Antibody combinations for detecting transforming growth factor-β1 and their applications

Abstract

This invention provides an antibody combination for detecting transforming growth factor-β1 (TGF-β1), 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 TGF-β1 antigen, high detection sensitivity, ease of use, and rapid and stable detection. The concentration of TGF-β1 in normal human serum is 28.4 ± 9.3 pg / ml. Existing conventional low-affinity antibodies are insufficient for effective detection in this situation. However, the high-affinity antibody combination and the TGF-β1 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 TGF-β1. Verification shows that the kit's sensitivity can reach 4.9 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 transforming growth factor-β1 and their applications. Technical Background

[0002] Transforming growth factor β (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily. Its key function is to regulate inflammatory processes, especially in the gut. TGF-β also plays a crucial role in stem cell differentiation and the regulation and differentiation of T cells.

[0003] TGF-β1 belongs to the TGF-β family and is the most abundant (>90%) and most active multifunctional cell activity regulator. In multicellular organisms, TGF-β1 is a potent growth inhibitor, significantly suppressing the growth and proliferation of lymphocytes, hematopoietic stem cells, epithelial cells, endothelial cells, hepatocytes, keratinocytes, and adipocytes. However, TGF-β1 can also promote fibroblast proliferation, extracellular matrix protein expression, and bone matrix protein synthesis.

[0004] Currently, TGF-β1 is considered a tumor suppressor gene; defects in its expression or activation can eliminate its growth-inhibiting effect, leading to malignant cell proliferation. Clinical data indicate that TGF-β1 is significantly associated with the development of cancers such as prostate cancer, breast cancer, and colorectal cancer. Furthermore, circulating TGF-β and Treg cell subsets are significantly reduced in the peripheral blood of patients with systemic lupus erythematosus (SLE). Increasing TGF-β levels in these patients can increase the proportion of Treg cell subsets and enhance immune tolerance. Therefore, accurate measurement of TGF-β1 levels in samples is of great significance for research on related cancers and other related diseases.

[0005] However, since the concentration of TGF-β1 in normal human serum is 29.92±7.66 ng / ml, most existing biological products targeting TGF-β1 are low-affinity antibodies, which have low accuracy and low sensitivity in detecting TGF-β1. Summary of the Invention

[0006] To address the aforementioned limitations, this invention proposes an antibody combination for detecting transforming growth factor-β1 and its application, overcoming the deficiencies and defects mentioned in the background art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The present invention provides an antibody combination for detecting transforming growth factor-β1 (TGF-β1), 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, 54-62, and 104-115 of SEQ ID No. 1, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 24-35, 51-59, and 92-102 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, 53-61, and 103-114 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 25-32, 52-61, and 103-116 of SEQ ID No. 4, respectively.

[0009] The primary antibody is 4G3, and the secondary antibody is 12H6.

[0010] The CDR1 of the variable region of the first antibody heavy chain is: AAFGTIFSN ;

[0011] The CDR2 of the variable region of the first antibody heavy chain is: KSRHSKKTA ;

[0012] The CDR3 of the variable region of the first antibody heavy chain is: NYTDPVAYGYQA ;

[0013] The CDR1 of the variable region of the first antibody light chain is: VKPQFNITSSVG ;

[0014] The CDR2 of the variable region of the first antibody light chain is: YWMSTMKSH ;

[0015] The CDR3 of the variable region of the first antibody light chain is: QPPSFYTVILT ;

[0016] The CDR1 of the variable region of the second antibody heavy chain is: GQWLNRRY ;

[0017] The CDR2 of the variable region of the second antibody heavy chain is: NCQRETPLI ;

[0018] The CDR3 of the variable region of the second antibody heavy chain is: SAEINRPDPSFN ;

[0019] The CDR1 of the variable region of the second antibody light chain is: GTPSDKYP ;

[0020] The CDR2 of the variable region of the second antibody light chain is: SWNQLVVPNY ;

[0021] The CDR3 of the variable region of the second antibody light chain is: DDLPLSDVYFGIIY .

[0022] In a preferred embodiment, the antibody combination for detecting transforming growth factor-β1 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.

