Antibody combinations for detecting interferon-gamma and their applications

Abstract

This invention provides an antibody combination for detecting interferon-gamma, comprising a first antibody and / or a second antibody, each antibody possessing a unique CDR fragment; it also provides specific applications of this antibody combination. The advantages of this invention are that the antibody combination exhibits strong affinity for interferon-gamma antigens, high detection sensitivity, ease of use, and rapid and stable detection. The concentration of IFN-γ in normal human blood is approximately 10.57 ± 3.58 ng / mL. Existing conventional low-affinity antibodies are insufficient for effective detection in this situation. However, the high-affinity antibody combination provided by this invention, along with the interferon-gamma quantitative detection kit containing this antibody combination using a double-antibody sandwich method, can effectively achieve quantitative detection of ultra-low concentrations of interferon-gamma. Verification shows that the kit's sensitivity can reach 2.8 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 gamma interferon and their applications. Technical Background

[0002] Interferon is a type of glycoprotein with high species specificity, so animal interferon is ineffective in humans. Interferon has antiviral, cell proliferation inhibitory, immune-regulating, and antitumor effects.

[0003] In 1957, Isaacs and Lindenmann first discovered the phenomenon of viral interference, namely that virus-infected cells can produce a factor that acts on other cells to interfere with viral replication, hence the name interferon. It is now known that interferons do not directly kill viruses, but rather induce host cells to produce several enzymes that interfere with viral gene transcription or the translation of viral protein components.

[0004] Based on the different cell sources that produce interferon, as well as differences in physicochemical properties and biological activities, interferon can be divided into α-interferon (IFN-α), β-interferon (IFN-β), and γ-interferon (IFN-γ).

[0005] Biological activities of IFN-γ:

[0006] The biological effects of IFN-γ exhibit strict species specificity; human IFN-γ acts only on human or primate cells, and its mechanism of action is as follows:

[0007] (1) It induces the expression of MHC class II antigens such as monocytes, macrophages, dendritic cells, skin fibroblasts, vascular endothelial cells, and stellate cells, enabling them to participate in antigen presentation and specific immune recognition processes; in addition, IFN-γ can upregulate the expression of ICAM-1 (CD54) in endothelial cells, promote the expression of FcγR in macrophages, synergistically induce TNF and promote macrophage killing of pathogenic microorganisms; secondly, in some immune diseases, the level of IFN-γ shows abnormalities, for example, the serum IFN-γ level in patients with lupus nephritis is significantly elevated, leading to tissue damage in patients.

[0008] (2) Promotes LPS-stimulated mouse B cell secretion and IgG2a, and reduces the production of IgG1, IgG2b, IgG3 and IgE; inhibits IL-4-induced mouse B cell proliferation, IgG1 and IgE production and FcεRII expression; promotes SAC-induced human B cell proliferation;

[0009] (3) Synergistically induces LAK activity with IL-2 and promotes IL-2R expression in T cells;

[0010] (4) Induces acute phase protein synthesis and induces myeloid cell differentiation.

[0011] However, since the concentration of IFN-γ in normal human blood is approximately 10.57±3.58 ng / mL, most existing biological products targeting IFN-γ are low-affinity antibodies, resulting in low accuracy and sensitivity in the detection of IFN-γ. Summary of the Invention

[0012] To address the aforementioned limitations, this invention proposes an antibody combination for detecting gamma interferon and its application, overcoming the deficiencies and defects mentioned in the background art.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] The present invention provides an antibody combination for detecting interferon-gamma (IFN-γ), 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 26-36, 56-63, and 105-116 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 23-36, 52-58, and 91-100 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, 54-61, and 103-113 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 23-35, 51-59, and 92-99 of SEQ ID No. 4, respectively.

[0015] The primary antibody is 29G2, and the secondary antibody is 25B6.

