy chain constant region amino acid sequence SEQ ID No.7 is:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
the amino acid sequence of the light chain constant region of the second antibody SEQ ID No.8 is:
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS。
the primary antibody and the secondary antibody are independently coated antibodies or detection antibodies.
The antibody combination is selected from any one of the following combinations: the first antibody is used as a detection antibody, the second antibody is used as a detection antibody, the first antibody is used as a coating antibody, the second antibody is used as a detection antibody, the second antibody is used as a coating antibody, and the first antibody is used as a detection antibody;
preferably, the primary antibody 11G10 is used as a coating antibody and the secondary antibody 7B9 is used as a detection antibody.
That is, the first antibody and the second antibody can be used as the detection antibody of interleukin 1-beta separately, at this time, the conventional monoclonal antibody detection method can be adopted to detect interleukin 1-beta by using the first antibody or the second antibody, and meanwhile, the first antibody can be used as the coating antibody and the second antibody can be used as the detection antibody, and the detection of interleukin 1-beta can be carried out by using a double antibody sandwich method; or the second antibody is used as a coating antibody and the first antibody is used as a detection antibody, and the detection of the interleukin 1-beta is carried out by a double-antibody sandwich method.
The invention also provides a polynucleotide for coding the heavy chain and the light chain of the antibody combination for detecting interleukin 1-beta, wherein the polynucleotide sequence for coding the heavy chain variable region of the first antibody is shown as SEQ ID No.9 or a nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No.9, and the polynucleotide sequence for coding the light chain variable region of the first antibody is shown as SEQ ID No.10 or a nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No. 10; the polynucleotide sequence of the heavy chain variable region of the second antibody is shown as SEQ ID No.11 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No.11, and the polynucleotide sequence of the light chain variable region of the second antibody is shown as SEQ ID No.12 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No. 12.
The polynucleotide sequence SEQ ID No.9 encoding the heavy chain variable region of the first antibody is:
gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcg gcgagcgattataaaaccgtggtggcgtttaccggcatgaactgggtgcgccaggcgccgggcaaaggcctggaatggatgggctggattaacgcggtgacctatggcggctatccggaaccgatttatgcggatagcgtgaaaggccgctttacctttagcctggataccagcaaaagcaccgcgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcggcatttatagccaggtgctgtatgattatgcgatgtggggccagggcaccctggtgaccgtgagcagc；
the polynucleotide sequence SEQ ID No.10 encoding the light chain variable region of the first antibody is:
gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcag ctttaccccgtttggccatgtggcgtggtatcagcagaaaccgggcaaagcgccgaaagcgctgatttatccgaaatttcatctgtatcatagcggcgtgccgtatcgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagattttgcgacctattattgctttttttatccgtatgcgcataaaacctttggccagggcaccaaagtggaaattaaacgcaccgtggcg；
the polynucleotide sequence SEQ ID No.11 encoding the heavy chain variable region of the second antibody is:
gatgtgcagctggtggaaagcggcggcggcctggtgcagccgggccgcagcctgaaactgagctgcgcg gcgagcggcatggcgagcctgattaactattatatggcgtgggtgcgccaggcgccgaccaaaggcctggaatgggtg gcgagcatttttggcaccgatggcaccaacaaaccggaaaccttttatcgcgatagcgtgaaaggccgctttaccgtgagccgcgataacgcgcgcagcagcctgtatctgcagatggatagcctgcgcagcgaagataccgcgacctattattgcaccaccgattatggcccgccgacccatggcgatgtgtattggggccagggcaccctggtgaccgtgagcagc；
the polynucleotide sequence SEQ ID No.12 encoding the light chain variable region of the second antibody is:
gatattcagatgacccagagcccggcgagcctgccggcgagcccggaagaaattgtgaccattacctgcca gggcggcctgccgcagtggaactggctgccgagctggtatcagcagaaaccgggcaaaagcccgcagctgctgatttataaccattataccaaactggaaaccgcggatggcgtgccgagccgctttagcgcgagccgcagcggcacccagtatagcctgaaaattagccgcctgcaggtggaagattttggcattttttattgcctggcggcgggcgtgaaaaacgataccacctttggcgcgggcaccaaactggaactgaaacgcaccgatgcg。
the polynucleotide for detecting the heavy chain and the light chain of the interleukin 1-beta antibody combination comprises a polynucleotide sequence of which the nucleotide sequence of the heavy chain constant region of the first antibody is shown as SEQ ID No.13 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.13, and a polynucleotide sequence of which the nucleotide sequence of the light chain constant region of the first antibody is shown as SEQ ID No.14 or a nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No. 14; the polynucleotide sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.15 or the nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.15, and the polynucleotide sequence of the light chain constant region of the second antibody is shown as SEQ ID No.16 or the nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No. 16.
