Antibody combination for detecting tumor necrosis factor-α and its application

Abstract

This invention provides an antibody combination for detecting tumor necrosis factor-α (TNF-α), comprising a first antibody and / or a second antibody, each possessing a unique CDR fragment. Specific applications of this antibody combination are also provided. The advantages of this invention are that the antibody combination exhibits strong affinity for TNF-α antigen, high detection sensitivity, ease of use, and rapid and stable detection. The concentration of TNF-α in normal human serum is 9.42 ± 2.91 ng / mL. Existing conventional low-affinity antibodies are insufficient for effective detection in this situation. However, the high-affinity antibody combination and the TNF-α quantitative detection kit containing this antibody combination using a double-antibody sandwich method provided by this invention can effectively achieve the quantitative detection of ultra-low concentrations of TNF-α. Verification shows that the kit's sensitivity can reach 2.2 pg / mL.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and more specifically to antibody combinations for detecting tumor necrosis factor-α and their applications. Technical Background

[0002] Tumor necrosis factor (TNF) is a small protein secreted by macrophages. TNF-α, mainly secreted by monocytes and macrophages, is an important inflammatory cytokine that can upregulate other inflammatory factors. For example, TNF-α is significantly elevated in patients with systemic lupus erythematosus (SLE), and serum TNF-α levels are correlated with disease activity. TNF-β is mainly secreted by activated T lymphocytes. TNF can stimulate the production of IL-1 both in vitro and in vivo. It is heat-sensitive, inactivated at 70°C for 30 minutes.

[0003] The biological functions of TNF-α and TNF-β are very similar, mainly including the following:

[0004] 1) Killing or inhibiting tumor cells: TNF can kill some tumor cells or inhibit their proliferation both in vivo and in vitro. Tumor cell lines have great differences in their sensitivity to TNF-α. TNF-α even has a stimulating effect on a very small number of tumor cells.

[0005] 2) Enhance the phagocytic capacity of neutrophils: increase the production of peroxide anions, enhance ADCC function, stimulate cell degranulation and secretion of myeloperoxidase;

[0006] 3) Anti-infection: It inhibits the growth of Plasmodium, inhibits viral replication (such as adenovirus type II and herpesvirus type II), inhibits viral protein synthesis, viral particle production and infectivity, and can kill virus-infected cells;

[0007] 4) Endogenous pyrogens: cause fever and induce the synthesis of acute-phase proteins in hepatocytes;

[0008] 5) Promote the differentiation of myeloid leukemia cells into macrophages: Promote the differentiation of myeloid leukemia cells ML-1, monocytic leukemia cells U937, and promyelocytic leukemia cells HL60;

[0009] 6) Promotes cell proliferation and differentiation: TNF promotes the expression of MHC class I antigens on T cells, enhances the proliferation capacity of IL-2-dependent thymocytes and T cells, promotes the production of lymphokines such as IL-2, CSF and IFN-γ, and enhances the proliferation and Ig secretion of B cells stimulated by mitogens or foreign antigens.

[0010] However, since the concentration in normal human serum is 9.42±2.91 ng / mL, most existing biological products targeting TNF-α are low-affinity antibodies, which have low accuracy and low sensitivity in detecting TNF-α. Summary of the Invention

[0011] To address the aforementioned limitations, this invention proposes an antibody combination for detecting tumor necrosis factor-α and its application, overcoming the deficiencies and defects mentioned in the background art.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] The present invention provides an antibody combination for detecting tumor necrosis factor-α (TNF-α), the antibody combination comprising a first antibody and / or a second antibody, wherein the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the first antibody are shown as amino acid sequences at positions 26-35, 55-63, and 105-114 of SEQ ID No. 1, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 24-32, 48-55, and 88-98 of SEQ ID No. 2, respectively; and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the second antibody are shown as amino acid sequences at positions 26-34, 54-63, and 105-117 of SEQ ID No. 3, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 24-32, 48-56, and 89-97 of SEQ ID No. 4, respectively.

[0014] The primary antibody is 18C5, and the secondary antibody is 3D5.

[0015] The CDR1 of the variable region of the first antibody heavy chain is: GMNDSLTSRN ;

[0016] The CDR2 of the variable region of the first antibody heavy chain is: KSAPEDTTF ;

[0017] The CDR3 of the variable region of the first antibody heavy chain is: AKRTSMFPTD ;

[0018] The CDR1 of the variable region of the first antibody light chain is: VPNKDFGAD ;

[0019] The CDR2 of the variable region of the first antibody light chain is: APNFNFQQ ;

[0020] The CDR3 of the variable region of the first antibody light chain is: MGTRLIPPSTV ;

[0021] The CDR1 of the variable region of the second antibody heavy chain is: GNFTGFFRP ;

[0022] The CDR2 of the variable region of the second antibody heavy chain is: TVVAFNFDTK ;

[0023] The CDR3 of the variable region of the second antibody heavy chain is: VSSLYPFGKLYQT ;

[0024] The CDR1 of the variable region of the second antibody light chain is: RTHVILTHA ;

[0025] The CDR2 of the variable region of the second antibody light chain is: LSAIRKPCQ ;

[0026] The CDR3 of the variable region of the second antibody light chain is: QRYNRAPYT .