[0023] The amino acid sequence of the variable region of the first antibody heavy chain, SEQ ID No. 1, is as follows:

[0024] EVKLEESGGGLVQPGGSMKLSCVAS AAFGTIFSN WMNWVRQSPEKGLEWVAEI KSRHSKKTA THYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSR NYTDPVAYGYQA WGQGTTLTVSS;

[0025] The amino acid sequence of the variable region of the first antibody light chain, SEQ ID No. 2, is as follows:

[0026] DILLTQSPAILSVSPGERVSFSC VKPQFNITSSVG WYQQRTNGSPRLLIK YWMSTMKSH GIPSRFSGSGSGTDFTLSINTVESEDIADYYC QPPSFYTVILT FGSGTNLEVKRTVA;

[0027] The amino acid sequence of the variable region of the second antibody heavy chain, SEQ ID No. 3, is as follows:

[0028] QVQLVESGGGLVQPGGSLRLSCAAS GQWLNRRY WMYWVRQAPGKGLEWVSEI NCQRETPLI TKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCAR SAEINRPDPSFN RGQGTQVTVSS;

[0029] The amino acid sequence of the variable region of the second antibody light chain, SEQ ID No. 4, is as follows:

[0030] VQLVESGGGLVQAGGSLSLSCSAS GTPSDKYP YMGWFRQAPGKERELLGNI SWNQLVVPNY IYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAA DDLPLSDVYFGIIY WGQGTQVTVS.

[0031] In a preferred embodiment, the antibody combination for detecting transforming growth factor-β1 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.

[0032] The amino acid sequence of the first antibody heavy chain constant region, SEQ ID No. 5, is as follows:

[0033] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0034] The amino acid sequence of the constant region of the light chain of the first antibody, SEQ ID No. 6, is as follows:

[0035] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;

[0036] The amino acid sequence of the constant region of the second antibody heavy chain, SEQ ID No. 7, is as follows:

[0037] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0038] The amino acid sequence of the constant region of the light chain of the second antibody, SEQ ID No. 8, is as follows:

[0039] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0040] An antibody combination for detecting transforming growth factor-β1, wherein the first and second antibodies are independently either coating antibodies or detection antibodies.

[0041] Furthermore, in the antibody combination described above for detecting transforming growth factor-β1, 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.

[0042] In other words, both the primary and secondary antibodies can be used individually as detection antibodies for transforming growth factor-β1. In this case, conventional monoclonal antibody detection methods can be used to detect transforming growth factor-β1 with either the primary or secondary antibody. Alternatively, the primary antibody can be used as the coating antibody and the secondary antibody as the detection antibody, using a double antibody sandwich method to detect transforming growth factor-β1; or the secondary antibody can be used as the coating antibody and the primary antibody as the detection antibody, using a double antibody sandwich method to detect transforming growth factor-β1.

[0043] 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 transforming growth factor-β1. The polynucleotide sequence encoding the variable region of the heavy chain 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 variable region of the light chain 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 variable region of the heavy chain 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; 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 a nucleotide sequence with greater than 90% homology to the sequence of SEQ ID No. 12.

[0044] The polynucleotide sequence encoding the variable region of the first antibody heavy chain, SEQ ID No. 9, is:

[0045] gaagtgaaactggaagaaagcggcggcggcctggtgcagccgggcggcagcatgaaactgagctgcgtggcgagcgcggcgtttggcaccattt ttagcaactggatgaactgggtgcgccagagcccggaaaaaggcctggaatgggtggcggaaattaaaagccgccatagcaaaaaaaccgcgacc cattatgcggaaagcgtgaaaggccgctttaccattagccgcgatgatagcaaaagcgcggtgtatctgcagatgaccgatctgcgcaccgaagataccggcgtgtattattgcagccgcaactataccgatccggtggcgtatggctatcaggcgtggggccagggcaccaccctgaccgtgagcagc;

[0046] The polynucleotide sequence encoding the variable region of the first antibody light chain, SEQ ID No. 10, is as follows:

[0047] gatattctgctgacccagagcccggcgattctgagcgtgagcccgggcgaacgcgtgagctttagctgcgtgaaaccgcagtttaacattaccagcagcgtgggctggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatattggatgagcaccatgaaaagc catggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacaccgtggaaagcgaagatattgcggattattattgccagccgccgagcttttataccgtgattctgacctttggcagcggcaccaacctggaagtgaaacgcaccgtggcg;

