Antibody combinations for detecting interleukin 6 and use thereof

Abstract

This invention provides an antibody combination for detecting interleukin-6 (IL-6), 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 the IL-6 antigen, high detection sensitivity, ease of use, and rapid and stable detection. The concentration of IL-6 in normal human blood is approximately 4.2 pg / mL. Existing conventional low-affinity antibodies are insufficient for effective detection in this situation. However, the high-affinity antibody combination and the IL-6 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 IL-6. Verification shows that the kit's sensitivity can reach 0.9 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 interleukin-6 and their applications. Background Technology

[0002] Interleukin-6 (IL-6) is a sensitive indicator for the early diagnosis of acute infections. It can serve as a differential diagnostic indicator for early inflammation and early sepsis, and is of significant reference value for developing patient treatment plans and improving prognosis. Elevated IL-6 levels are positively correlated with disease severity, with the magnitude of increase reflecting the severity of the condition. In critically ill patients, sustained high levels of IL-6 expression can serve as a sensitive indicator for assessing the severity and prognosis of sepsis and MODS, and can more quickly reflect the effectiveness of antibiotic treatment. For inflammatory and infectious diseases, PCT combined with IL-6 testing can improve the early diagnosis rate of infection, avoid missed diagnoses, and reduce misdiagnoses. It can serve as a first-line test for early warning and rapid diagnosis of sepsis, and effectively guide the rational use of antibiotics. Especially in newborns with rapid onset, rapid disease progression, and subtle clinical symptoms, the use of a combined inflammatory test (IL-6 + PCT) is particularly necessary. IL-6 levels are significantly elevated in autoimmune diseases, tumors, postoperative conditions, burns, and other stress responses, and show a positive correlation with the severity of the corresponding diseases (e.g., serum IL-6 levels are significantly elevated in patients with lupus nephritis, and are positively correlated with disease activity).

[0003] However, since the concentration of IL6 in the blood of normal people is about 4.2 pg / mL, most of the existing biological products targeting IL6 are low-affinity antibodies, which have low accuracy and low sensitivity in detecting IL6.

[0004] Patent application No. 201110322423.1, entitled "Preparation and Application of High Affinity Anti-hIL6R Antibody", discloses a high-affinity human interleukin-6 receptor (hIL6R) mouse monoclonal antibody, and the detection sensitivity of the established hIL6R enzyme-linked immunosorbent assay method reaches 0.031 ng / ml.

[0005] Patent application No. 202110618231.9, entitled "An ELISA kit for detecting canine IL-6 and its application", prepared monoclonal and polyclonal antibodies against canine IL-6. The two antibodies were used as detection antibodies and capture vectors respectively in an ELISA double antibody sandwich kit, which can quantitatively detect IL-6 in samples. The minimum detection value is 1.17 ng / mL, and the detection range is 1.17 ng / mL to 300 ng / mL.

[0006] Patent application No. 202111448761.X, entitled "Monoclonal Antibody for Detecting Mouse Interleukin-6 and Its Preparation Method and Application", discloses a detection antibody obtained by labeling an antibody secreted by hybridoma cell line 20-A4-D5 with acrid ester, and a capture antibody obtained by labeling an antibody secreted by hybridoma cell line 10-B8-C9 with biotin. The detection antibody and the capture antibody are then applied to a chemiluminescent kit for detecting mouse interleukin-6. The sensitivity of this kit is <1.5 pg / mL, and the linear range for detecting mouse interleukin-6 is 6.25-400 pg / mL. Summary of the Invention

[0007] To address the aforementioned limitations, this invention proposes an antibody combination for detecting interleukin-6 and its application, overcoming the deficiencies and defects mentioned in the background art.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The inventive point of this invention is to provide an antibody combination for detecting interleukin-6, 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 26-34, 54-60, and 102-111 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, 52-60, and 95-103 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 26-33, 54-61, and 103-112 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 24-34, 50-57, and 90-98 of SEQ ID NO:4, respectively.

[0010] The primary antibody is 4E5, and the secondary antibody is 8F2.