The polynucleotide sequence SEQ ID No.13 encoding the heavy chain constant region of the first antibody is:
gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgc ggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa；
the polynucleotide sequence SEQ ID No.14 encoding the light chain constant region of the first antibody is:
ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaa gcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtggaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc；
the polynucleotide sequence SEQ ID No.15 encoding the heavy chain constant region of the second antibody is:
gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgc ggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa；
the polynucleotide sequence SEQ ID No.16 encoding the light chain constant region of the second antibody is:
ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaa gcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtggaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc。
an expression system for detecting polynucleotides of heavy and light chains of an antibody combination of interleukin 1-beta, the expression system being a mammalian expression vector.
The antigen expression system of interleukin 1-beta (IL-1 beta) is the Expi293 expression system.
The antibody expression system is a CHO expression system.
Host cells comprising the expression system described above.
Use of an antibody combination for detecting interleukin 1-beta in the preparation of a biological product for in vitro quantitative detection of interleukin 1-beta content.
In vitro quantitative detection refers to the detection of the content of interleukin 1-beta in serum, plasma, whole blood and/or peripheral blood.
The biological product is a kit.
The kit is a double-antibody sandwich method detection kit.
Example 2
The first antibody and the second antibody are both obtained by screening by a phage display method, purified proteins are marked by NHS-Biotin reagent, interleukin 1-beta (IL-1 beta) antigen is expressed by an Expi293 expression system, and the expression sequence is shown as SEQ ID No.17 or an amino acid sequence with the sequence homology of more than 90% with SEQ ID No. 17.
SEQ ID No.17 sequence:
AEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGG IQLRISDHHYSKGFRQAASVVVAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS。
the nucleotide sequence of the interleukin 1-beta (IL-1 beta) antigen is shown as SEQ ID No.18 or the nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No. 18.
SEQ ID No.18 sequence:
gcggaagtgccggaactggcgagcgaaatgatggcgtattatagcggcaacgaagatgatctgttttttgaag cggatggcccgaaacagatgaaatgcagctttcaggatctggatctgtgcccgctggatggcggcattcagctgcgcattagcgatcatcattatagcaaaggctttcgccaggcggcgagcgtggtggtggcgatggataaactgcgcaaaatgctggtgccgtgcccgcagacctttcaggaaaacgatctgagcaccttttttccgtttatttttgaagaagaaccgattttttttgatacctgggataacgaagcgtatgtgcatgatgcgccggtgcgcagcctgaactgcaccctgcgcgatagccagcagaaaagcctggtgatgagcggcccgtatgaactgaaagcgctgcatctgcagggccaggatatggaacagcaggtggtgtttagcatgagctttgtgcagggcgaagaaagcaacgataaaattccggtggcgctgggcctgaaagaaaaaaacctgtatctgagctgcgtgctgaaagatgataaaccgaccctgcagctggaaagcgtggatccgaaaaactatccgaaaaaaaaaatgga aaaacgctttgtgtttaacaaaattgaaattaacaacaaactggaatttgaaagcgcgcagtttccgaactggtatattagcaccagccaggcggaaaacatgccggtgtttctgggcggcaccaaaggcggccaggatattaccgattttaccatgcagtttgtgagcagc。
example 3
The specific compositions and specific concentrations of the respective compositions of the kits containing 2 antibodies are shown in table 1.