[0048] The polynucleotide sequence encoding the variable region of the second antibody heavy chain, SEQ ID No. 11, is as follows:

[0049] caggtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggccagtggctgaaccgcc gctattggatgtattgggtgcgccaggcgccgggcaaaggcctggaatgggtgagcgaaattaactgccagcgcgaaaccccgctgattaccaa atatccggatagcgtgaaaggccgctttaccattagccgcgataacgcgaaaaacaccctgtatctgcagatgaacagcctgaaaccggaagat accgcgctgtattattgcgcgcgcagcgcggaaattaaccgcccggatccgagctttaaccgcggccagggcacccaggtgaccgtgagcagc;

[0050] The polynucleotide sequence encoding the variable region of the second antibody light chain, SEQ ID No. 12, is as follows:

[0051] gtgcagctggtggaaagcggcggcggcctggtgcaggcgggcggcagcctgagcctgagctgcagcgcgagcggcaccccgagcgataaatatc cgtatatgggctggtttcgccaggcgccgggcaaagaacgcgaactgctgggcaacattagctggaaccagctggtggtgccgaactatatttat tataaagatagcgtgaaaggccgctttaccattagccgcgatgatgcgaaaaacaccatttatctgcagatgaaccgcctgaaaccggaagatac cgcggtgtattattgcgcggcggatgatctgccgctgagcgatgtgtattttggcattatttattggggccagggcacccaggtgaccgtgagc.

[0052] In a preferred embodiment, the heavy and light chain polynucleotides of the antibody combination for detecting transforming growth factor-β1 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.

[0053] The polynucleotide sequence encoding the constant region of the first antibody heavy chain, SEQ ID No. 13, is as follows:

[0054] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0055] The polynucleotide sequence encoding the constant region of the first antibody light chain, SEQ ID No. 14, is as follows:

[0056] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;

[0057] The polynucleotide sequence encoding the constant region of the second antibody heavy chain, SEQ ID No. 15, is as follows:

[0058] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0059] The polynucleotide sequence encoding the constant region of the light chain of the second antibody, SEQ ID No. 16, is as follows:

[0060] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.

[0061] The third inventive aspect of this invention is to provide an expression system for the heavy and light chain polynucleotides of the antibody combination described above for detecting transforming growth factor-β1, wherein the expression system is a mammalian expression vector.

[0062] In a preferred embodiment, the expression system described above, specifically the transforming growth factor-β1 antigen expression system, is the Expi 293 expression system.

[0063] In a preferred embodiment, the expression system described above, the antibody expression system is a CHO expression system.

[0064] The fourth inventive point of this invention is to provide a host cell containing the above-described expression system.

[0065] 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 in vitro quantitative detection of transforming growth factor-β1 content.

[0066] In a preferred embodiment, the above-described application, wherein the in vitro quantitative detection refers to the detection of transforming growth factor-β1 content in serum, plasma, whole blood, and / or peripheral blood.

[0067] In a preferred embodiment, the above-described application uses a reagent kit as the biological product.

[0068] In a preferred embodiment, the above-described application refers to a double-antibody sandwich assay kit.

[0069] Compared with the prior art, the present invention has the following advantages:

[0070] This invention provides an antibody combination for detecting transforming growth factor-β1 and its application. The antibody combination has strong affinity for the transforming growth factor-β1 antigen, high detection sensitivity, is easy to use, and provides rapid and stable detection. The concentration of transforming growth factor-β1 in normal human serum is 28.4±9.3 pg / ml. Existing conventional low-affinity antibodies are difficult to effectively detect this concentration. However, the high-affinity antibody combination and the transforming growth factor-β1 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 transforming growth factor-β1. The sensitivity of the kit has been verified to reach 4.9 pg / mL.

[0071] The double-antibody sandwich method used in this invention to determine the TGF-β1 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 TGF-β1 in the sample. This structure is more conducive to the detection of TGF-β1. Furthermore, this invention effectively utilizes an avidin-biotin-enzyme complex to achieve high detection sensitivity.