[0016] The CDR1 of the variable region of the first antibody heavy chain is: GVARSITTRSY ;

[0017] The CDR2 of the variable region of the first antibody heavy chain is: NDYNYDGD ;

[0018] The CDR3 of the variable region of the first antibody heavy chain is: DPDAATGFHPDY ;

[0019] The CDR1 of the variable region of the first antibody light chain is: AGANNDIGTYAYVS ;

[0020] The CDR2 of the variable region of the first antibody light chain is: KVTTRAS ;

[0021] The CDR3 of the variable region of the first antibody light chain is: ASYRNFNNAV ;

[0022] The CDR1 of the variable region of the second antibody heavy chain is: GTFDQTSSY ;

[0023] The CDR2 of the variable region of the second antibody heavy chain is: SAVVPTGS ;

[0024] The CDR3 of the variable region of the second antibody heavy chain is: RAGNYNTYGDY ;

[0025] The CDR1 of the variable region of the second antibody light chain is: GLSVTASTDYPPG ;

[0026] The CDR2 of the variable region of the second antibody light chain is: SVPNDAWSS ;

[0027] The CDR3 of the variable region of the second antibody light chain is: ALQIRKTE .

[0028] In a preferred embodiment, the antibody combination for detecting interferon-gamma has the following amino acid sequence: 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 light chain variable region has the following amino acid sequence: 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 light chain variable region has the following amino acid sequence: the light chain variable region of the second antibody 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.

[0029] The amino acid sequence of the variable region of the first antibody heavy chain, SEQ ID No. 1, is as follows:

[0030] EVQLQESGPGLVKPSQTLSLTCTVS GVARSITTRSY AWSWIRQPPGKGLEWMGVI NDYNYDGD TYYSPSLKSRTSISWDTSKNQFSLQLSSVTPEDTAVYYCAR DPDAATGFHPDY WGQGTQVTVSS;

[0031] The amino acid sequence of the variable region of the first antibody light chain, SEQ ID No. 2, is as follows:

[0032] QAVLTQPPLVSGTPGQTVTISC AGANNDIGTYAYVSWYQQLPGTAPKLLIY KVTTRAS GIPSRFSGSKSGNTASLTISGLQSEDEADYYC ASYRNFNNAV FGRGTHLTVLGQPKA;

[0033] The amino acid sequence of the variable region of the second antibody heavy chain, SEQ ID No. 3, is as follows:

[0034] EVQLVESGGGLVQPGGSLRLSCAAS GTFDQTSSY RMYWVRQPPGKGLEWVSAI SAVVPTGS TYYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAN RAGNYNTYGDY WGQGTQVTVSS;

[0035] The amino acid sequence of the variable region of the second antibody light chain, SEQ ID No. 4, is as follows:

[0036] QTVVTQEPSLSVSPGGTVTLTC GLSVTASTDYPPG WFQQTPGQAPRALIY SVPNDAWSS GVPSRFSGSISGNKAALTITGAQPEDEADYYC ALQIRKTE FGGGTHLTVLGQPKA.

[0037] In a preferred embodiment, the antibody combination for detecting gamma interferon includes 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.

[0038] The amino acid sequence of the first antibody heavy chain constant region, SEQ ID No. 5, is as follows:

[0039] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0040] The amino acid sequence of the constant region of the light chain of the first antibody, SEQ ID No. 6, is as follows:

[0041] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;

[0042] The amino acid sequence of the constant region of the second antibody heavy chain, SEQ ID No. 7, is as follows:

[0043] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0044] The amino acid sequence of the constant region of the light chain of the second antibody, SEQ ID No. 8, is as follows:

[0045] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0046] The antibody combination used to detect interferon-gamma, wherein the first and second antibodies are independently either coating antibodies or detection antibodies.

[0047] Furthermore, in the above-mentioned antibody combination for detecting gamma interferon, 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.

[0048] In other words, both the primary and secondary antibodies can be used individually as detection antibodies for interferon-gamma. In this case, conventional monoclonal antibody detection methods can be used to detect interferon-gamma using 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, employing a double-antibody sandwich method for interferon-gamma detection; or the secondary antibody can be used as the coating antibody and the primary antibody as the detection antibody, employing a double-antibody sandwich method for interferon-gamma detection.

[0049] The second inventive aspect of this invention is to provide a polynucleotide encoding the heavy and light chains of the aforementioned antibody combination for detecting interferon-gamma, 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.