TABLE 1
The reagents are as follows:
(1) ELISA assay coating buffer (pH 9.6.05M carbonate buffer):
NaHCO 3 1.59 grams of the total weight of the product,
NaHCO 3 2.93 g of the total weight of the product,
distilled water is added to 1000ml;
(2) ELISA assay washing buffer (pH 7.4 PBS): 0.15M
KH 2 PO 4 0.2 g of the total weight of the mixture,
Na 2 HPO 4 ·12H 2 o2.9 g of the total weight of the mixture,
8.0 g of NaCl, and the concentration of the sodium chloride in the solution is higher than that of the sodium chloride,
0.2 g of KCl, which is prepared from the raw materials of the formula,
Tween-20 0.05％0.5ml，
distilled water is added to 1000ml;
(3) ELISA sample dilution:
bovine Serum Albumin (BSA) 0.1 g,
adding a washing buffer solution to 100ml;
(4) ELISA experiment termination liquid (2M H) 2 SO 4 )：
178.3ml of distilled water and 21.7ml of concentrated sulfuric acid (98%) are added dropwise;
(5) ELISA substrate buffer (pH 5.0 disodium phosphate citrate):
0.2M Na 2 HPO 4 (28.4 g/L) 25.7ml,
0.1M citric acid (19.2 g/L) 24.3ml,
adding 50ml of distilled water;
(6) ELISA experiment TMB (tetramethylbenzidine) use solution:
TMB (10 mg/5ml absolute ethanol) 0.5ml,
10ml of substrate buffer (pH 5.5),
0.75％H 2 O 2 32μl；
(7) ELISA test blocking solution:
bovine Serum Albumin (BSA) 5 g,
wash buffer was added to 100ml.
Example 4
1. The specific procedures for the use of the kit containing 2 antibodies for interleukin 1-beta detection are shown below.
The steps are as follows:
1) Antibody pre-coating: diluting 11G10 antibody with coating buffer to a concentration of 1 μg/mL, adding 100 μl per well to the ELISA plate, and standing overnight at 4deg.C;
2) Closing: adding 100 mu L of sealing liquid into each hole, sealing for 0.5-1 hour at 37 ℃, washing the plate 3 times with washing buffer liquid, and beating to dry each time for 30 s;
3) Dilution and sample addition of standard: the method comprises the steps of arranging 16 holes of a standard substance hole and a blank control hole on an enzyme-labeled coating plate, carrying out gradient dilution on hIL-1 beta antigen by using sample diluent, wherein each concentration is 200 mu L, and the concentrations are respectively 500pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.6pg/mL, 7.8pg/mL and 3.9pg/mL, each concentration is two complex holes, the sample adding amount of each hole is 100 mu L, and each blank hole is added with 100 mu L of sample diluent;
4) Diluting and adding samples to be tested: 240 μl of sample diluent is added, then 60 μl of sample to be detected (the final dilution of the sample is 5 times) is added, three compound wells are added, and 100 μl of sample is added to the bottom of the enzyme-labeled plate hole;
5) Incubation: incubating for 30 min at 37deg.C with sealing plate membrane, carefully removing sealing plate membrane, discarding liquid, spin-drying, washing the plate with washing buffer solution for 3 times each for 30s, and drying;
6) Incubation of biotin-labeled antibody: adding a fresh diluted biotin-labeled antibody into each reaction well, washing the plate with a washing buffer solution for 3 times at a temperature of between 0.5 and 1 hour at a temperature of between 37 ℃ and 100 mu L per well for 30 seconds each time, and beating to dry;
7) Color development: adding 50 mu l of TMB color developing agent into each hole, gently shaking and mixing uniformly, and developing color for 15 minutes at 37 ℃ in a dark place;
8) And (3) terminating: adding 50 μl of stop solution into each well to stop the reaction (blue changes vertically to yellow);
9) And (3) measuring: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with blank air-conditioner zero, and the measurement was performed within 15 minutes after the addition of the stop solution.
2. Sample requirements:
serum is collected by a sterile tube, the blood is naturally coagulated at room temperature for 10-20 minutes, and centrifuged at 2-8deg.C for about 20 minutes (2000-3000 rpm), and the supernatant is collected carefully and centrifuged again if precipitation occurs during storage.
The results of the kit containing the antibody combination for in vitro quantitative determination of the content of interleukin 1-beta (IL-1β) in human serum, plasma, whole blood, peripheral blood are shown in Table 2 below.