[0072] The detection kit of the present invention can not only rapidly and accurately determine the content of TGF-β1 in serum, providing reliable clinical reference value for the early diagnosis and early treatment of cancer (especially lung cancer); but also achieves a significant improvement in the detection sensitivity of TGF-β1 due to the use of a combination of two specific monoclonal antibodies. Attached Figure Description

[0073] Figure 1 This is a standard curve for an ELISA kit containing an antibody combination for detecting transforming growth factor-β1, provided in an embodiment of the present invention. Detailed Implementation

[0074] 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.

[0075] 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.

[0076] To further understand the present invention, the present invention will be described in detail below with reference to the preferred embodiments.

[0077] Example 1

[0078] An antibody combination for detecting transforming growth factor-β1 (TGF-β1) 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 26-34, 54-62, and 104-115 of SEQ ID No. 1, respectively, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 24-35, 51-59, and 92-102 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 26-33, 53-61, and 103-114 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 25-32, 52-61, and 103-116 of SEQ ID No. 4, respectively.

[0079] The primary antibody is 4G3, and the secondary antibody is 12H6.

[0080] The CDR1 of the variable region of the first antibody heavy chain is: AAFGTIFSN ;

[0081] The CDR2 of the variable region of the first antibody heavy chain is: KSRHSKKTA ;

[0082] The CDR3 of the variable region of the first antibody heavy chain is: NYTDPVAYGYQA ;

[0083] The CDR1 of the variable region of the first antibody light chain is: VKPQFNITSSVG ;

[0084] The CDR2 of the variable region of the first antibody light chain is: YWMSTMKSH ;

[0085] The CDR3 of the variable region of the first antibody light chain is: QPPSFYTVILT ;

[0086] The CDR1 of the variable region of the second antibody heavy chain is: GQWLNRRY ;

[0087] The CDR2 of the variable region of the second antibody heavy chain is: NCQRETPLI ;

[0088] The CDR3 of the variable region of the second antibody heavy chain is: SAEINRPDPSFN ;

[0089] The CDR1 of the variable region of the second antibody light chain is: GTPSDKYP ;

[0090] The CDR2 of the variable region of the second antibody light chain is: SWNQLVVPNY ;

[0091] The CDR3 of the variable region of the second antibody light chain is: DDLPLSDVYFGIIY .

[0092] 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.

[0093] The amino acid sequence of the variable region of the first antibody heavy chain, SEQ ID No. 1, is as follows:

[0094] EVKLEESGGGLVQPGGSMKLSCVAS AAFGTIFSN WMNWVRQSPEKGLEWVAEI KSRHSKKTA THYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSR NYTDPVAYGYQA WGQGTTLTVSS;

[0095] The amino acid sequence of the variable region of the first antibody light chain, SEQ ID No. 2, is as follows:

[0096] DILLTQSPAILSVSPGERVSFSC VKPQFNITSSVG WYQQRTNGSPRLLIK YWMSTMKSH GIPSRFSGSGSGTDFTLSINTVESEDIADYYC QPPSFYTVILT FGSGTNLEVKRTVA;

[0097] The amino acid sequence of the variable region of the second antibody heavy chain, SEQ ID No. 3, is as follows:

[0098] QVQLVESGGGLVQPGGSLRLSCAAS GQWLNRRY WMYWVRQAPGKGLEWVSEI NCQRETPLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCAR SAEINRPDPSFN RGQGTQVTVSS;

[0099] The amino acid sequence of the variable region of the second antibody light chain, SEQ ID No. 4, is as follows:

[0100] VQLVESGGGLVQAGGSLSLSCSAS GTPSDKYP YMGWFRQAPGKERELLGNI SWNQLVVPNY IYYKDSVKGRFTISRDDAKNTIYLQMNRLKPEDTAVYYCAA DDLPLSDVYFGIIY WGQGTQVTVS.

[0101] 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.

[0102] The amino acid sequence of the first antibody heavy chain constant region, SEQ ID No. 5, is as follows:

[0103] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0104] The amino acid sequence of the constant region of the light chain of the first antibody, SEQ ID No. 6, is as follows:

[0105] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS;

[0106] The amino acid sequence of the constant region of the second antibody heavy chain, SEQ ID No. 7, is as follows:

[0107] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0108] The amino acid sequence of the constant region of the light chain of the second antibody, SEQ ID No. 8, is as follows:

[0109] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0110] The primary and secondary antibodies are either coating antibodies or detection antibodies, which are independent of each other.