[0027] In a preferred embodiment, the antibody combination for detecting tumor necrosis factor-α comprises the following: the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID No. 1 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 1; the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 2 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 2; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID No. 3 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 3; the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 4 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 4.

[0028] The amino acid sequence of the variable region of the first antibody heavy chain, SEQ ID No. 1, is as follows:

[0029] EVQLVESGGGLVQPGGSLRLSCAVS GMNDSLTSRN SVNWIRQAPGKGLEWVGLI KSAPEDTTF TDYNSAIKSRFTISRDTSKSTVYLQMNSLRAEDTAVYYCAR AKRTSMFPTD WGQGTLVTVSS;

[0030] The amino acid sequence of the variable region of the first antibody light chain, SEQ ID No. 2, is as follows:

[0031] DIQMTQSPSSLSASVGDRVTITC VPNKDFGAD WYQQKPGKAPKLLIY APNFNFQQ GVPSRFSGSGSATDYTLTISSLQPEDFATYYC MGTRLIPPSTV FGQGTKVEVKRTVA;

[0032] The amino acid sequence of the variable region of the second antibody heavy chain, SEQ ID No. 3, is as follows:

[0033] EVQLVESGGGLVQPGRSLRLSCAAS GNFTGFFRP AMHWVRQAPGKGLEWVSAI TVVAFNFDTK IDYADSVEGRFTISRDNAKNSLYLDMNSLRAEDTAVYYCAK VSSLYPFGKLYQT WGQGTLVTVSS;

[0034] The amino acid sequence of the variable region of the second antibody light chain, SEQ ID No. 4, is as follows:

[0035] DIQMTQSPSSLSASVGDRVTITC RTHVILTHA WYQQKPGKAPKLLIY LSAIRKPCQ GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QRYNRAPYT FGQGTKVEIKRTVA.

[0036] In a preferred embodiment, the antibody combination for detecting tumor necrosis factor-α comprises the following: the amino acid sequence of the heavy chain constant region of the first antibody is as shown in SEQ ID No. 5 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 5; the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 6 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 6; the amino acid sequence of the heavy chain constant region of the second antibody is as shown in SEQ ID No. 7 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 7; the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 8 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 8.

[0037] The amino acid sequence of the first antibody heavy chain constant region, SEQ ID No. 5, is as follows:

[0038] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0039] The amino acid sequence of the constant region of the light chain of the first antibody, SEQ ID No. 6, is as follows:

[0040] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;

[0041] The amino acid sequence of the constant region of the second antibody heavy chain, SEQ ID No. 7, is as follows:

[0042] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0043] The amino acid sequence of the constant region of the light chain of the second antibody, SEQ ID No. 8, is as follows:

[0044] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0045] An antibody combination for detecting tumor necrosis factor-α, wherein the first and second antibodies are independently either coating antibodies or detection antibodies.

[0046] Furthermore, in the above-mentioned antibody combination for detecting tumor necrosis factor-α, the antibody combination is selected from any of the following combinations: a first antibody as a detection antibody, a second antibody as a detection antibody, a first antibody as a coating antibody + a second antibody as a detection antibody, or a second antibody as a coating antibody + a first antibody as a detection antibody; preferably, the first antibody as a coating antibody + the second antibody as a detection antibody.

[0047] In other words, both the primary and secondary antibodies can be used independently as detection antibodies for tumor necrosis factor-α. In this case, conventional monoclonal antibody detection methods can be used to detect tumor necrosis factor-α with either the primary or secondary antibody. Alternatively, the primary antibody can be used as the coating antibody and the secondary antibody as the detection antibody, using a double antibody sandwich method to detect tumor necrosis factor-α; or the secondary antibody can be used as the coating antibody and the primary antibody as the detection antibody, using a double antibody sandwich method to detect tumor necrosis factor-α.

[0048] The second inventive point of this invention is to provide a polynucleotide encoding the heavy and light chains of the antibody combination described above for detecting tumor necrosis factor-α, wherein the polynucleotide sequence encoding the variable region of the heavy chain of the first antibody is as shown in SEQ ID No. 9 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 9; the polynucleotide sequence encoding the variable region of the light chain of the first antibody is as shown in SEQ ID No. 10 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 10; the polynucleotide sequence encoding the variable region of the heavy chain of the second antibody is as shown in SEQ ID No. 11 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 11; and the polynucleotide sequence encoding the variable region of the light chain of the second antibody is as shown in SEQ ID No. 12 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 12.

[0049] The polynucleotide sequence encoding the variable region of the first antibody heavy chain, SEQ ID No. 9, is:

[0050] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggtgagcggcatgaacgatagcctgaccagccgcaacagcgtgaactggattcgccaggcgccgggcaaaggcctggaatgggtgggcctgattaaaagcgcgccggaagataccacctttaccgattataacagcgcgattaaaagccgctttaccattagccgcgataccagcaaaagcaccgtgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcgcgaaacgcaccagcatgtttccgaccgattggggccagggcaccctggtgaccgtgagcagc;

[0051] The polynucleotide sequence SEQ ID No.10 encoding the variable region of the light chain of the first antibody is:

[0052] gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcgtgccgaacaaagattttggcgcggattggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatgcgccgaactttaactttcagcagggcgtgccgagccgctttagcggcagcggcagcgcgaccgattataccctgaccattagcagcctgcagccggaagattttgcgacctattattgcatgggcacccgcctgattccgccgagcaccgtgtttggccagggcaccaaagtggaagtgaaacgcaccgtggcg;