TABLE 2
| IL-1β(ng/ml) | Clinical application advice |
| ＜0.005 | Reference value for 95% locus in apparent healthy population |
| 0.005-0.100 | Normal, or in the presence of minor inflammation, minor infection |
| 0.100-0.200 | Suggesting a general bacterial infection or systemic inflammatory response |
| ＞0.200 | The indication may be sepsis |
The standard curve of the kit of the invention is shown in FIG. 1.
Example 5
Two commercial IL-1 beta detection kits were used as comparative products 1-2, and samples were detected separately from the kit provided in example 4 of the present invention.
The commercial IL-1 beta detection kit comprises the following two types:
comparative product 1:
product name: human interleukin 1beta (IL-1 beta) detection kit (ELISA method);
english name: human Interleukin-1beta ELISAKIT;
product number: ZN2236;
the product specification is as follows: 96T;
purchased from beijing berlaibo technologies limited.
Comparative product 2:
product name: IL-1 beta (human) ELISA detection kit;
english name: interlukin-1 beta (human) EIAKit;
product number: 583311-480;
the product specification is as follows: 480 analyses;
purchased from eimeria technologies ltd.
In the detection process, the basic characteristics of the kit are compared, and the basic characteristics are shown in the following table 3.
TABLE 3 Table 3
| Example 4 | Comparative product 1 | Comparative product 2 | |
| Antigens | Human IL-1 beta | Human IL-1 beta | Human IL-1 beta |
| Coated antibodies | 11G10 | IL-1 beta monoclonal antibodies | Monoclonal antibodies |
| Detection antibodies | 7B9 | Biotin-labeled IL-1 beta monoclonal antibodies | Acetylcholinesterase-labeled Fab |
| Method | ELISA | ELISA | ELISA |
| Sensitivity of | 0.3pg/mL | ＜0.15pg/ml | 3.9pg/ml |
| Detection range | 2pg/mL-500pg/mL | 3.9pg/mL-250pg/mL | 0-250pg/ml |
As can be seen from Table 3, the basic characteristics of each kit are already different, and the samples for which example 4, comparative product 1 and comparative product 2 are directed are interleukin 1-beta (IL-1 beta) in human serum; however, the coated antibody and the detection antibody of example 4 are completely different from those of comparative product 1 and comparative product 2, and on the premise that the detection sensitivity of the kit provided in example 4 is greatly higher than that of comparative product 2 and slightly lower than that of comparative product 1 (because the monoclonal antibody adopted in comparative document 1 is used for coating and detection), but the detection range is far higher than that of both. In general, the receptor subjected to artificial recombination must have higher sensitivity than the normal antigen due to the strong recombination purpose, and the kit of example 4 actually reaches the highest sensitivity of the similar products.
Further, IL-1 beta detection is performed by using a blood sample, and the detection method specifically comprises the following steps: the serum samples of the patients suffering from inflammation or infection and normal persons were collected in 10 cases, and IL-1β detection was performed using the kit of this patent example 4 and commercially available IL-1β detection kits (comparative product 1 and comparative product 2), respectively, and the results are shown in Table 4 below, wherein the H group is healthy body serum, and the P group is inflammatory or infectious body serum.
TABLE 4 Table 4
By combining the criteria in Table 2, it can be determined that if the serum IL-1β concentration is >5pg/mL, it is possible to have inflammation or other infection, and in the comparison of the detection data in Table 4, it can be seen that the commercial kit (comparative product 1) has a condition of missing detection due to low sensitivity in the detection of P1, P8 and P9, and the missing detection rate reaches 30%, which is a higher level of missing detection rate; in the detection of P1 and P8, the detection omission ratio of the commercial kit (comparative product 2) is 20% because of low sensitivity, and the detection omission ratio is slightly lower than that of comparative product 1 but is also at a medium level, whereas the detection result of the kit in the embodiment 4 of the invention is that the detection omission ratio is not 0, which indicates that the sensitivity of the kit containing the antibody combination is far higher than that of the existing product.