[0111] 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.

[0112] The preferred method is to use the first antibody 4G3 as the coating antibody and the second antibody 12H6 as the detection antibody.

[0113] In other words, both the primary and secondary antibodies can be used individually as detection antibodies for transforming growth factor-β1. In this case, conventional monoclonal antibody detection methods can be used to detect transforming growth factor-β1 with either the primary or secondary antibody. Alternatively, the primary antibody can be used as the coating antibody and the secondary antibody as the detection antibody, using a double antibody sandwich method to detect transforming growth factor-β1; or the secondary antibody can be used as the coating antibody and the primary antibody as the detection antibody, using a double antibody sandwich method to detect transforming growth factor-β1.

[0114] The present invention also provides polynucleotides encoding the heavy and light chains of the antibody combination for detecting transforming growth factor-β1 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.

[0115] The polynucleotide sequence encoding the variable region of the first antibody heavy chain, SEQ ID No. 9, is:

[0116] gaagtgaaactggaagaaagcggcggcggcctggtgcagccgggcggcagcatgaaactgagctgcgtggcgagcgcggcgtttggcaccattt ttagcaactggatgaactgggtgcgccagagcccggaaaaaggcctggaatgggtggcggaaattaaaagccgccatagcaaaaaaaccgcgacc cattatgcggaaagcgtgaaaggccgctttaccattagccgcgatgatagcaaaagcgcggtgtatctgcagatgaccgatctgcgcaccgaagataccggcgtgtattattgcagccgcaactataccgatccggtggcgtatggctatcaggcgtggggccagggcaccaccctgaccgtgagcagc;

[0117] The polynucleotide sequence encoding the variable region of the first antibody light chain, SEQ ID No. 10, is as follows:

[0118] gatattctgctgacccagagcccggcgattctgagcgtgagcccgggcgaacgcgtgagctttagctgcgtgaaaccgcagtttaacattaccagcagcgtgggctggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatattggatgagcaccatgaaaagc catggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacaccgtggaaagcgaagatattgcggattattattgccagccgccgagcttttataccgtgattctgacctttggcagcggcaccaacctggaagtgaaacgcaccgtggcg;

[0119] The polynucleotide sequence encoding the variable region of the second antibody heavy chain, SEQ ID No. 11, is as follows:

[0120] caggtgcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggccagtggctgaaccgcc gctattggatgtattgggtgcgccaggcgccgggcaaaggcctggaatgggtgagcgaaattaactgccagcgcgaaaccccgctgattaccaa atatccggatagcgtgaaaggccgctttaccattagccgcgataacgcgaaaaacaccctgtatctgcagatgaacagcctgaaaccggaagat accgcgctgtattattgcgcgcgcagcgcggaaattaaccgcccggatccgagctttaaccgcggccagggcacccaggtgaccgtgagcagc;

[0121] The polynucleotide sequence encoding the variable region of the second antibody light chain, SEQ ID No. 12, is as follows:

[0122] gtgcagctggtggaaagcggcggcggcctggtgcaggcgggcggcagcctgagcctgagctgcagcgcgagcggcaccccgagcgataaatatc cgtatatgggctggtttcgccaggcgccgggcaaagaacgcgaactgctgggcaacattagctggaaccagctggtggtgccgaactatatttat tataaagatagcgtgaaaggccgctttaccattagccgcgatgatgcgaaaaacaccatttatctgcagatgaaccgcctgaaaccggaagatac cgcggtgtattattgcgcggcggatgatctgccgctgagcgatgtgtattttggcattatttattggggccagggcacccaggtgaccgtgagc.

[0123] The heavy and light chain polynucleotides of the antibody combination for detecting transforming growth factor-β1 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.