[0053] The polynucleotide sequence SEQ ID No.11 encoding the variable region of the heavy chain of the second antibody is:

[0054] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggccgcagcctgcgcctgagctgcgcggcgagcggcaactttaccggcttttttcgcccggcgatgcattgggtgcgccaggcgccgggcaaaggcctggaatgggtgagcgcgattaccgtggtggcgtttaactttgataccaaaattgattatgcggatagcgtggaaggccgctttaccattagccgcgataacgcgaaaaacagcctgtatctggatatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgaaagtgagcagcctgtatccgtttggcaaactgtatcagacctggggccagggcaccctggtgaccgtgagcagc;

[0055] The polynucleotide sequence SEQ ID No.12 encoding the variable region of the light chain of the second antibody is:

[0056] gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgccgcacccatgtgattctgacccatgcgtggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatctgagcgcgattcgcaaaccgtgccagggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagatgtggcgacctattattgccagcgctataaccgcgcgccgtatacctttggccagggcaccaaagtggaaattaaacgcaccgtggcg。

[0057] In a preferred embodiment, the heavy and light chain polynucleotides of the antibody combination for detecting tumor necrosis factor-α include: a polynucleotide sequence encoding the heavy chain constant region of the first antibody as shown in SEQ ID No. 13 or a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 13; a polynucleotide sequence encoding the light chain constant region of the first antibody as shown in SEQ ID No. 14 or a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 14; a polynucleotide sequence encoding the heavy chain constant region of the second antibody as shown in SEQ ID No. 15 or a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 15; and a polynucleotide sequence encoding the light chain constant region of the second antibody as shown in SEQ ID No. 16 or a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 16.

[0058] The polynucleotide sequence encoding the constant region of the first antibody heavy chain, SEQ ID No. 13, is as follows:

[0059] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0060] The polynucleotide sequence encoding the constant region of the first antibody light chain, SEQ ID No. 14, is as follows:

[0061] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;

[0062] The polynucleotide sequence encoding the constant region of the second antibody heavy chain, SEQ ID No. 15, is as follows:

[0063] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0064] The polynucleotide sequence encoding the constant region of the light chain of the second antibody, SEQ ID No. 16, is as follows:

[0065] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.

[0066] The third inventive aspect of this invention is to provide an expression system for the heavy and light chain polynucleotides of the antibody combination described above for detecting tumor necrosis factor-α, wherein the expression system is a mammalian expression vector.

[0067] In a preferred embodiment, the expression system described above, specifically the tumor necrosis factor-α antigen expression system, is the Expi 293 expression system.

[0068] In a preferred embodiment, the above-described expression system, the antibody expression system, is a CHO expression system.

[0069] The fourth inventive point of this invention is to provide a host cell containing the above-described expression system.

[0070] The fifth inventive point of this invention is to provide the application of the above-described antibody combination in the preparation of a biological product for the in vitro quantitative detection of tumor necrosis factor-α content.

[0071] In a preferred embodiment, the above-described application, wherein the in vitro quantitative detection refers to the detection of the content of tumor necrosis factor-α in serum, plasma, whole blood and / or peripheral blood.

[0072] In a preferred embodiment, the above-described application uses a reagent kit as the biological product.

[0073] In a preferred embodiment, the above-described application refers to a double-antibody sandwich assay kit.

[0074] Compared with the prior art, the present invention has the following advantages:

[0075] This invention provides an antibody combination for detecting tumor necrosis factor-α and its application. The antibody combination has strong affinity for the tumor necrosis factor-α antigen, high detection sensitivity, is easy to use, and provides rapid and stable detection. The concentration of tumor necrosis factor-α in normal human serum is 9.42±2.91 ng / mL. Existing conventional low-affinity antibodies are difficult to effectively detect in this situation. However, the high-affinity antibody combination and the tumor necrosis factor-α quantitative detection kit containing this antibody combination using a double antibody sandwich method provided by this invention can effectively achieve the quantitative detection of ultra-low concentrations of tumor necrosis factor-α. The sensitivity of the kit has been verified to reach 2.2 pg / mL.

[0076] The double-antibody sandwich method used in this invention to determine TNF-α levels in samples involves a sandwich complex structure of "coated antibody-antigen-detection antibody" formed by the antibody coated on the ELISA plate, the detection antibody, and the TNF-α in the sample. This structure is more conducive to the detection of TNF-α. Furthermore, this invention effectively utilizes an avidin-biotin-enzyme complex to achieve high detection sensitivity.

[0077] The detection kit of the present invention can not only rapidly and accurately determine the content of TNF-α in serum, providing reliable clinical reference value for the early diagnosis and early treatment of inflammation; but also achieves a significant improvement in the detection sensitivity of TNF-α due to the use of a combination of two specific monoclonal antibodies. Attached Figure Description

[0078] Figure 1 This is a standard curve for an ELISA kit containing an antibody combination for detecting tumor necrosis factor-α, provided in an embodiment of the present invention. Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the description herein is merely illustrative and not intended to limit the scope of the invention.

[0080] Unless otherwise defined, 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 pertains. The terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. All reagents and instruments used herein are commercially available, and the characterization methods involved are described in relevant prior art and will not be repeated herein.