[0011] The CDR1 of the variable region of the first antibody heavy chain is: GFAGFSKDY;

[0012] The CDR2 of the variable region of the first antibody heavy chain is: NGDAGDS;

[0013] The CDR3 of the variable region of the heavy chain of the first antibody is: DLLYAFWGDS;

[0014] The CDR1 of the variable region of the first antibody light chain is: TLRSGINVPTYRIY;

[0015] The CDR2 of the variable region of the light chain of the first antibody is: YKSDSDKGS;

[0016] The CDR3 of the variable region of the first antibody light chain is: AIWHSSAWV;

[0017] The CDR1 of the variable region of the second antibody heavy chain is: GFERKFNR;

[0018] The CDR2 of the variable region of the heavy chain of the second antibody is: NYRNKQYG;

[0019] The CDR3 of the variable region of the second antibody heavy chain is: ESYGYTDTSY;

[0020] The CDR1 of the variable region of the light chain of the second antibody is: NVTQAIGTSLS;

[0021] The CDR2 of the variable region of the second antibody light chain is: NADATIAR;

[0022] The CDR3 of the variable region of the second antibody light chain is: LQPNSTPYT.

[0023] In a preferred embodiment, the antibody combination for detecting interleukin-6 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.

[0024] The amino acid sequence of the variable region of the first antibody heavy chain, SEQ ID NO:1, is:

[0025] QVQLVETGGDLVRPGGSLRLSCAASGFAGFSKDYAMTWVRQGPGKR LEWVSSI NGDAGDS TYYADSVKGRFTISRDNFKNTVDLHLSSLRPEDTALYY CAK DLLYAFWGDS RGQGTLVTVSS;

[0026] The amino acid sequence of the variable region of the first antibody light chain, SEQ ID NO:2, is:

[0027] QAVLTQPSSLSASPGASVSLTC TLRSGINVPTYRIY WYQQKPGSPPQFL LR YKSDSDKGS GVPSRFSGSRDASANAGILLISGLRSEDEADYYC AIWHSSA WV FGGGTQLEIK;

[0028] The amino acid sequence of the variable region of the second antibody heavy chain, SEQ ID NO:3, is:

[0029] EVQLVESGGGLVQPGGSLRLSCAAS GFERKFNR YFMNWVRQAPGKG LEWVAQM NYRNKQYG TYYAESLEGRFTISRDDSKNSLYLQMNSLKTEDTAV YYCAR ESYGYTDTSY WGQGTLVTVSS;

[0030] The amino acid sequence of the variable region of the second antibody light chain, SEQ ID NO:4, is:

[0031] DIQMTQSPSSLSASVGDRVTITC NVTQAIGTSLS WYQQKPGKAPKLLI Y NADATIAR GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQPNSTPYT FGQG TKLEIK.

[0032] In a preferred embodiment, the antibody combination for detecting interleukin-6 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.

[0033] The amino acid sequence of the constant region of the first antibody heavy chain, SEQ ID NO:5, is:

[0034] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0035] The amino acid sequence of the light chain constant region of the first antibody, SEQ ID NO:6, is:

[0036] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS;

[0037] The amino acid sequence of the constant region of the second antibody heavy chain, SEQ ID NO:7, is:

[0038] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0039] The amino acid sequence of the constant region of the light chain of the second antibody, SEQ ID NO:8, is:

[0040] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS.

[0041] The antibody combination used to detect interleukin-6, wherein the first and second antibodies are independently either coating antibodies or detection antibodies.

[0042] Furthermore, the antibody combination for detecting interleukin-6 described above 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.

[0043] In other words, both the primary and secondary antibodies can be used individually as detection antibodies for interleukin-6. In this case, the conventional monoclonal antibody detection method can be used to detect interleukin-6 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 for interleukin-6 detection; 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 for interleukin-6 detection.

[0044] 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 interleukin-6. 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.