[0050] The polynucleotide sequence encoding the variable region of the first antibody heavy chain, SEQ ID No. 9, is:

[0051] gaagtgcagctgcaggaaagcggcccgggcctggtgaaaccgagccagaccctgagcctgacctgcaccgtgagcggcgtggcgcgcagcattaccacccgcagctatgcgtggagctggattcgccagccgccgggcaaaggcctggaatggatgggcgtgattaacgattataactatgatggcgatacctattatagcccgagcctgaaaagccgcaccagcattagctgggataccagcaaaaaccagtttagcctgcagctgagcagcgtgaccccggaagataccgcggtgtattattgcgcgcgcgatccggatgcggcgaccggctttcatccggattattggggccagggcacccaggtgaccgtgagcagc;

[0052] The polynucleotide sequence SEQ ID No.10 encoding the variable region of the light chain of the first antibody is:

[0053] caggcggtgctgacccagccgccgctggtgagcggcaccccgggccagaccgtgaccattagctgcgcgggcgcgaacaacgatattggcacctatgcgtatgtgagctggtatcagcagctgccgggcaccgcgccgaaactgctgatttataaagtgaccacccgcgcgagcggcattccgagccgctttagcggcagcaaaagcggcaacaccgcgagcctgaccattagcggcctgcagagcgaagatgaagcggattattattgcgcgagctatcgcaactttaacaacgcggtgtttggccgcggcacccatctgaccgtgctgggccagccgaaagcg;

[0054] The polynucleotide sequence SEQ ID No.11 encoding the variable region of the heavy chain of the second antibody is:

[0055] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggcacctttgatcagaccagcagctatcgcatgtattgggtgcgccagccgccgggcaaaggcctggaatgggtgagcgcgattagcgcggtggtgccgaccggcagcacctattatggcgatagcgtgaaaggccgctttaccattagccgcgataacgcgaaaaacaccgtgtatctgcagatgaacagcctgaaaccggaagataccgcggtgtattattgcgcgaaccgcgcgggcaactataacacctatggcgattattggggccagggcacccaggtgaccgtgagcagc;

[0056] The polynucleotide sequence SEQ ID No.12 encoding the variable region of the light chain of the second antibody is:

[0057] cagaccgtggtgacccaggaaccgagcctgagcgtgagcccgggcggcaccgtgaccctgacctgcggcctgagcgtgaccgcgagcaccgattatccgccgggctggtttcagcagaccccgggccaggcgccgcgcgcgctgatttatagcgtgccgaacgatgcgtggagcagcggcgtgccgagccgctttagcggcagcattagcggcaacaaagcggcgctgaccattaccggcgcgcagccggaagatgaagcggattattattgcgcgctgcagattcgcaaaaccgaatttggcggcggcacccatctgaccgtgctgggccagccgaaagcg。

[0058] In a preferred embodiment, the heavy and light chain polynucleotides of the antibody combination for detecting interferon-gamma include: a polynucleotide sequence encoding the constant region of the heavy chain 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 constant region of the light chain 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 constant region of the heavy chain 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 constant region of the light chain 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.

[0059] The polynucleotide sequence encoding the constant region of the first antibody heavy chain, SEQ ID No. 13, is as follows:

[0060] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0061] The polynucleotide sequence encoding the constant region of the first antibody light chain, SEQ ID No. 14, is as follows:

[0062] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;

[0063] The polynucleotide sequence encoding the constant region of the second antibody heavy chain, SEQ ID No. 15, is as follows:

[0064] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0065] The polynucleotide sequence encoding the constant region of the light chain of the second antibody, SEQ ID No. 16, is as follows:

[0066] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.

[0067] The third inventive aspect of this invention is to provide an expression system for the heavy and light chain polynucleotides of the antibody combination for detecting gamma interferon, wherein the expression system is a mammalian expression vector.

[0068] In a preferred embodiment, the expression system described above, wherein the antigen expression system for interferon γ is the Expi293 expression system.

[0069] In a preferred embodiment, the above-described expression system, the antibody expression system, is a CHO expression system.