[0081] To further understand the present invention, the present invention will be further described in detail below with reference to the preferred embodiments.

[0082] Example 1

[0083] An antibody combination for detecting tumor necrosis factor-α (TNF-α) comprises a first antibody and / or a second antibody. The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the first antibody are shown as amino acid sequences at positions 26-35, 55-63, and 105-114 of SEQ ID No. 1, respectively, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 24-32, 48-55, and 88-98 of SEQ ID No. 2, respectively. The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the second antibody are shown as amino acid sequences at positions 26-34, 54-63, and 105-117 of SEQ ID No. 3, respectively, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 24-32, 48-56, and 89-97 of SEQ ID No. 4, respectively.

[0084] The primary antibody is 18C5, and the secondary antibody is 3D5.

[0085] The CDR1 of the variable region of the first antibody heavy chain is: GMNDSLTSRN ;

[0086] The CDR2 of the variable region of the first antibody heavy chain is: KSAPEDTTF ;

[0087] The CDR3 of the variable region of the first antibody heavy chain is: AKRTSMFPTD ;

[0088] The CDR1 of the variable region of the first antibody light chain is: VPNKDFGAD ;

[0089] The CDR2 of the variable region of the first antibody light chain is: APNFNFQQ ;

[0090] The CDR3 of the variable region of the first antibody light chain is: MGTRLIPPSTV ;

[0091] The CDR1 of the variable region of the second antibody heavy chain is: GNFTGFFRP ;

[0092] The CDR2 of the variable region of the second antibody heavy chain is: TVVAFNFDTK ;

[0093] The CDR3 of the variable region of the second antibody heavy chain is: VSSLYPFGKLYQT ;

[0094] The CDR1 of the variable region of the second antibody light chain is: RTHVILTHA ;

[0095] The CDR2 of the variable region of the second antibody light chain is: LSAIRKPCQ ;

[0096] The CDR3 of the variable region of the second antibody light chain is: QRYNRAPYT .

[0097] In the antibody combination, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID No. 1 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 2 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 2; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID No. 3 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 4 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 4.

[0098] The amino acid sequence of the variable region of the first antibody heavy chain, SEQ ID No. 1, is as follows:

[0099] EVQLVESGGGLVQPGGSLRLSCAVS GMNDSLTSRN SVNWIRQAPGKGLEWVGLI KSAPEDTTF TDYNSAIKSRFTISRDTSKSTVYLQMNSLRAEDTAVYYCAR AKRTSMFPTD WGQGTLVTVSS;

[0100] The amino acid sequence of the variable region of the first antibody light chain, SEQ ID No. 2, is as follows:

[0101] DIQMTQSPSSLSASVGDRVTITC VPNKDFGAD WYQQKPGKAPKLLIY APNFNFQQ GVPSRFSGSGSATDYTLTISSLQPEDFATYYC MGTRLIPPSTV FGQGTKVEVKRTVA;

[0102] The amino acid sequence of the variable region of the second antibody heavy chain, SEQ ID No. 3, is as follows:

[0103] EVQLVESGGGLVQPGRSLRLSCAAS GNFTGFFRP AMHWVRQAPGKGLEWVSAI TVVAFNFDTK IDYADSVEGRFTISRDNAKNSLYLDMNSLRAEDTAVYYCAK VSSLYPFGKLYQT WGQGTLVTVSS;

[0104] The amino acid sequence of the variable region of the second antibody light chain, SEQ ID No. 4, is as follows:

[0105] DIQMTQSPSSLSASVGDRVTITC RTHVILTHA WYQQKPGKAPKLLIY LSAIRKPCQ GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QRYNRAPYT FGQGTKVEIKRTVA.

[0106] In the antibody combination, the amino acid sequence of the heavy chain constant region of the first antibody is as shown in SEQ ID No. 5 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 5, and the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 6 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 6; the amino acid sequence of the heavy chain constant region of the second antibody is as shown in SEQ ID No. 7 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 7, and the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 8 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 8.

[0107] The amino acid sequence of the first antibody heavy chain constant region, SEQ ID No. 5, is as follows:

[0108] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0109] The amino acid sequence of the constant region of the light chain of the first antibody, SEQ ID No. 6, is as follows:

[0110] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;

[0111] The amino acid sequence of the constant region of the second antibody heavy chain, SEQ ID No. 7, is as follows:

[0112] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0113] The amino acid sequence of the constant region of the light chain of the second antibody, SEQ ID No. 8, is as follows:

[0114] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0115] The primary and secondary antibodies are either coating antibodies or detection antibodies, which are independent of each other.

[0116] The antibody combination can be selected from any of the following combinations: the first antibody as the detection antibody, the second antibody as the detection antibody, the first antibody as the coating antibody + the second antibody as the detection antibody, or the second antibody as the coating antibody + the first antibody as the detection antibody.

[0117] The preferred method is to use the first antibody 18C5 as the coating antibody and the second antibody 3D5 as the detection antibody.

[0118] In other words, both the primary and secondary antibodies can be used independently as detection antibodies for tumor necrosis factor-α. In this case, conventional monoclonal antibody detection methods can be used to detect tumor necrosis factor-α with either the primary or secondary antibody. Alternatively, the primary antibody can be used as the coating antibody and the secondary antibody as the detection antibody, using a double antibody sandwich method to detect tumor necrosis factor-α; or the secondary antibody can be used as the coating antibody and the primary antibody as the detection antibody, using a double antibody sandwich method to detect tumor necrosis factor-α.