[0124] The polynucleotide sequence encoding the constant region of the first antibody heavy chain, SEQ ID No. 13, is as follows:

[0125] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0126] The polynucleotide sequence encoding the constant region of the first antibody light chain, SEQ ID No. 14, is as follows:

[0127] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc;

[0128] The polynucleotide sequence encoding the constant region of the second antibody heavy chain, SEQ ID No. 15, is as follows:

[0129] gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagcctgagcccgggcaaa;

[0130] The polynucleotide sequence encoding the constant region of the light chain of the second antibody, SEQ ID No. 16, is as follows:

[0131] ggccagccgaaagcggcgccgagcgtgaccctgtttccgccgagcagcgaagaactgcaggcgaacaaagcgaccctggtgtgcctgattagcgatttttatccgggcgcggtgaccgtggcgtggaaagcggatagcagcccggtgaaagcgggcgtg gaaaccaccaccccgagcaaacagagcaacaacaaatatgcggcgagcagctatctgagcctgaccccggaacagtggaaaagccatcgcagctatagctgccaggtgacccatgaaggcagcaccgtggaaaaaaccgtggcgccgaccgaatgcagc.

[0132] An expression system for heavy and light chain polynucleotides of an antibody combination for detecting transforming growth factor-β1, the expression system being a mammalian expression vector.

[0133] The antigen expression system for transforming growth factor-β1 (TGF-β1) is the Expi 293 expression system.

[0134] The antibody expression system is the CHO expression system.

[0135] Host cells containing the above expression system.

[0136] Application of antibody combinations for detecting transforming growth factor-β1 in the preparation of biopharmaceuticals for in vitro quantitative detection of transforming growth factor-β1 content.

[0137] In vitro quantitative detection refers to the detection of transforming growth factor-β1 content in serum, plasma, whole blood and / or peripheral blood.

[0138] The biological product is a reagent kit.

[0139] The kit is a double-antibody sandwich assay kit.

[0140] Example 2

[0141] 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 transforming growth factor-β1 (TGF-β1) 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.

[0142] SEQ ID No.17 sequence:

[0143] ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

[0144] The nucleotide sequence of the transforming growth factor-β1 (TGF-β1) 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.

[0145] SEQ ID No.18 sequence:

[0146] gcgctggataccaactattgctttagcagcaccgaaaaaaactgctgcgtgcgccagctgtatattgattttcgcaaagatctgggctggaaatggattcatgaaccgaaaggctatcatgcgaacttttgcctgggcccgtgcccgtatatttggagcctggatacc cagtatagcaaagtgctggcgctgtataaccagcataacccgggcgcgagcgcggcgccgtgctgcgtgccgcaggcgctggaaccgctgccgattgtgtattatgtgggccgcaaaccgaaagtggaacagctgagcaacatgattgtgcgcagctgcaaatgcagc.

[0147] Example 3

[0148] The specific composition of the kit containing two antibodies and the specific concentration of each component are shown in Table 1.

[0149] Table 1

[0150]

[0151] The reagents are shown below:

[0152] (1) ELISA coating buffer (pH 9.6 0.05M carbonate buffer):

[0153] 1.59 grams of NaHCO3

[0154] NaHCO3 2.93 g,

[0155] Add distilled water to a final volume of 1000 ml;

[0156] (2) ELISA washing buffer (pH 7.4 PBS): 0.15M

[0157] 0.2 g of KH2PO4

[0158] 2.9 grams of Na2HPO4·12H2O

[0159] 8.0 grams of NaCl

[0160] 0.2 grams of KCl

[0161] Tween-20 0.05% 0.5ml

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

[0163] (3) ELISA sample dilution solution:

[0164] Bovine serum albumin (BSA) 0.1 g,

[0165] Add washing buffer to 100 ml;

[0166] (4) ELISA test stop solution (2M H2SO4):

[0167] Add 178.3 ml of distilled water dropwise to 21.7 ml of concentrated sulfuric acid (98%).

[0168] (5) ELISA substrate buffer (pH 5.0, disodium hydrogen phosphate, citrate):

[0169] 0.2M Na₂HPO₄ (28.4 g / L) 25.7 ml,

[0170] 0.1M citric acid (19.2 g / L) 24.3 ml

[0171] Add 50ml of distilled water;

[0172] (6) ELISA assay TMB (tetramethylbenzidine) solution:

[0173] TMB (10mg / 5ml anhydrous ethanol) 0.5ml,

[0174] 10 ml of substrate buffer (pH 5.5)

[0175] 0.75% H2O2 32μl;

[0176] (7) ELISA blocking solution:

[0177] 5 grams of bovine serum albumin (BSA)

[0178] Add washing buffer to 100 ml.