[0070] The fourth inventive point of this invention is to provide a host cell containing the above-described expression system.

[0071] The fifth inventive point of this invention is to provide the application of the above-described antibody combination in the preparation of biological products for in vitro quantitative detection of interferon-gamma content.

[0072] In a preferred embodiment, the above-described application, wherein the in vitro quantitative detection refers to the detection of the content of gamma interferon in serum, plasma, whole blood and / or peripheral blood.

[0073] In a preferred embodiment, the above-described application uses a reagent kit as the biological product.

[0074] In a preferred embodiment, the above-described application refers to a double-antibody sandwich assay kit.

[0075] Compared with the prior art, the present invention has the following advantages:

[0076] The present invention provides an antibody combination for detecting interferon-gamma and its application. The antibody combination has a strong affinity for interferon-gamma antigen, high detection sensitivity, is easy to use, and provides rapid and stable detection. The concentration of IFN-γ in normal human blood is approximately 10.57±3.58 ng / mL. Existing conventional low-affinity antibodies are difficult to effectively detect in this situation. However, the high-affinity antibody combination and the interferon-gamma quantitative detection kit containing this antibody combination using a double-antibody sandwich method provided by the present invention can effectively achieve the quantitative detection of ultra-low concentrations of interferon-gamma. The sensitivity of the kit has been verified to reach 2.8 pg / mL.

[0077] The double-antibody sandwich method used in this invention to determine the IFN-γ level in a sample 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 IFN-γ in the sample. This structure is more conducive to the detection of IFN-γ. Furthermore, this invention effectively utilizes an avidin-biotin-enzyme complex to achieve high detection sensitivity.

[0078] The detection kit of the present invention can not only rapidly and accurately determine the IFN-γ content 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 IFN-γ due to the use of a combination of two specific monoclonal antibodies. Attached Figure Description

[0079] Figure 1 This is a standard curve for an ELISA kit containing an antibody combination for detecting gamma interferon, provided in an embodiment of the present invention. Detailed Implementation

[0080] 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.

[0081] 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.

[0082] To further understand the present invention, the present invention will be described in detail below with reference to the preferred embodiments.

[0083] Example 1

[0084] An antibody combination for detecting interferon-gamma (IFN-γ) 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 26-36, 56-63, and 105-116 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 23-36, 52-58, and 91-100 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, 54-61, and 103-113 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 23-35, 51-59, and 92-99 of SEQ ID No. 4, respectively.

[0085] The primary antibody is 29G2, and the secondary antibody is 25B6.

[0086] The CDR1 of the variable region of the first antibody heavy chain is: GVARSITTRSY ;

[0087] The CDR2 of the variable region of the first antibody heavy chain is: NDYNYDGD ;

[0088] The CDR3 of the variable region of the first antibody heavy chain is: DPDAATGFHPDY ;

[0089] The CDR1 of the variable region of the first antibody light chain is: AGANNDIGTYAYVS ;

[0090] The CDR2 of the variable region of the first antibody light chain is: KVTTRAS ;

[0091] The CDR3 of the variable region of the first antibody light chain is: ASYRNFNNAV ;

[0092] The CDR1 of the variable region of the second antibody heavy chain is: GTFDQTSSY ;

[0093] The CDR2 of the variable region of the second antibody heavy chain is: SAVVPTGS ;

[0094] The CDR3 of the variable region of the second antibody heavy chain is: RAGNYNTYGDY ;

[0095] The CDR1 of the variable region of the second antibody light chain is: GLSVTASTDYPPG ;

[0096] The CDR2 of the variable region of the second antibody light chain is: SVPNDAWSS ;

[0097] The CDR3 of the variable region of the second antibody light chain is: ALQIRKTE .

[0098] 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.