[0119] The present invention also provides polynucleotides encoding the heavy and light chains of the antibody combination described above for detecting tumor necrosis factor-α. The polynucleotide sequence encoding the variable region of the heavy chain of the first antibody is shown in SEQ ID No. 9 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 9. The polynucleotide sequence encoding the variable region of the light chain of the first antibody is shown in SEQ ID No. 10 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 10. The polynucleotide sequence encoding the variable region of the heavy chain of the second antibody is shown in SEQ ID No. 11 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 11. The polynucleotide sequence encoding the variable region of the light chain of the second antibody is shown in SEQ ID No. 12 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 12.

[0120] The polynucleotide sequence encoding the variable region of the first antibody heavy chain, SEQ ID No. 9, is:

[0121] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggtgagcggcatgaacgatagcctga ccagccgcaacagcgtgaactggattcgccaggcgccgggcaaaggcctggaatgggtgggcctgattaaaagcgcgccggaagataccacctt taccgattataacagcgcgattaaaagccgctttaccattagccgcgataccagcaaaagcaccgtgtatctgcagatgaacagcctgcgcgcg gaagataccgcggtgtattattgcgcgcgcgcgaaacgcaccagcatgtttccgaccgattggggccagggcaccctggtgaccgtgagcagc;

[0122] The polynucleotide sequence encoding the variable region of the first antibody light chain, SEQ ID No. 10, is as follows:

[0123] gatattcagatgacccagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcgtgccgaacaaagattttggcgcggattggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatgcgccgaactttaactttcagcagggc gtgccgagccgctttagcggcagcggcagcgcgaccgattataccctgaccattagcagcctgcagccggaagattttgcgacctattattgcatgggcacccgcctgattccgccgagcaccgtgtttggccagggcaccaaagtggaagtgaaacgcaccgtggcg;

[0124] The polynucleotide sequence encoding the variable region of the second antibody heavy chain, SEQ ID No. 11, is as follows:

[0125] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggccgcagcctgcgcctgagctgcgcggcgagcggcaactttaccggctttttt cgcccggcgatgcattgggtgcgccaggcgccgggcaaaggcctggaatgggtgagcgcgattaccgtggtggcgtttaactttgataccaaaaatt gattatgcggatagcgtggaaggccgctttaccattagccgcgataacgcgaaaaacagcctgtatctggatatgaacagcctgcgcgcggaagat accgcggtgtattattgcgcgaaagtgagcagcctgtatccgtttggcaaactgtatcagacctggggccagggcaccctggtgaccgtgagcagc;

[0126] The polynucleotide sequence encoding the variable region of the second antibody light chain, SEQ ID No. 12, is as follows:

[0127] gatattcagatgacccagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgccgcacccatgtgattctgacccatgcgtggtatcagcagaaaccgggcaaagcgccgaaactgctgatttatctgagcgcgattcgcaaaccgtgcca gggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagatgtggcgacctattattgccagcgctataaccgcgcgccgtatacctttggccagggcaccaaagtggaaattaaacgcaccgtggcg.

[0128] The heavy and light chain polynucleotides of the antibody combination for detecting tumor necrosis factor-α, wherein the polynucleotide sequence encoding the constant region of the heavy chain of the first antibody is as shown in SEQ ID No. 13 or a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 13; the polynucleotide sequence encoding the constant region of the light chain of the first antibody is as shown in SEQ ID No. 14 or a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 14; the polynucleotide sequence encoding the constant region of the heavy chain of the second antibody is as shown in SEQ ID No. 15 or a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 15; and the polynucleotide sequence encoding the constant region of the light chain of the second antibody is as shown in SEQ ID No. 16 or a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 16.

[0129] The polynucleotide sequence encoding the constant region of the first antibody heavy chain, SEQ ID No. 13, is as follows:

[0130] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0131] The polynucleotide sequence encoding the constant region of the first antibody light chain, SEQ ID No. 14, is as follows:

[0132] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;

[0133] The polynucleotide sequence encoding the constant region of the second antibody heavy chain, SEQ ID No. 15, is as follows:

[0134] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0135] The polynucleotide sequence encoding the constant region of the light chain of the second antibody, SEQ ID No. 16, is as follows:

[0136] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.

[0137] An expression system for heavy and light chain polynucleotides of an antibody combination for detecting tumor necrosis factor-α, the expression system being a mammalian expression vector.

[0138] The antigen expression system for tumor necrosis factor-α (TNF-α) is the Expi 293 expression system.

[0139] The antibody expression system is the CHO expression system.

[0140] Host cells containing the above expression system.

[0141] Application of antibody combinations for detecting tumor necrosis factor-α in the preparation of biopharmaceuticals for in vitro quantitative detection of tumor necrosis factor-α levels.

[0142] In vitro quantitative detection refers to the detection of the content of tumor necrosis factor-α in serum, plasma, whole blood and / or peripheral blood.

[0143] The biological product is a reagent kit.

[0144] The kit is a double-antibody sandwich assay kit.

[0145] Example 2

[0146] The first and second antibodies were both obtained by screening using phage display and then the purified proteins were labeled with NHS-Biotin reagent. The tumor necrosis factor-α (TNF-α) antigen was expressed using the Expi293 expression system, and its expression sequence is shown in SEQ ID No. 17 or is an amino acid sequence with greater than 90% homology to the sequence in SEQ ID No. 17.