[0045] The polynucleotide sequence encoding the variable region of the first antibody heavy chain, SEQ ID NO:9, is:

[0046] caggtgcagctggtggagaccggcggcgacctggtgaggcccggcggcagcctgaggctgagctgcgccgccagcggcttcgccggcttcagcaaggactacgccatgacctgggtgaggcagggccccggcaagaggctggagtgggtgagcagcatcaacggcgacgccggcgacagcacctactacgccgacagcgtgaagggcaggttcaccatcagcagggacaacttcaagaacaccgtggacctgcacctgagcagcctgaggcccgaggacaccgccctgtactactgcgccaaggacctgctgtacgccttctggggcgacagcaggggccagggcaccctggtgaccgtgagcagc;

[0047] The polynucleotide sequence SEQ ID NO:10 encoding the variable region of the light chain of the first antibody is:

[0048] caggcggtgctgacccagccgagcagcctgagcgcgagcccgggcgcgagcgtgagcctgacctgcaccctgcgcagcggcattaacgtgccgacctatcgcatttattggtatcagcagaaaccgggcagcccgccgcagtttctgctgcgctataaaagcgatagcgataaaggcagcggcgtgccgagccgctttagcggcagccgcgatgcgagcgcgaacgcgggcattctgctgattagcggcctgcgcagcgaagatgaagcggattattattgcgcgatttggcatagcagcgcgtgggtgtttggcggcggcacccagctggaaattaaa;

[0049] The polynucleotide sequence SEQ ID NO:11 encoding the variable region of the heavy chain of the second antibody is:

[0050] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggctttgaacgcaaatttaaccgctattttatgaactgggtgcgccaggcgccgggcaaaggcctggaatgggtggcgcagatgaactatcgcaacaaacagtatggcacctattatgcggaaagcctggaaggccgctttaccattagccgcgatgatagcaaaaacagcctgtatctgcagatgaacagcctgaaaaccgaagataccgcggtgtattattgcgcgcgcgaaagctatggctataccgataccagctattggggccagggcaccctggtgaccgtgagcagc;

[0051] The polynucleotide sequence SEQ ID NO:12 encoding the variable region of the light chain of the second antibody is as follows:

[0052] gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcaa cgtgacccaggcgattggcaccagcctgagctggtatcagcagaaaccgggcaaagcgccgaaactgctgatttataacgcggatgcgaccattgcgcgcggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagattttgcgacctattattgcctgcagccgaacagcaccccgtatacctttggccagggcaccaaactggaaattaaa。

[0053] In a preferred embodiment, the heavy and light chain polynucleotides of the antibody combination for detecting interleukin-6 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.

[0054] The polynucleotide sequence encoding the constant region of the first antibody heavy chain, SEQ ID NO:13, is:

[0055] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0056] The polynucleotide sequence encoding the constant region of the first antibody light chain, SEQ ID NO:14, is:

[0057] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;

[0058] The polynucleotide sequence encoding the constant region of the second antibody heavy chain, SEQ ID NO:15, is:

[0059] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0060] The polynucleotide sequence encoding the constant region of the second antibody light chain, SEQ ID NO:16, is:

[0061] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.

[0062] 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 interleukin-6, wherein the expression system is a mammalian expression vector.

[0063] In a preferred embodiment, the expression system described above, wherein the interleukin-6 antigen expression system is the Expi293 expression system.

[0064] In a preferred embodiment, the above-described expression system, the antibody expression system, is a CHO expression system.

[0065] The fourth inventive point of this invention is to provide a host cell containing the above-described expression system.

[0066] 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 interleukin-6 content.

[0067] In a preferred embodiment, the above-described application, wherein the in vitro quantitative detection refers to the detection of the interleukin-6 content in serum, plasma, whole blood, and / or peripheral blood.

[0068] In a preferred embodiment, the above-described application uses a reagent kit as the biological product.

[0069] In a preferred embodiment, the above-described application refers to a double-antibody sandwich assay kit.

[0070] Compared with the prior art, the present invention has the following advantages:

[0071] This invention provides an antibody combination for detecting interleukin-6 and its application. The antibody combination has a strong affinity for the interleukin-6 antigen, high detection sensitivity, is easy to use, and provides rapid and stable detection. The concentration of IL6 in normal human blood is approximately 4.2 pg / mL. Existing conventional low-affinity antibodies are difficult to effectively detect in this situation. However, the high-affinity antibody combination and the interleukin-6 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 interleukin-6. The sensitivity of the kit has been verified to reach 0.9 pg / mL.