[0099] The amino acid sequence of the variable region of the first antibody heavy chain, SEQ ID No. 1, is as follows:

[0100] EVQLQESGPGLVKPSQTLSLTCTVS GVARSITTRSY AWSWIRQPPGKGLEWMGVI NDYNYDGD TYYSPSLKSRTSISWDTSKNQFSLQLSSVTPEDTAVYYCAR DPDAATGFHPDY WGQGTQVTVSS;

[0101] The amino acid sequence of the variable region of the first antibody light chain, SEQ ID No. 2, is as follows:

[0102] QAVLTQPPLVSGTPGQTVTISC AGANNDIGTYAYVS WYQQLPGTAPKLLIY KVTTRAS GIPSRFSGSKSGNTASLTISGLQSEDEADYYC ASYRNFNNAV FGRGTHLTVLGQPKA;

[0103] The amino acid sequence of the variable region of the second antibody heavy chain, SEQ ID No. 3, is as follows:

[0104] EVQLVESGGGLVQPGGSLRLSCAAS GTFDQTSSY RMYWVRQPPGKGLEWVSAI SAVVPTGS TYYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAN RAGNYNTYGDY WGQGTQVTVSS;

[0105] The amino acid sequence of the variable region of the second antibody light chain, SEQ ID No. 4, is as follows:

[0106] QTVVTQEPSLSVSPGGTVTLTC GLSVTASTDYPPG WFQQTPGQAPRALIY SVPNDAWSS GVPSRFSGSISGNKAALTITGAQPEDEADYYC ALQIRKTE FGGGTHLTVLGQPKA.

[0107] 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.

[0108] The amino acid sequence of the first antibody heavy chain constant region, SEQ ID No. 5, is as follows:

[0109] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0110] The amino acid sequence of the constant region of the light chain of the first antibody, SEQ ID No. 6, is as follows:

[0111] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;

[0112] The amino acid sequence of the constant region of the second antibody heavy chain, SEQ ID No. 7, is as follows:

[0113] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0114] The amino acid sequence of the constant region of the light chain of the second antibody, SEQ ID No. 8, is as follows:

[0115] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0116] The primary and secondary antibodies are either coating antibodies or detection antibodies, which are independent of each other.

[0117] 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.

[0118] The preferred method is to use the first antibody 29G2 as the coating antibody and the second antibody 25B6 as the detection antibody.

[0119] In other words, both the primary and secondary antibodies can be used individually as detection antibodies for interferon-gamma. In this case, conventional monoclonal antibody detection methods can be used to detect interferon-gamma using 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, employing a double-antibody sandwich method for interferon-gamma detection; or the secondary antibody can be used as the coating antibody and the primary antibody as the detection antibody, employing a double-antibody sandwich method for interferon-gamma detection.

[0120] The present invention also provides polynucleotides encoding the heavy and light chains of the antibody combination for detecting interferon gamma described above. 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.

[0121] The polynucleotide sequence encoding the variable region of the first antibody heavy chain, SEQ ID No. 9, is:

[0122] gaagtgcagctgcaggaaagcggcccgggcctggtgaaaccgagccagaccctgagcctgacctgcaccgtgagcggcgtggcgcgcagcattac cacccgcagctatgcgtggagctggattcgccagccgccgggcaaaggcctggaatggatgggcgtgattaacgattataactatgatggcgatac ctattatagcccgagcctgaaaagccgcaccagcattagctgggataccagcaaaaaccagtttagcctgcagctgagcagcgtgaccccggaagataccgcggtgtattattgcgcgcgcgatccggatgcggcgaccggctttcatccggattattggggccagggcacccaggtgaccgtgagcagc;

[0123] The polynucleotide sequence encoding the variable region of the first antibody light chain, SEQ ID No. 10, is as follows:

[0124] caggcggtgctgacccagccgccgctggtgagcggcaccccgggccagaccgtgaccattagctgcgcgggcgcgaacaacgatattggcacctatgcgtatgtgagctggtatcagcagctgccgggcaccgcgccgaaactgctgatttataaagtgaccacccgcgcgag cggcattccgagccgctttagcggcagcaaaagcggcaacaccgcgagcctgaccattagcggcctgcagagcgaagatgaagcggattattattgcgcgagctatcgcaactttaacaacgcggtgtttggccgcggcacccatctgaccgtgctgggccagccgaaagcg;

[0125] The polynucleotide sequence encoding the variable region of the second antibody heavy chain, SEQ ID No. 11, is as follows:

[0126] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggcacctttgatcagacc agcagctatcgcatgtattgggtgcgccagccgccgggcaaaggcctggaatgggtgagcgcgattagcgcggtggtgccgaccggcagcacc tattatggcgatagcgtgaaaggccgctttaccattagccgcgataacgcgaaaaacaccgtgtatctgcagatgaacagcctgaaaccggaagataccgcggtgtattattgcgcgaaccgcgcgggcaactataacacctatggcgattattggggccagggcacccaggtgaccgtgagcagc;

[0127] The polynucleotide sequence encoding the variable region of the second antibody light chain, SEQ ID No. 12, is as follows:

[0128] cagaccgtggtgacccaggaaccgagcctgagcgtgagcccgggcggcaccgtgaccctgacctgcggcctgagcgtgaccgcgagcaccgattatccgccgggctggtttcagcagaccccgggccaggcgccgcgcgcgctgatttatagcgtgccgaacgatgcgtgg agcagcggcgtgccgagccgctttagcggcagcattagcggcaacaaagcggcgctgaccattaccggcgcgcagccggaagatgaagcggattattattgcgcgctgcagattcgcaaaaccgaatttggcggcggcacccatctgaccgtgctgggccagccgaaagcg.

[0129] The heavy and light chain polynucleotides of the antibody combination for detecting interferon-gamma include: a polynucleotide sequence encoding the constant region of the heavy chain 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 constant region of the light chain 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 constant region of the heavy chain 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 constant region of the light chain 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.

[0130] The polynucleotide sequence encoding the constant region of the first antibody heavy chain, SEQ ID No. 13, is as follows:

[0131] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0132] The polynucleotide sequence encoding the constant region of the first antibody light chain, SEQ ID No. 14, is as follows:

[0133] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;

[0134] The polynucleotide sequence encoding the constant region of the second antibody heavy chain, SEQ ID No. 15, is as follows:

[0135] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0136] The polynucleotide sequence encoding the constant region of the light chain of the second antibody, SEQ ID No. 16, is as follows:

[0137] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.

[0138] An expression system for heavy and light chain polynucleotides of an antibody combination for detecting interferon-gamma, the expression system being a mammalian expression vector.

[0139] The antigen expression system for interferon-gamma (IFN-γ) is the Expi 293 expression system.

[0140] The antibody expression system is the CHO expression system.

[0141] Host cells containing the above expression system.

[0142] Application of antibody combinations for detecting interferon-gamma in the preparation of biopharmaceuticals for in vitro quantitative detection of interferon-gamma levels.

[0143] In vitro quantitative detection refers to the detection of the content of gamma interferon in serum, plasma, whole blood and / or peripheral blood.

[0144] The biological product is a reagent kit.

[0145] The kit is a double-antibody sandwich assay kit.

[0146] Example 2

[0147] 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 interferon-gamma (IFN-γ) 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.

[0148] SEQ ID No.17 sequence:

[0149] QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ.

[0150] The nucleotide sequence of the interferon-gamma (IFN-γ) antigen is as shown in SEQ ID No. 18 or is a nucleotide sequence with greater than 90% homology to the sequence in SEQ ID No. 18.

[0151] SEQ ID No.18 sequence:

[0152] caggatccgtatgtgaaagaagcggaaaacctgaaaaaatattttaacgcgggccatagcgatgtggcggataacggcaccctgtttctgggcattctgaaaaactg gaaagaagaaagcgatcgcaaaattatgcagagccagattgtgagcttttattttaaactgtttaaaaactttaaagatgatcagagcattcagaaaagcgtggaaac cattaaagaagatatgaacgtgaaattttttaacagcaacaaaaaaaacgcgatgattttgaaaaactgaccaactatagcgtgaccgatctgaacgtgcagcgca aagcgattcatgaactgattcaggtgatggcggaactgagcccggcggcgaaaaccggcaaacgcaaacgcagccagatgctgtttcgcggccgccgcgcgagccag.

[0153] Example 3

[0154] The specific composition of the kit containing two antibodies and the specific concentration of each component are shown in Table 1.