[0179] Example 4

[0180] 1. The specific operating procedure for the kit containing two antibodies for the detection of transforming growth factor-β1 is shown below.

[0181] step:

[0182] 1) Antibody pre-coating: Dilute 4G3 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;

[0183] 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.

[0184] 3) Dilution and loading of standards: Set up 16 wells for standards and blank control on the enzyme-labeled plate. Serially dilute hTGF-β1 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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);

[0190] 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.

[0191] 2. Sample requirements:

[0192] 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.

[0193] Normally, the TGF-β1 level in lung cancer patients is 37.2±9.4 pg / ml, while the TGF-β1 level in normal samples is 28.4±9.3 pg / ml.

[0194] The criteria for interpreting the results of the antibody-containing kit for the in vitro quantitative detection of transforming growth factor-β1 (TGF-β1) in human serum, plasma, whole blood, and peripheral blood are shown in Table 2 below.

[0195] Table 2

[0196] TGF-β1 (pg / ml) Clinical application recommendations ＜19 Reference values ​​for 95% of sites in seemingly healthy individuals 19-27 Normal, or belongs to the intermediate risk group for cancer. 27-37 This indicates that they belong to a high-risk group for cancer. ＞37 This could be a sign of lung cancer.

[0197] The standard curve of the reagent kit of the present invention is as follows: Figure 1 As shown.

[0198] Example 5

[0199] A commercially available TGF-β1 detection kit was used as a comparison product 1, and the samples were tested using the kit provided in Example 4 of this invention.

[0200] Commercially available TGF-β1 detection kits are:

[0201] Comparison with Product 1:

[0202] Product Name: Human TGF beta 1 ELISA Kit;

[0203] Product code: ab108912;

[0204] Product specifications: 96T;

[0205] Purchased from Abcam (Shanghai) Trading Co., Ltd.

[0206] During the testing process, the basic characteristics of the reagent kits were first compared, as shown in Table 3 below.

[0207] Table 3

[0208]

[0209]

[0210] As can be seen from Table 3, the basic characteristics of each kit are different. The samples targeted by Example 4 and Comparative Product 1 are both transforming growth factor-β1 (TGF-β1) in human serum; however, the coating antibody and detection antibody of Example 4 are completely different from those of Comparative Product 1. Under this premise, the detection sensitivity of the kit provided by Example 4 is much higher than that of Comparative Product 1. The kit of Example 4 has actually reached the highest sensitivity that can be achieved by similar products.

[0211] Furthermore, TGF-β1 was detected using blood samples. The specific detection method was as follows: 10 serum samples were collected from lung cancer patients and 10 from healthy individuals. TGF-β1 was detected using the kit described in Example 4 of this patent and a commercially available TGF-β1 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 lung cancer patients.

[0212] Table 4

[0213]

[0214]

[0215] Based on the criteria in Table 2, it can be determined that if the serum TGF-β1 concentration is >19 pg / mL, it indicates the possible presence of cancers such as lung cancer. In the comparison of detection data in Table 4, it can be seen that the commercially available kit (comparison product 1) showed false negatives / false positives due to low sensitivity in the detection of H3, H9, P2, P4, P7, and P10, with a false negative / false positive rate of 30%, which is considered a high level. In contrast, the kit of this invention showed false negatives / false positives in the detection of H5 and P7, with a false negative / false positive rate of 10%. This demonstrates that the kit containing the antibody combination of this invention has a much higher sensitivity than existing products.

[0216] Example 6

[0217] This invention provides two antibodies for detecting transforming growth factor-β1 in human serum samples: a first antibody 4G3 and a second antibody 12H6. 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 transforming growth factor-β1 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.

[0218] The basic characteristics of the kits consisting of a single primary antibody 4G3, a single secondary antibody 12H6, an antibody combination of 4G3 (coating) + 12H6 (detection), and an antibody combination of 12H6 (coating) + 4G3 (detection) are compared in Table 5.

[0219] Table 5

[0220]

[0221]

[0222] As shown in Table 5, both the first antibody 4G3 and the second antibody 12H6 can effectively detect transforming growth factor-β1 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 (compare product 2 in Table 3). The antibody combination 12H6 (coating) + 4G3 (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 4G3 + 12H6 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.