Example 6
The invention provides two antibodies for detecting interleukin 1-beta in a human serum sample, namely a first antibody 11G10 and a second antibody 7B9, which are combined and then detected by adopting a double antibody sandwich method, but in practice, the two antibodies obtained by screening by a phage display method also have the function of detecting interleukin 1-beta in the human serum sample when used independently and used in a replacement way, and in order to verify the effect of the antibodies when used independently and used in a replacement way, the applicant adopts the similar method of examples 1-5 (the monoclonal antibody is detected by adopting a conventional method and the double antibody is detected by adopting a sandwich method) and compares the results, in particular as follows.
The basic characteristic comparison results of the kit composed of the primary antibody 11G10 alone, the secondary antibody 7B9 alone, the antibody combination 11G10 (coating) +7b9 (detection), and the antibody combination 7B9 (coating) +11g10 (detection) are shown in table 5.
TABLE 5
| 11G10 alone | 7B9 alone | 7B9+11G10 | 11G10+7B9 (example 4) | |
| Antigens | Human IL-1 beta | Human IL-1 beta | Human IL-1 beta | Human IL-1 beta |
| Coated antibodies | — | — | 7B9 (second antibody) | 11G10 (first antibody) |
| Detection antibodies | 11G10 (first antibody) | 7B9 (second antibody) | 11G10 (first antibody) | 7B9 (second antibody) |
| Method | ELISA | ELISA | ELISA | ELISA |
| Sensitivity of | 1.0pg/mL | 0.9pg/mL | 0.5pg/mL | 0.3pg/mL |
| Detection range | 10-100pg/mL | 10-100pg/mL | 5-300pg/mL | 2pg/mL-500pg/mL |
As can be seen from Table 5, the single use of the first antibody 11G10 and the single use of the second antibody 7B9 can effectively detect interleukin 1-beta in a human serum sample, and the detection sensitivity and the detection range of the single use of the two antibodies are also superior to those of the existing commercial product (comparative product 2 in Table 3), while the effect of the first antibody and the second antibody is better than that of the single use of the first antibody and the second antibody, namely, the combination of the first antibody and the second antibody 7B9 (coating) +11G10 (detection), and furthermore, the combination of the antibodies 11G10+7B9 disclosed by the invention has the best detection sensitivity and detection range in all choices, which are superior to those of the existing product, and are also superior to the mode of single use and replacement.
To test the actual use effect, the applicant also performed IL-1β detection on random human blood samples, similar to the detection method used to draw the conclusions in Table 4, with specific results shown in Table 6.
TABLE 6
Also, by combining the criteria in table 2, it can be determined that if the serum IL-1β concentration is >5pg/mL, it is possible to have inflammation or other infection, and in the comparison of the detection data in table 6, it can be seen that the detection result of the antibody combination 11g10+7b9 kit does not have missed detection, that is, the missed detection rate is 0, and the detection result of the antibody combination 7b9+111g10 kit has missed detection in the detection of P8, that is, the missed detection rate is 10%; the second antibody 7B9 alone showed a missing detection of 10% in the detection against P1; the use of the primary antibody 11G10 alone caused missed detection in the detection against P8, while false detection occurred in the detection against H8; from this comparison result, it can be demonstrated that the primary antibody and the secondary antibody are used alone, and the detection sensitivity is slightly lower than that of the combination of the antibodies.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (15)
1. An antibody combination for detecting interleukin 1-beta, characterized in that the antibody combination comprises a first antibody and a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of a heavy chain variable region of the first antibody are respectively shown as 26-34, 54-63 and 105-116 amino acid sequences of SEQ ID No.1, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of a light chain variable region are respectively shown as 24-32, 48-56 and 89-98 amino acid sequences of SEQ ID No. 2; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the second antibody are respectively shown as the 26 th-33, 53 th-62 th and 104 th-114 th amino acid sequences of SEQ ID No.3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as the 24 th-35 th, 51 th-60 th and 93 th-102 th amino acid sequences of SEQ ID No. 4; the antibody combination is selected from any one of the following combinations: the first antibody is used as a coating antibody and the second antibody is used as a detection antibody, the second antibody is used as a coating antibody and the first antibody is used as a detection antibody.
