Bispecific antibodies for improving the sensitivity of immunoassays and uses thereof

Abstract

The application relates to a bispecific antibody for improving the sensitivity of immune detection and application thereof. The bispecific antibody comprises a first binding region with specific binding capacity for an antigen; and a second binding region comprising a tracer or a tracer-containing protein. The bispecific antibody can not only specifically bind to the antigen, but also specifically bind to the tracer, and the influence generated in the labeling process is avoided and the interference of excessive labels is avoided. The bispecific antibody can amplify the signal by directly binding to the tracer, so that the sensitivity is improved, and the specific binding capacity and stability are also improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a bispecific antibody for improving the sensitivity of immune detection and its application. Background Technology

[0002] Bispecific antibodies (BsAbs) are artificial antibodies that can simultaneously and specifically bind to two antigens or epitopes. Since the concept of bispecific antibodies was first proposed by Nisonoff and his collaborators in 1960, the construction concepts, technological platforms, and product development of bispecific antibodies have continuously innovated with the rapid progress in related fields such as genetic engineering antibodies and immunology. To date, more than 100 bispecific antibody structures have been reported, and over 85 bispecific antibodies are in clinical trials, with approximately 86% of these projects focusing on oncology. Compared to ordinary antibodies, bispecific antibodies have advantages such as high specificity, strong targeting, high yield, good stability, low dosage, and fewer toxic side effects, showing broad application prospects in cancer treatment and autoimmune diseases.

[0003] Bispecific antibodies contain two different antigen-binding domains, which do not exist in nature and can only be prepared artificially. The preparation methods of bispecific antibodies are as follows: (1) Chemical conjugation method: This method was first used in 1985. The principle is to couple different specific antibody molecules or F(ab')2 antibody fragments into a bispecific antibody by using a chemical cross-linking agent. The bispecific antibodies prepared by this method can directly utilize existing antibodies and have a high yield, but their activity may be affected by damage to the antigen-binding site; (2) Double hybridoma fusion method: Two hybridoma cells are fused to synthesize a double hybridoma cell line, and a stable target cell line with the functions of the two antibodies is screened out. The preparation of bispecific antibodies using the hybridoma method has a large degree of randomness and low efficiency, but the bispecific antibodies have better biological activity and more stable antibody structure.

[0004] Traditional labeling methods require antibodies with high purity, high titer, and strong antigen affinity. The use of strong alkaline reagents during the labeling process results in a high pH, ​​which makes the protein easily inactivated and causes significant protein loss. Furthermore, it can damage the conformation or function of the protein to some extent, leading to poor stability and low sensitivity of the labeled protein. Summary of the Invention

[0005] Based on this, this application provides a bispecific antibody with good sensitivity and specificity and high batch-to-batch consistency.

[0006] The technical solution to the above-mentioned technical problems in this application is as follows:

[0007] This application provides a bispecific antibody for improving the sensitivity of immunoassay, the bispecific antibody comprising a first binding region capable of specifically binding to an antigen; and

[0008] The second binding region includes a tracer or a protein containing a tracer.

[0009] The bispecific antibody of this application can not only specifically bind to antigens, but also specifically bind to tracers, eliminating the influence of the labeling process and avoiding interference when there are too many labels. The bispecific antibody can amplify the signal by directly binding to the tracer, thereby improving its sensitivity, as well as its specific binding ability and stability.

[0010] In some embodiments, the second binding region is a tracer, which includes any one of radionuclides, fluorescent substances, enzymes, chemiluminescent agents, bioluminescent agents, micron and nanoparticles, and biotin.

[0011] In one embodiment, the first binding region includes a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region is H-CDR1 with an amino acid sequence as shown in SEQ ID NO:1, and the first light chain variable region is L-CDR1 with an amino acid sequence as shown in SEQ ID NO:2; or

[0012] The first heavy chain variable region is H-CDR2 with an amino acid sequence as shown in SEQ ID NO:13, and the first light chain variable region is L-CDR2 with an amino acid sequence as shown in SEQ ID NO:14.

[0013] In some embodiments, the second binding region includes a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region is H-CDR3 with an amino acid sequence as shown in SEQ ID NO:3, and the second light chain variable region is L-CDR3 with an amino acid sequence as shown in SEQ ID NO:4, or the second heavy chain variable region is H-CDR4 with an amino acid sequence as shown in SEQ ID NO:15, and the second light chain variable region is L-CDR4 with an amino acid sequence as shown in SEQ ID NO:16.