[0223] To test the actual usage effect, the applicant also conducted TGF-β1 testing on random human blood samples. The testing method was similar to the method that led to the conclusion in Table 4, and the specific results are shown in Table 6.

[0224] Table 6

[0225]

[0226]

[0227] Similarly, based on the judgment criteria in Table 2, it can be determined that if the serum TGF-β1 concentration is >19 pg / mL, it indicates the possible presence of cancers such as lung cancer. Comparing the detection data in Table 6 above, it can be seen that the antibody combination 4G3+12H6 kit showed false negatives / false positives in the detection of H5 and P7, with a false negative / false positive rate of 10%. The antibody combination 12H6+4G3 kit showed false negatives / false positives in the detection of H3, P7, and P8, with a false negative / false positive rate of 15%. Using the second antibody 12H6 alone showed false negatives / false positives in the detection of H5, P2, and P4, with a false negative / false positive rate of 15%. Using the first antibody 4G3 alone showed false negatives / false positives in the detection of H3, H7, P2, and P6, with a false negative / false positive rate of 20%. These comparisons indicate that the detection sensitivity of using the first and second antibodies alone is slightly lower than that of using the antibody combination.

[0228] 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 transforming growth factor-β1, characterized in that, The antibody combination is a first antibody and a second antibody, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the first antibody are respectively shown in SEQ ID No. 1, 26-34, 54-62, 104-115, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown in SEQ ID No. 2, 24-35, 51-59, 92-102; the amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the second antibody are respectively shown in SEQ ID No. 3, 26-33, 53-61, 103-114, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown in SEQ ID No. 4, 25-32, 52-61, 103-116.

2. The antibody combination for detecting transforming growth factor-β1 according to claim 1, characterized by, The amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID No. 1, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID No. 3, the amino acid sequence of the light chain variable region is shown in SEQ ID No.

4.

3. The antibody combination for detecting transforming growth factor-β1 according to claim 2, characterized by, The amino acid sequence of the heavy chain constant region of the first antibody is shown in SEQ ID No. 5 or an amino acid sequence with more than 90% homology with SEQ ID No. 5, the amino acid sequence of the light chain constant region is shown in SEQ ID No. 6 or an amino acid sequence with more than 90% homology with SEQ ID No. 6; the amino acid sequence of the heavy chain constant region of the second antibody is shown in SEQ ID No. 7 or an amino acid sequence with more than 90% homology with SEQ ID No. 7, the amino acid sequence of the light chain constant region is shown in SEQ ID No. 8 or an amino acid sequence with more than 90% homology with SEQ ID No.

8.

4. The antibody panel for detecting transforming growth factor-β1 according to any one of claims 1 to 3, characterized in that, The antibody combination is selected from any one of the following combinations: 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.

5. The antibody panel for detecting transforming growth factor-β1 according to any one of claims 1 to 3, characterized in that, The antibody combination is selected as the first antibody as a coating antibody + the second antibody as a detection antibody.

6. A polynucleotide encoding the heavy chain variable region and the light chain variable region of the antibody pair for detecting transforming growth factor-β1 according to any one of claims 1 to 5, characterized in that, The polynucleotide sequence encoding the heavy chain variable region of the first antibody is shown in SEQ ID No. 9, the polynucleotide sequence encoding the light chain variable region of the first antibody is shown in SEQ ID No. 10; the polynucleotide sequence encoding the heavy chain variable region of the second antibody is shown in SEQ ID No. 11, the polynucleotide sequence encoding the light chain variable region of the second antibody is shown in SEQ ID No.

12.

7. Use of the antibody combination of any one of claims 1-5 in the preparation of a biological product for in vitro quantitative detection of the content of transforming growth factor-β1.

8. Use according to claim 7, characterized in that, The in vitro quantitative detection refers to the detection of the content of transforming growth factor-β1 in serum, plasma, whole blood and / or peripheral blood. The in vitro quantitative detection refers to the detection of the content of transforming growth factor-β1 in serum, plasma, whole blood and / or peripheral blood.

9. Use according to claim 8, characterized in that, The biological product is a kit.

10. Use according to claim 9, characterized in that, The kit is a double antibody sandwich method detection kit.

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