[0072] The double-antibody sandwich method used in this invention to determine the IL-6 level 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 IL-6 in the sample. This structure is more conducive to the detection of IL-6. Furthermore, this invention effectively utilizes an avidin-biotin-enzyme complex to achieve high detection sensitivity.

[0073] The detection kit of the present invention can not only rapidly and accurately determine the level of IL-6 in serum, providing reliable clinical reference value for the early diagnosis and early treatment of inflammation; but also achieves a significant improvement in the sensitivity of IL-6 detection due to the use of a combination of two specific monoclonal antibodies. Attached Figure Description

[0074] Figure 1 This is a standard curve for an ELISA kit containing an antibody combination for detecting interleukin-6, provided in an embodiment of the present invention. Detailed Implementation

[0075] 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.

[0076] 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.

[0077] To further understand the present invention, the present invention will be further described in detail below with reference to the preferred embodiments.

[0078] Example 1

[0079] An antibody combination for detecting interleukin-6, 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 26-34, 54-60, and 102-111 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, 52-60, and 95-103 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 26-33, 54-61, and 103-112 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 24-34, 50-57, and 90-98 of SEQ ID NO:4, respectively.

[0080] The first antibody can be used alone to detect interleukin-6, and the second antibody can also be used alone to detect interleukin-6, but using the first and second antibodies together provides the best accuracy and sensitivity for detecting interleukin-6.

[0081] The primary antibody is 4E5, and the secondary antibody is 8F2.

[0082] The CDR1 of the variable region of the first antibody heavy chain is: GFAGFSKDY;

[0083] The CDR2 of the variable region of the first antibody heavy chain is: NGDAGDS;

[0084] The CDR3 of the variable region of the heavy chain of the first antibody is: DLLYAFWGDS;

[0085] The CDR1 of the variable region of the first antibody light chain is: TLRSGINVPTYRIY;

[0086] The CDR2 of the variable region of the light chain of the first antibody is: YKSDSDKGS;

[0087] The CDR3 of the variable region of the first antibody light chain is: AIWHSSAWV;

[0088] The CDR1 of the variable region of the second antibody heavy chain is: GFERKFNR;

[0089] The CDR2 of the variable region of the heavy chain of the second antibody is: NYRNKQYG;

[0090] The CDR3 of the variable region of the second antibody heavy chain is: ESYGYTDTSY;

[0091] The CDR1 of the variable region of the light chain of the second antibody is: NVTQAIGTSLS;

[0092] The CDR2 of the variable region of the second antibody light chain is: NADATIAR;

[0093] The CDR3 of the variable region of the second antibody light chain is: LQPNSTPYT.

[0094] 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.

[0095] The amino acid sequence of the variable region of the first antibody heavy chain, SEQ ID NO:1, is:

[0096] QVQLVETGGDLVRPGGSLRLSCAASGFAGFSKDYAMTWVRQGPGKR LEWVSSI NGDAGDS TYYADSVKGRFTISRDNFKNTVDLHLSSLRPEDTALYY CAK DLLYAFWGDS RGQGTLVTVSS;

[0097] The amino acid sequence of the variable region of the first antibody light chain, SEQ ID NO:2, is:

[0098] QAVLTQPSSLSASPGASVSLTC TLRSGINVPTYRIY WYQQKPGSPPQFL LR YKSDSDKGS GVPSRFSGSRDASANAGILLISGLRSEDEADYYC AIWHSSA WV FGGGTQLEIK;

[0099] The amino acid sequence of the variable region of the second antibody heavy chain, SEQ ID NO:3, is:

[0100] EVQLVESGGGLVQPGGSLRLSCAAS GFERKFNR YFMNWVRQAPGKG LEWVAQM NYRNKQYG TYYAESLEGRFTISRDDSKNSLYLQMNSLKTEDTAV YYCAR ESYGYTDTSYWGQGTLVTVSS;

[0101] The amino acid sequence of the variable region of the second antibody light chain, SEQ ID NO:4, is:

[0102] DIQMTQSPSSLSASVGDRVTITC NVTQAIGTSLS WYQQKPGKAPKLLI Y NADATIAR GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQPNSTPYT FGQG TKLEIK.