2. The antibody combination for detecting interleukin 1- β according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID No.1 or is an amino acid sequence having a homology of more than 90% with SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.2 or is an amino acid sequence having a homology of more than 90% with SEQ ID No. 2; the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID No.3 or the amino acid sequence with the homology of more than 90% with the SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.4 or the amino acid sequence with the homology of more than 90% with the SEQ ID No. 4.
3. The antibody combination for detecting interleukin 1- β according to claim 2, wherein the amino acid sequence of the heavy chain constant region of the first antibody is shown as SEQ ID No.5 or is an amino acid sequence having a homology of more than 90% with SEQ ID No.5, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.6 or is an amino acid sequence having a homology of more than 90% with SEQ ID No. 6; the amino acid sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.7 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No.7, and the amino acid sequence of the light chain constant region is shown as SEQ ID No.8 or the amino acid sequence with the homology of more than 90% with the sequence of SEQ ID No. 8.
4. An antibody combination for detecting interleukin 1-beta according to any one of claims 1-3, wherein the antibody combination is a primary antibody as a coating antibody and a secondary antibody as a detecting antibody.
5. A polynucleotide encoding the heavy and light chain of the antibody combination for detecting interleukin 1- β of any one of claims 1-4, wherein the polynucleotide encoding the heavy chain variable region of said first antibody has a nucleotide sequence shown in SEQ ID No.9 or having a nucleotide sequence having greater than 90% sequence homology to SEQ ID No.9, and the polynucleotide encoding the light chain variable region of said first antibody has a nucleotide sequence shown in SEQ ID No.10 or having a nucleotide sequence having greater than 90% sequence homology to SEQ ID No. 10; the polynucleotide sequence for encoding the heavy chain variable region of the second antibody is shown as SEQ ID No.11 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No.11, and the polynucleotide sequence for encoding the light chain variable region of the second antibody is shown as SEQ ID No.12 or the nucleotide sequence with the sequence homology of more than 90 percent with SEQ ID No. 12.
6. The polynucleotide for detecting the heavy chain and light chain of an antibody combination of interleukin 1- β according to claim 5, wherein the polynucleotide sequence encoding the heavy chain constant region of said first antibody is shown in SEQ ID No.13 or is a nucleotide sequence having a sequence homology of more than 90% with SEQ ID No.13, and the polynucleotide sequence encoding the light chain constant region of said first antibody is shown in SEQ ID No.14 or is a nucleotide sequence having a sequence homology of more than 90% with SEQ ID No. 14; the polynucleotide sequence of the heavy chain constant region of the second antibody is shown as SEQ ID No.15 or the nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No.15, and the polynucleotide sequence of the light chain constant region of the second antibody is shown as SEQ ID No.16 or the nucleotide sequence with the sequence homology of more than 90 percent with the SEQ ID No. 16.
7. An expression system comprising a polynucleotide for detecting the heavy and light chains of the antibody combination of interleukin 1-beta according to any one of claims 5-6, characterized in that said expression system is a mammalian expression vector.
8. The expression system of claim 7, wherein the antibody expression system is a CHO expression system.
9. A host cell comprising the expression system of any one of claims 7-8.
10. Use of an antibody combination according to any one of claims 1-4 for the preparation of a biological product for the in vitro quantitative detection of interleukin 1-beta content.
11. The use according to claim 10, wherein the in vitro quantitative detection is the detection of the content of interleukin 1- β in serum, plasma, whole blood and/or peripheral blood.
12. The use according to claim 11, wherein the biological product is a kit.
13. The use according to claim 12, wherein the kit is a double antibody sandwich assay kit.
14. The antibody for detecting interleukin 1-beta is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the antibody are respectively shown as the amino acid sequences of 26-34, 54-63 and 105-116 of SEQ ID No.1, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as the amino acid sequences of 24-32, 48-56 and 89-98 of SEQ ID No. 2.
15. The antibody for detecting interleukin 1-beta is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the antibody are respectively shown as 26-33, 53-62 and 104-114 amino acid sequences of SEQ ID No.3, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as 24-35, 51-60 and 93-102 amino acid sequences of SEQ ID No. 4.
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| EP2746293A1 (en) * | 2012-12-21 | 2014-06-25 | Novo Nordisk A/S | Anti-IL21 antibodies for use in the treatment of cardiovascular diseases |
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