[0147] SEQ ID No.17 sequence:

[0148] VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL*.

[0149] The nucleotide sequence of the tumor necrosis factor-α (TNF-α) antigen is as shown in SEQ ID No. 18 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 18.

[0150] SEQ ID No.18 sequence:

[0151] gtgcgcagcagcagccgcaccccgagcgataaaccggtggcgcatgtggtggcgaacccgcaggcggaaggccagctgcagtggctgaaccgccgcgcgaacgcgctgctggcgaacg gcgtggaactgcgcgataaccagctggtggtgccgagcgaaggcctgtatctgatttatagccaggtgctgtttaaaggccagggctgcccgagcacccatgtgctgctgacccatacc attagccgcattgcggtgagctatcagaccaaagtgaacctgctgagcgcgattaaaagcccgtgccagcgcgaaaccccggaaggcgcggaagcgaaaccgtggtatgaaccgattta tctgggcggcgtgtttcagctggaaaaaggcgatcgcctgagcgcggaaattaaccgcccggattatctggattttgcggaaagcggccaggtgtattttggcattattgcgctgtaa.

[0152] Example 3

[0153] The specific composition of the kit containing two antibodies and the specific concentration of each component are shown in Table 1.

[0154] Table 1

[0155]

[0156] The reagents are shown below:

[0157] (1) ELISA coating buffer (pH 9.6 0.05M carbonate buffer):

[0158] 1.59 grams of NaHCO3

[0159] NaHCO3 2.93 g,

[0160] Add distilled water to a final volume of 1000 ml;

[0161] (2) ELISA washing buffer (pH 7.4 PBS): 0.15M

[0162] 0.2 g of KH2PO4

[0163] 2.9 grams of Na2HPO4·12H2O

[0164] 8.0 grams of NaCl

[0165] 0.2 grams of KCl

[0166] Tween-20 0.05% 0.5ml

[0167] Add distilled water to a final volume of 1000 ml;

[0168] (3) ELISA sample dilution solution:

[0169] Bovine serum albumin (BSA) 0.1 g,

[0170] Add washing buffer to 100 ml;

[0171] (4) ELISA test stop solution (2M H2SO4):

[0172] Add 178.3 ml of distilled water dropwise to 21.7 ml of concentrated sulfuric acid (98%).

[0173] (5) ELISA substrate buffer (pH 5.0, disodium hydrogen phosphate, citrate):

[0174] 0.2M Na₂HPO₄ (28.4 g / L) 25.7 ml,

[0175] 0.1M citric acid (19.2 g / L) 24.3 ml

[0176] Add 50ml of distilled water;

[0177] (6) ELISA assay TMB (tetramethylbenzidine) solution:

[0178] TMB (10mg / 5ml anhydrous ethanol) 0.5ml,

[0179] 10 ml of substrate buffer (pH 5.5)

[0180] 0.75% H2O2 32μl;

[0181] (7) ELISA blocking solution:

[0182] 5 grams of bovine serum albumin (BSA)

[0183] Add washing buffer to 100 ml.

[0184] Example 4

[0185] 1. The specific operation procedure for using a kit containing two antibodies for the detection of tumor necrosis factor-α is shown below.

[0186] step:

[0187] 1) Antibody pre-coating: Dilute 18C5 antibody with coating buffer to a concentration of 1 μg / mL, add 100 μL to each well of the microplate, and incubate overnight at 4°C;

[0188] 2) Blocking: Add 100 μL of blocking solution to each well, block at 37°C for 0.5-1 hour, wash the plate 3 times with washing buffer for 30 seconds each time, and pat dry.

[0189] 3) Dilution and loading of standards: Set up 16 wells for standards and blank control on the enzyme-labeled plate. Serially dilute hTNF-α antigen with sample diluent at 200 μL each concentration, resulting in concentrations of 500 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.6 pg / mL, 7.8 pg / mL, and 3.9 pg / mL. Each concentration is tested in duplicate. Load 100 μL of sample diluent into each well. Add 100 μL of sample diluent to each blank well.

[0190] 4) Dilution and addition of the sample to be tested: Add 240 μl of sample diluent and then 60 μl of the sample to be tested (the final dilution of the sample is 5 times). Repeat this process in three replicates, adding 100 μl of the sample to the bottom of each well of the microplate.

[0191] 5) Incubation: After sealing the plate with the sealing film, incubate at 37°C for 30 minutes. Carefully remove the sealing film, discard the liquid, shake dry, wash the plate 3 times with washing buffer for 30 seconds each time, and pat dry.

[0192] 6) Biotin-labeled antibody incubation: Add freshly diluted biotin-labeled antibody to each well, 100 μL per well, incubate at 37°C for 0.5-1 h, wash the plate 3 times with washing buffer for 30 s each time, and pat dry.

[0193] 7) Color development: Add 50 μl of TMB color developer to each well, gently shake to mix, and develop color at 37°C in the dark for 15 minutes.

[0194] 8) Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow at this point);

[0195] 9) Measurement: Zero the blank air conditioner and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0196] 2. Sample requirements:

[0197] Collect serum using sterile tubes. Allow the blood to clot naturally at room temperature for 10-20 minutes, then centrifuge at 2-8°C for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If precipitation occurs during storage, centrifuge again.