[0103] 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.

[0104] The amino acid sequence of the constant region of the first antibody heavy chain, SEQ ID NO:5, is:

[0105] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0106] The amino acid sequence of the light chain constant region of the first antibody, SEQ ID NO:6, is:

[0107] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS;

[0108] The amino acid sequence of the constant region of the second antibody heavy chain, SEQ ID NO:7, is:

[0109] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0110] The amino acid sequence of the constant region of the light chain of the second antibody, SEQ ID NO:8, is:

[0111] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS.

[0112] The polynucleotides encoding the heavy and light chains of the antibody combination for detecting interleukin-6 described above are as follows: 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; and 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.

[0113] The polynucleotide sequence encoding the variable region of the first antibody heavy chain, SEQ ID NO:9, is:

[0114] caggtgcagctggtggagaccggcggcgacctggtgaggcccggcggcagcctgaggctgagctgcgccgccagcggcttcgccggcttcagcaaggactacgccatgacctgggtgaggcagggccccggcaagaggctggagtgggtgagcagcatcaacggcgacgccggcgacagcacc tactacgccgacagcgtgaagggcaggttcaccatcagcagggacaacttcaagaacaccgtggacctgcacctgagcagcctgaggcccgaggacaccgcctgtactgcgccaaggacctgctgtacgccttctggggcgacagcaggggccagggcaccctggtgaccgtgagcagc;

[0115] The polynucleotide sequence encoding the variable region of the first antibody light chain, SEQ ID NO:10, is:

[0116] caggcggtgctgacccagccgagcagcctgagcgcgagcccgggcgcgagcgtgagcctgacctgcaccctgcgcagcggcattaacgtgccgacctatcgcatttattggtatcagcagaaaccgggcagcccgccgcagtttctgctgcgctataaaagcgatagcga taaaggcagcggcgtgccgagccgctttagcggcagccgcgatgcgagcgcgaacgcgggcattctgctgattagcggcctgcgcagcgaagatgaagcggattattattgcgcgatttggcatagcagcgcgtgggtgtttggcggcggcacccagctggaaattaaa;

[0117] The polynucleotide sequence encoding the variable region of the second antibody heavy chain, SEQ ID NO:11, is:

[0118] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggctttgaacgcaaatt taaccgctattttatgaactgggtgcgccaggcgccgggcaaaggcctggaatgggtggcgcagatgaactatcgcaacaaacagtatggcac ctattatgcggaaagcctggaaggccgctttaccattagccgcgatgatagcaaaaacagcctgtatctgcagatgaacagcctgaaaaccgaagataccgcggtgtattattgcgcgcgcgaaagctatggctataccgataccagctattggggccagggcaccctggtgaccgtgagcagc;

[0119] The polynucleotide sequence encoding the variable region of the second antibody light chain, SEQ ID NO:12, is:

[0120] gatattcagatgacccagcccgagcagcctgagcgcgagcgtgggcgatcgcgtgaccattacctgcaa cgtgacccaggcgattggcaccagcctgagctggtatcagcagaaaccgggcaaagcgccgaaactgctgatttataacgcggatgcgaccattgcgcgcggcgtgccgagccgctttagcggcagc ggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagattttgcgacctattattgcctgcagccgaacagcaccccgtatacctttggccagggcaccaaactggaaattaaa.

[0121] 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.

[0122] The polynucleotide sequence encoding the constant region of the first antibody heavy chain, SEQ ID NO:13, is:

[0123] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0124] The polynucleotide sequence encoding the constant region of the first antibody light chain, SEQ ID NO:14, is:

[0125] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;

[0126] The polynucleotide sequence encoding the constant region of the second antibody heavy chain, SEQ ID NO:15, is:

[0127] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0128] The polynucleotide sequence encoding the constant region of the second antibody light chain, SEQ ID NO:16, is:

[0129] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.

[0130] An expression system for heavy and light chain polynucleotides used to detect an antibody combination of interleukin-6, the expression system being a mammalian expression vector.

[0131] The antigen expression system for interleukin-6 (IL6) is the Expi 293 expression system.

[0132] The antibody expression system is the CHO expression system.