[0155] Table 1

[0156]

[0157]

[0158] The reagents are shown below:

[0159] (1) ELISA coating buffer (pH 9.6 0.05M carbonate buffer):

[0160] 1.59 grams of NaHCO3

[0161] NaHCO3 2.93 g,

[0162] Add distilled water to a final volume of 1000 ml;

[0163] (2) ELISA washing buffer (pH 7.4 PBS): 0.15M

[0164] 0.2 g of KH2PO4

[0165] 2.9 grams of Na2HPO4·12H2O

[0166] 8.0 grams of NaCl

[0167] 0.2 grams of KCl

[0168] Tween-20 0.05% 0.5ml

[0169] Add distilled water to a final volume of 1000 ml;

[0170] (3) ELISA sample dilution solution:

[0171] Bovine serum albumin (BSA) 0.1 g,

[0172] Add washing buffer to 100 ml;

[0173] (4) ELISA test stop solution (2M H2SO4):

[0174] Add 178.3 ml of distilled water dropwise to 21.7 ml of concentrated sulfuric acid (98%).

[0175] (5) ELISA substrate buffer (pH 5.0, disodium hydrogen phosphate, citrate):

[0176] 0.2M Na₂HPO₄ (28.4 g / L) 25.7 ml

[0177] 0.1M citric acid (19.2 g / L) 24.3 ml,

[0178] Add 50ml of distilled water;

[0179] (6) ELISA assay TMB (tetramethylbenzidine) solution:

[0180] TMB (10mg / 5ml anhydrous ethanol) 0.5ml,

[0181] 10 ml of substrate buffer (pH 5.5)

[0182] 0.75% H2O2 32μl;

[0183] (7) ELISA blocking solution:

[0184] 5 grams of bovine serum albumin (BSA)

[0185] Add washing buffer to 100 ml.

[0186] Example 4

[0187] 1. The specific operating procedure for using a kit containing two antibodies for gamma interferon detection is shown below.

[0188] step:

[0189] 1) Antibody pre-coating: Dilute 29G2 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;

[0190] 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.

[0191] 3) Dilution and loading of standards: Set up 16 wells for standards and blank control on the enzyme-labeled plate. Serially dilute hIFN-γ 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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);

[0197] 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.

[0198] 2. Sample requirements:

[0199] 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.

[0200] The criteria for interpreting the results of the antibody-containing kit for the in vitro quantitative detection of interferon-gamma (IFN-γ) in human serum, plasma, whole blood, and peripheral blood are shown in Table 2 below.

[0201] Table 2

[0202] IFN-γ (ng / ml) Clinical application recommendations ＜0.003 Reference values ​​for 95% of sites in seemingly healthy individuals 0.003-0.060 Normal, or may indicate mild inflammation or mild infection. 0.060-0.100 This suggests a common bacterial infection or a systemic inflammatory response. ＞0.100 This could be a sign of sepsis.

[0203] The standard curve of the reagent kit of the present invention is as follows: Figure 1 As shown.

[0204] Example 5

[0205] A commercially available IFN-γ detection kit was used as a comparison product 1, and the samples were tested using the kit provided in Example 4 of this invention.

[0206] Commercially available IFN-γ detection kits are:

[0207] Comparison with Product 1:

[0208] Product Name: Human IFN-γ ELISA Kit;

[0209] Product code: PI511;

[0210] Product specifications: 96T;

[0211] Purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0212] During the testing process, the basic characteristics of the reagent kits were first compared, as shown in Table 3 below.

[0213] Table 3

[0214]

[0215]

[0216] As can be seen from Table 3, the basic characteristics of each kit have differed. Both Example 4 and Comparative Product 1 target human serum interferon-gamma (IFN-γ) as the sample; however, the coating antibody and detection antibody in Example 4 are completely different from those in Comparative Product 1. Under this premise, the detection sensitivity of the kit provided in Example 4 significantly exceeds that of Comparative Product 1, and the detection range is also wider. The kit in Example 4 has actually achieved the highest sensitivity achievable by similar products.

[0217] Furthermore, IFN-γ 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. IFN-γ was detected using the kit described in Example 4 of this patent and a commercially available IFN-γ 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.