[0198] The criteria for interpreting the results of the antibody-containing kit for the in vitro quantitative detection of tumor necrosis factor-α (TNF-α) in human serum, plasma, whole blood, and peripheral blood are shown in Table 2 below.

[0199] Table 2

[0200] TNF-α (ng / ml) Clinical application recommendations ＜0.006 Reference values ​​for 95% of sites in seemingly healthy individuals 0.006-0.120 Normal, or may indicate mild inflammation or mild infection. 0.120-0.200 This suggests a common bacterial infection or a systemic inflammatory response. ＞0.200 This could be a sign of sepsis.

[0201] The standard curve of the reagent kit of the present invention is as follows: Figure 1 As shown.

[0202] Example 5

[0203] A commercially available TNF-α detection kit was used as a comparison product 1, and the samples were tested using the kit provided in Example 4 of this invention.

[0204] Commercially available TNF-α detection kits are:

[0205] Comparison with Product 1:

[0206] Product Name: Human TNF-α ELISA Kit (Human Tumor Necrosis Factor-α Enzyme-Linked Immunosorbent Assay Kit);

[0207] Product code: PT518;

[0208] Product specifications: 96T;

[0209] Purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0210] During the testing process, the basic characteristics of the reagent kits were first compared, as shown in Table 3 below.

[0211] Table 3

[0212] Example 4 Comparison Product 1 antigen Human TNF-α Human TNF-α Coated antibody 18C5 Human TNF-α antibody Antibody detection 3D5 Human TNF-α biotinylated antibody method ELISA ELISA Sensitivity 2.2 pg / mL 4.3 pg / mL Detection range 10 pg / mL - 1000 pg / mL 50 pg / mL - 200 pg / mL

[0213] As can be seen from Table 3, the basic characteristics of each kit differ. Both Example 4 and Comparative Product 1 target tumor necrosis factor-α (TNF-α) in human serum; however, the coating antibody and detection antibody in Example 4 are completely different from those in Comparative Product 1. Under these circumstances, the detection sensitivity of the kit provided in Example 4 significantly exceeds that of Comparative Product 1, and its detection range is also wider. The kit in Example 4 has actually achieved the highest sensitivity achievable by similar products.

[0214] Furthermore, TNF-α was detected using blood samples. The specific detection method was as follows: 10 serum samples were collected from patients with inflammation or infection and 10 from normal individuals. TNF-α was detected using the kit described in Example 4 of this patent and a commercially available TNF-α detection kit (comparative product 1). The results are shown in Table 4 below. In Table 4, group H represents serum from healthy individuals, and group P represents serum from individuals with inflammation or infection.

[0215] Table 4

[0216]

[0217]

[0218] Based on the criteria in Table 2, it can be determined that if the serum TNF-α concentration is >6 pg / mL, it indicates the possible presence of inflammation or other infections. In the comparison of detection data in Table 4, it can be seen that the commercially available kit (comparison product 1) showed false negatives / false positives due to low sensitivity in the detection of H4, H7, P2, P9, and P10, with a false negative / false positive rate of 25%, which is considered a high level. In contrast, the kit of this invention showed a false positive in the detection of H10, with a false negative / false positive rate of 5%. This demonstrates that the kit containing the antibody combination of this invention has a much higher sensitivity than existing products.

[0219] Example 6

[0220] This invention provides two antibodies for detecting tumor necrosis factor-α in human serum samples: a first antibody 18C5 and a second antibody 3D5. These antibodies are combined and detected using a double-antibody sandwich method. However, the two antibodies obtained by phage display screening also possess the function of detecting tumor necrosis factor-α in human serum samples when used alone or interchangeably. To verify their effectiveness when used alone or interchangeably, the applicant conducted detection using methods similar to those in Examples 1-5 (the monoclonal antibody was detected using a conventional method, and the double antibody was detected using a sandwich method), and the results were compared as follows.

[0221] The basic characteristics of the kits consisting of a single primary antibody 18C5, a single secondary antibody 3D5, an antibody combination of 18C5 (coating) + 3D5 (detection), and an antibody combination of 3D5 (coating) + 18C5 (detection) are compared in Table 5.

[0222] Table 5

[0223] 18C5 alone 3D5 standalone 3D5+18C5 18C5+3D5 antigen Human TNF-α Human TNF-α Human TNF-α Human TNF-α Coated antibody — — 3D5 (second antibody) 18C5 (primary antibody) Antibody detection 18C5 (primary antibody) 3D5 (second antibody) 18C5 (primary antibody) 3D5 (second antibody) method ELISA ELISA ELISA ELISA Sensitivity 3.2 pg / mL 3.0 pg / mL 2.5 pg / mL 2.2 pg / mL Detection range 50 pg / mL - 500 pg / mL 50 pg / mL - 500 pg / mL 20 pg / mL - 800 pg / mL 10 pg / mL - 1000 pg / mL

[0224] As shown in Table 5, both the first antibody 18C5 and the second antibody 3D5 alone can effectively detect tumor necrosis factor-α in human serum samples. The detection sensitivity and detection range of the two antibodies alone are also better than those of existing commercially available products (compare product 1 in Table 3). The antibody combination 3D5 (coating) + 18C5 (detection), which swaps the first and second antibodies, is even better than the use of the first and second antibodies alone. Furthermore, the antibody combination 18C5 + 3D5 described in this invention has the best detection sensitivity and detection range among all options, which is not only better than existing products, but also better than the methods of using them alone and swapping them.