[0133] Host cells containing the above expression system.

[0134] Application of antibody combinations for detecting interleukin-6 in the preparation of biopharmaceuticals for in vitro quantitative detection of interleukin-6 levels.

[0135] In vitro quantitative detection refers to the detection of interleukin-6 levels in serum, plasma, whole blood, and / or peripheral blood.

[0136] The biological product is a reagent kit.

[0137] The kit is a double-antibody sandwich assay kit.

[0138] Example 2

[0139] 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 interleukin-6 (IL6) 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.

[0140] SEQ ID NO:17 sequence:

[0141] VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMC ESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQ NRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQ NQWLQDMTTHLILRSFKEFLQSSLRALRQM.

[0142] The nucleotide sequence of the interleukin-6 (IL6) antigen is shown in SEQ ID NO:18 or is a nucleotide sequence with greater than 90% homology to the sequence in SEQ ID NO:18.

[0143] SEQ ID NO:18 sequence:

[0144] .

[0145] Example 3

[0146] The specific composition of the kit containing two antibodies and the specific concentration of each component are shown in Table 1.

[0147] Table 1

[0148]

[0149] The reagents are shown below:

[0150] (1) ELISA coating buffer (pH 9.6 0.05M carbonate buffer):

[0151] 1.59 grams of NaHCO3

[0152] NaHCO3 2.93 g,

[0153] Add distilled water to a final volume of 1000 ml;

[0154] (2) ELISA washing buffer (pH 7.4 PBS): 0.15M

[0155] 0.2 g of KH2PO4

[0156] 2.9 grams of Na2HPO4·12H2O

[0157] 8.0 grams of NaCl

[0158] 0.2 grams of KCl

[0159] Tween-20 0.05% 0.5ml

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

[0161] (3) ELISA sample dilution solution:

[0162] Bovine serum albumin (BSA) 0.1 g,

[0163] Add washing buffer to 100 ml;

[0164] (4) ELISA test stop solution (2M H2SO4):

[0165] Add 178.3 ml of distilled water dropwise to 21.7 ml of concentrated sulfuric acid (98%).

[0166] (5) ELISA substrate buffer (pH 5.0, disodium hydrogen phosphate, citrate):

[0167] 0.2M Na₂HPO₄ (28.4 g / L) 25.7 ml,

[0168] 0.1M citric acid (19.2 g / L) 24.3 ml,

[0169] Add 50ml of distilled water;

[0170] (6) ELISA assay TMB (tetramethylbenzidine) solution:

[0171] TMB (10mg / 5ml anhydrous ethanol) 0.5ml,

[0172] 10 ml of substrate buffer (pH 5.5)

[0173] 0.75% H2O2 32μl;

[0174] (7) ELISA blocking solution:

[0175] 5 grams of bovine serum albumin (BSA)

[0176] Add washing buffer to 100 ml.

[0177] Example 4

[0178] 1. The specific operating procedure for using a kit containing two antibodies for interleukin-6 detection is shown below.

[0179] step:

[0180] 1) Antibody pre-coating: Dilute 4E5 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;

[0181] 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.

[0182] 3) Dilution and loading of standards: Set up 16 wells for standards and blank control on the enzyme-labeled plate. Serially dilute hIL6 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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);

[0188] 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.

[0189] 2. Sample requirements:

[0190] 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.

[0191] The criteria for interpreting the results of the antibody-containing kit for the in vitro quantitative detection of interleukin-6 (IL-6) in human serum, plasma, whole blood, and peripheral blood are shown in Table 2 below.

[0192] Table 2

[0193] IL-6 (ng / ml) Clinical application recommendations ＜0.007 Reference values ​​for 95% of sites in seemingly healthy individuals 0.007-0.150 Normal, or may indicate mild inflammation or mild infection. 0.150-0.250 This suggests a common bacterial infection or a systemic inflammatory response. ＞0.250 This could be a sign of sepsis.

[0194] The standard curve of the reagent kit of the present invention is as follows: Figure 1 As shown.