[0218] Table 4

[0219]

[0220]

[0221] Based on the judgment criteria in Table 2, it can be determined that if the serum IFN-γ concentration is >3 pg / mL, it indicates that inflammation or other infections may be present. In the comparison of the detection data in Table 4 above, it can be seen that the commercially available kit (comparison product 1) showed cases of missed detection or false detection due to low sensitivity in the detection of H1, P4, P7, and P8, with a missed detection (false detection) rate of 20%, which is considered a high level of missed detection. In contrast, the detection results of the kit of the present invention showed no missed detection / false detection, that is, the missed detection / false detection rate was 0, indicating that the kit of the present invention containing antibody combination has a much higher sensitivity than existing products.

[0222] Example 6

[0223] This invention provides two antibodies for detecting interferon-gamma in human serum samples: a first antibody 29G2 and a second antibody 25B6. These antibodies are combined and detected using a double-antibody sandwich method. However, the two antibodies obtained by phage display screening also have the function of detecting interferon-gamma 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.

[0224] The basic characteristics of the kits composed of single primary antibody 29G2, single secondary antibody 25B6, antibody combination 29G2 (coating) + 25B6 (detection), and antibody combination 25B6 (coating) + 29G2 (detection) are compared in Table 5.

[0225] Table 5

[0226] 29G2 standalone 25B6 alone 25B6+29G2 29G2+25B6 (Example 4) antigen Human IFN-γ Human IFN-γ Human IFN-γ Human IFN-γ Coated antibody — — 25B6 (Second Antibody) 29G2 (primary antibody) Antibody detection 29G2 (primary antibody) 25B6 (Second Antibody) 29G2 (primary antibody) 25B6 (Second Antibody) method ELISA ELISA ELISA ELISA Sensitivity 6.0 pg / mL 6.0 pg / mL 3.5 pg / mL 2.8 pg / mL Detection range 20 pg / mL - 500 pg / mL 20 pg / mL - 500 pg / mL 10 pg / mL - 500 pg / mL 10 pg / mL - 500 pg / mL

[0227] As shown in Table 5, both the first antibody 29G2 and the second antibody 25B6 alone can effectively detect gamma interferon 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 25B6 (coating) + 29G2 (detection), which swaps the first and second antibodies, is even more effective than the use of the first and second antibodies alone. Furthermore, the antibody combination 29G2 + 25B6 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.

[0228] To test the actual usage effect, the applicant also conducted IFN-γ detection on random human blood samples. The detection method was similar to the one that led to the conclusion in Table 4, and the specific results are shown in Table 6.

[0229] Table 6

[0230]

[0231]

[0232] Similarly, based on the judgment criteria in Table 2, it can be determined that if the serum IFN-γ concentration is >3 pg / mL, it indicates possible inflammation or other infection. In the comparison of the detection data in Table 6 above, it can be seen that the antibody combination 29G2+25B6 kit showed no false negatives, i.e., a false negative rate of 0%. The antibody combination 25B6+29G2 kit showed false negatives / false positives in the detection of H3 and P4, i.e., a false negative / false positive rate of 10%. Using the second antibody 25B6 alone showed false negatives / false positives in the detection of H9 and P5, with a false negative / false positive rate of 10%. Using the first antibody 29G2 alone showed false negatives / false positives in the detection of H3, H4, and P8, with a false negative / false positive rate of 15%. 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.

[0233] 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 gamma interferon, 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 26-36, 56-63, and 105-116 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 23-36, 52-58, and 91-100 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, 54-61, and 103-113 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 23-35, 51-59, and 92-99 of SEQ ID No. 4, respectively.

2. The antibody composition for detecting gamma interferon 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 gamma interferon 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 gamma interferon 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 gamma interferon 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 gamma interferon 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 gamma interferon as described in 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 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 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 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 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 gamma interferon 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 for the heavy and light chains of the antibody composition for detecting interferon gamma 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 the γ-interferon 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 interferon-gamma content.

14. The application according to claim 13, characterized in that, The in vitro quantitative detection refers to the detection of the content of gamma interferon 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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