[0225] To test the actual usage effect, the applicant also conducted TNF-α testing on random human blood samples. The testing method was similar to the one that yielded the conclusions in Table 4, and the specific results are shown in Table 6.

[0226] Table 6

[0227]

[0228]

[0229] Similarly, based on the judgment criteria in Table 2, it can be determined that if the serum TNF-α concentration is >6 pg / mL, it indicates the possible presence of inflammation or other infections. In the comparison of the detection data in Table 6, it can be seen that the antibody combination 18C5+3D5 kit showed false negatives in the detection of H10, with a false negative rate of 5%; the antibody combination 3D5+18C5 kit showed false negatives in the detection of H4, H8, and P5, with a false negative rate of 15%; using the second antibody 3D5 alone showed false negatives in the detection of H5, P3, and P7, with a false negative rate of 15%; using the first antibody 18C5 alone showed false negatives in the detection of H8, P1, P4, and P7, with a false negative rate of 20%. These comparison results indicate that the detection sensitivity of using the first and second antibodies alone is slightly lower than that of using the antibody combination.

[0230] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibody composition for detecting tumor necrosis factor-α, characterized in that, The antibody composition comprises a first antibody and a second antibody. The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the first antibody are shown as amino acid sequences at positions 26-35, 55-63, and 105-114 of SEQ ID No. 1, respectively, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 24-32, 48-55, and 88-98 of SEQ ID No. 2, respectively. The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the second antibody are shown as amino acid sequences at positions 26-34, 54-63, and 105-117 of SEQ ID No. 3, respectively, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 24-32, 48-56, and 89-97 of SEQ ID No. 4, respectively.

2. The antibody composition for detecting tumor necrosis factor-α according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID No. 1 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 2 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 2; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID No. 3 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 4 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No.

4.

3. The antibody composition for detecting tumor necrosis factor-α according to claim 2, characterized in that, The amino acid sequence of the heavy chain constant region of the first antibody is as shown in SEQ ID No. 5 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 5, and the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 6 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 6; the amino acid sequence of the heavy chain constant region of the second antibody is as shown in SEQ ID No. 7 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No. 7, and the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 8 or is an amino acid sequence with greater than 90% homology to the sequence of SEQ ID No.

8.

4. The antibody composition for detecting tumor necrosis factor-α according to any one of claims 1-3, characterized in that, The first antibody and the second antibody are independently either coating antibodies or detection antibodies.

5. The antibody composition for detecting tumor necrosis factor-α according to claim 4, characterized in that, The antibody composition is selected from any of the following combinations: a first antibody as a coating antibody and a second antibody as a detection antibody, or a second antibody as a coating antibody and a first antibody as a detection antibody.

6. The antibody composition for detecting tumor necrosis factor-α according to claim 5, characterized in that, The antibody composition is selected with a first antibody as the coating antibody and a second antibody as the detection antibody.

7. A polynucleotide encoding the heavy and light chains of the antibody composition for detecting tumor necrosis factor-α according to any one of claims 1-6, characterized in that, The polynucleotide sequence encoding the heavy chain variable region of the first antibody is as shown in SEQ ID No. 9 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 9; the polynucleotide sequence encoding the light chain variable region of the first antibody is as shown in SEQ ID No. 10 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 10; the polynucleotide sequence encoding the heavy chain variable region of the second antibody is as shown in SEQ ID No. 11 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 11; the polynucleotide sequence encoding the light chain variable region of the second antibody is as shown in SEQ ID No. 12 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No.

12.

8. The heavy and light chain polynucleotides of the antibody composition for detecting tumor necrosis factor-α according to claim 7, characterized in that, The polynucleotide sequence encoding the heavy chain constant region of the first antibody is as shown in SEQ ID No. 13 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 13; the polynucleotide sequence encoding the light chain constant region of the first antibody is as shown in SEQ ID No. 14 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 14; the polynucleotide sequence encoding the heavy chain constant region of the second antibody is as shown in SEQ ID No. 15 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 15; the polynucleotide sequence encoding the light chain constant region of the second antibody is as shown in SEQ ID No. 16 or is a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No.

16.

9. The expression system of heavy and light chain polynucleotides of the antibody composition for detecting tumor necrosis factor-α according to any one of claims 7-8, characterized in that, The expression system is a mammalian expression vector.

10. The expression system according to claim 9, characterized in that, The antigen expression system for tumor necrosis factor-α is the Expi 293 expression system.

11. The expression system according to claim 9, characterized in that, The antibody expression system is a CHO expression system.

12. A host cell containing the expression system according to any one of claims 9-11.

13. The use of the antibody composition according to any one of claims 1-6 in the preparation of a biological product for the in vitro quantitative detection of tumor necrosis factor-α content.

14. The application according to claim 13, characterized in that, The in vitro quantitative detection refers to the detection of the content of tumor necrosis factor-α in serum, plasma or peripheral blood.

15. The application according to claim 14, characterized in that, The biological product in question is a reagent kit.

16. The application according to claim 15, characterized in that, The kit is a double-antibody sandwich assay kit.

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