[0195] Example 5

[0196] The technical solutions described in the three related patent applications in the background section were used as comparative examples 1-3 (i.e., the three patent documents with application numbers 201110322423.1, 202110618231.9 and 202111448761.X), and a commercially available IL-6 detection kit was used as comparative example 4. The samples were tested using both the kit provided in Example 4 of this invention.

[0197] During the testing process, the basic characteristics of the reagent kits were first compared, as shown in Table 3 below.

[0198] Table 3

[0199]

[0200]

[0201] As can be seen from Table 3, the basic characteristics of each kit are different. Example 4 targets interleukin-6 (IL6) in human serum, while Comparative Examples 2 and 3 target canine and mouse serum, respectively. Although Comparative Example 1 targets human serum samples, its antigen is the recombinant protein receptor for interleukin-6 (hIL6R), and its purpose is to "bind recombinant hIL6R and recombinant IL6". Only Comparative Example 4 targets the same human IL6 antigen. The coating antibody and detection antibody of Example 4 are also completely different from those of Comparative Examples 1-4. Under these conditions, the detection sensitivity of the kit provided by Example 4 is much higher than that of Comparative Example 4, and its detection range is also much higher than that of Comparative Example 4. Although the sensitivity of the kit of Comparative Example 1 is higher than that of Example 4, the detection antigen it targets is different from that of Example 4. Moreover, under normal circumstances, the sensitivity of artificially recombined receptors is necessarily higher than that of ordinary antigens due to the strong purpose of their recombination.

[0202] Furthermore, IL6 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 healthy individuals. IL6 was detected using the kit of this patent and a commercially available IL6 detection kit (Comparative Example 4). 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. Since Comparative Example 1 targets the IL6 antigen receptor (hIL6R) of different human serum samples, and Comparative Examples 2-3 target the IL6 antigen of canine and mouse serum samples, a unified comparison cannot be made among the three. Therefore, only the detection results of Comparative Example 4, which uses the same commercially available kit for IL6 detection, are provided for comparison.

[0203] Table 4

[0204]

[0205]

[0206] Based on the criteria in Table 2, it can be determined that if the serum IL6 concentration is >7 pg / mL, it indicates the possible presence of inflammation or other infections. In the comparison of the detection data in Table 4, it can be seen that the commercially available Dakota kit (Comparative Example 4) showed false negatives due to low sensitivity in the detection of P1, P8, and P9, with a false negative rate of 30%, which is considered high. In contrast, the kit of this invention showed no false negatives, i.e., a false negative rate of 0%, indicating that the kit containing the antibody combination of this invention has a much higher sensitivity than existing products.

[0207] Example 6

[0208] This invention provides two antibodies for detecting interleukin-6 in human serum samples: a first antibody 4E5 and a second antibody 8F2. These antibodies are combined and detected using a double-antibody sandwich method. However, the two antibodies obtained by phage display screening can also detect interleukin-6 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 1-6 (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.

[0209] The basic characteristics of the kits composed of single primary antibody 4E5, single secondary antibody 8F2, antibody combination 4E5 (coating) + 8F2 (detection), and antibody combination 8F2 (coating) + 4E5 (detection) are compared in Table 5.

[0210] Table 5

[0211]

[0212] As shown in Table 5, both the first antibody 4E5 and the second antibody 8F2 can effectively detect interleukin-6 in human serum samples when used alone. The detection sensitivity and detection range of the two antibodies when used alone are also better than those of existing commercially available products (Dakewei-IL6 kit). The antibody combination 8F2 (coating) + 4E5 (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 4E5 + 8F2 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.

[0213] To test the actual usage effect, the applicant also conducted IL6 testing on random human blood samples. The testing method was similar to the method that led to the conclusions in Table 4, and the specific results are shown in Table 6.

[0214] Table 6

[0215]

[0216]

[0217] Similarly, based on the judgment criteria in Table 2, it can be determined that if the serum IL6 concentration is >7 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 commercially available Dakota kit (Comparative Example 4) showed false negatives due to low sensitivity in the detection of P1, P8, and P9, with a false negative rate reaching 30%, which is considered a relatively high level. In contrast, the antibody combination 4E5+8F2 kit and the antibody combination 8F2+4E5 kit showed no false negatives, meaning a false negative rate of 0%. Furthermore, the detection results of the antibody combination 4E5+8F2 were further away from the judgment standard (i.e., 7 pg / mL), indicating that this antibody combination was relatively more effective. Using the first antibody 4E5 alone resulted in a false negative rate of 20% for the detection of P1 and P8; using the second antibody 8F2 alone resulted in a false negative rate of 10% for the detection of P8. These results demonstrate that using the first and second antibodies alone provides slightly lower detection sensitivity than using the antibody combination, but it is still superior to the currently available Daktronics kits.

[0218] 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. A combination of antibodies for detecting interleukin 6, characterized in that, The antibody combination comprises a first antibody and a second antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the first antibody are respectively as shown in SEQ ID NO: 1, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively as shown in SEQ ID NO: 2; and the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the second antibody are respectively as shown in SEQ ID NO: 3, and the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively as shown in SEQ ID NO:

4.

2. The antibody combination for detecting interleukin 6 according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO: 1 or an amino acid sequence having more than 90% sequence homology with SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2 or an amino acid sequence having more than 90% sequence homology with SEQ ID NO: 2; and the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO: 3 or an amino acid sequence having more than 90% sequence homology with SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 4 or an amino acid sequence having more than 90% sequence homology with SEQ ID NO:

4.

3. The antibody combination for detecting interleukin 6 according to claim 2, characterized by, The amino acid sequence of the heavy chain constant region of the first antibody is as shown in SEQ ID NO: 5 or an amino acid sequence having more than 90% sequence homology with SEQ ID NO: 5, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 6 or an amino acid sequence having more than 90% sequence homology with SEQ ID NO: 6; and the amino acid sequence of the heavy chain constant region of the second antibody is as shown in SEQ ID NO: 7 or an amino acid sequence having more than 90% sequence homology with SEQ ID NO: 7, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 8 or an amino acid sequence having more than 90% sequence homology with SEQ ID NO:

8.

4. The antibody combination for detecting interleukin 6 according to any one of claims 1 to 3, characterized by, The first antibody and the second antibody are independently a coating antibody or a detection antibody.

5. The antibody combination for detecting interleukin 6 according to claim 4, characterized by, The antibody combination is selected from any one of the following combination forms: the first antibody as a detection antibody, the second antibody as a detection antibody, the first antibody as a coating antibody + the second antibody as a detection antibody, the second antibody as a coating antibody + the first antibody as a detection antibody.

6. The antibody combination for detecting interleukin 6 according to claim 4, wherein The antibody combination is selected from the following combination form: the first antibody as a coating antibody + the second antibody as a detection antibody.

7. A polynucleotide encoding the antibody combination of any one of claims 1-6. 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 having more than 90% sequence homology with SEQ ID NO: 9, and 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 having more than 90% sequence homology with 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 having more than 90% sequence homology with SEQ ID NO: 11, and 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 having more than 90% sequence homology with SEQ ID NO:

12.

8. The polynucleotide of claim 7, wherein, The polynucleotide sequence encoding the heavy chain constant region of the first antibody is as shown in SEQ ID NO: 13 or a nucleotide sequence having more than 90% sequence homology with SEQ ID NO: 13, and the polynucleotide sequence encoding the light chain constant region of the first antibody is as shown in SEQ ID NO: 14 or a nucleotide sequence having more than 90% sequence homology with 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 a nucleotide sequence having more than 90% sequence homology with SEQ ID NO: 15, and the polynucleotide sequence encoding the light chain constant region of the second antibody is as shown in SEQ ID NO: 16 or a nucleotide sequence having more than 90% sequence homology with SEQ ID NO:

16.

9. An expression system comprising the polynucleotide according to claim 7 or 8, characterized in that, The expression system is a mammalian expression vector.

10. A host cell containing the expression system of claim 9.

11. Use of the antibody combination of any one of claims 1-6 in the preparation of a biological product for in vitro quantitative detection of interleukin 6.

12. Use according to claim 11, characterized in that, The in vitro quantitative detection refers to detection of the content of interleukin 6 in serum, plasma, whole blood and / or peripheral blood; The biological product is a kit.

13. Use according to claim 12, characterized in that, The kit is a double antibody sandwich assay kit.

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