[0014] In one embodiment, the first binding region includes a first heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 (H-CDR1), and a first light chain variable region having an amino acid sequence as shown in SEQ ID NO:2 (L-CDR1); and

[0015] The second binding region includes a second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:3 (H-CDR3) and a second light chain variable region having an amino acid sequence as shown in SEQ ID NO:4 (L-CDR3).

[0016] In one embodiment, the first binding region includes a first heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:13 (H-CDR2), and a first light chain variable region having an amino acid sequence as shown in SEQ ID NO:14 (L-CDR2); and

[0017] The second binding region includes a second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:15 (H-CDR4) and a second light chain variable region having an amino acid sequence as shown in SEQ ID NO:16 (L-CDR4).

[0018] In one embodiment, the bispecific antibody further includes a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region being shown in SEQ ID NO:5; and the amino acid sequence of the light chain constant region being shown in SEQ ID NO:6.

[0019] This application also provides an application of the above-mentioned bispecific antibody in the preparation of immunoassay reagents.

[0020] In some embodiments, the immunoassay reagent is used to perform at least one of the following detection methods: enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunogold assay, immunochemiluminescence assay, immunoturbidimetric assay, immunoblotting, and dot blot assay.

[0021] This application also provides a detection kit comprising the above-mentioned bispecific antibody. Attached Figure Description

[0022] Figure 1 The image shows the purification process of the bispecific antibody in Example 1, where lane 1 is the protein molecular weight standard, lane 2 is the bispecific antibody reducing SDS-PAGE, and lane 3 is the bispecific antibody non-reducing SDS-PAGE.

[0023] Figure 2 The image shows the purification process of the bispecific antibody in Example 2, where lane 1 is the protein molecular weight standard, lane 2 is the bispecific antibody reducing SDS-PAGE, and lane 3 is the bispecific antibody non-reducing SDS-PAGE.

[0024] Figure 3 This is a graph showing the protein chip detection results in Example 1;

[0025] Figure 4 The image shows the protein chip detection results in Comparative Example 1. Detailed Implementation

[0026] To facilitate understanding of this application, a more complete description will be provided below. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be more thorough and complete.

[0027] 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0028] Terminology Explanation:

[0029] Antibodies are a class of immunoglobulins that specifically bind to antigens. Generally, antibodies exist as one or more Y-shaped monomers. Each Y-shaped monomer consists of four polypeptide chains, including two identical heavy chains and two identical light chains, named according to their molecular weight. The apex of the Y-shaped structure is the variable region, the antigen-binding site. Each heavy chain has two regions: a constant region and a variable region. All antibodies of the same type share the same constant region, while different types of antibodies differ. Each light chain also has two connected domains: a constant region and a variable region.

[0030] A "vector" is a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0031] "Host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0032] As shown in the background section, traditional immunoassays often use horseradish peroxidase (HRP) or alkaline phosphatase (ALP) to directly label antibodies. Although the experiment is simple and low-cost, the traditional labeling method has several problems: the antibodies required for labeling are of high quality, requiring high activity, purity, titer, and strong affinity; protein loss is significant during the labeling process, there are large differences between labeling batches, and high uncertainty and randomness; and the stability and sensitivity of the secondary antibody after labeling are poor.

[0033] Based on this, one embodiment of this application provides a bispecific antibody, wherein the bispecific antibody includes a first binding region having specific binding ability to an antigen; and

[0034] The second binding region includes a tracer or a tracer-containing protein.

[0035] The bispecific antibody of this application can not only specifically bind to antigens, but also specifically bind to tracers, eliminating the influence of the labeling process and avoiding interference when there are too many labels. The bispecific antibody can amplify the signal by directly binding to the tracer, thereby improving its sensitivity, as well as its specific binding ability and stability.

[0036] It is understandable that the tracer-containing proteins in the above-mentioned bispecific antibodies mainly amplify the signal through the tracer, thereby improving their sensitivity, as well as their specific binding ability and stability.

[0037] In some embodiments, the second binding region is a tracer, which includes any one of radionuclides, fluorescent substances, enzymes, chemiluminescent agents, bioluminescent agents, micron and nanoparticles, and biotin.

[0038] In some embodiments, the aforementioned radionuclide includes carbon-14 (… 14 C), Phosphorus 32 ( 32 P), sulfur 35 ( 35 S), Iodine-125 ( 125 I), hydrogen 3( 3 At least one of the radioactive isotopes hydrogen (H).

[0039] In some embodiments, the fluorescent material includes at least one of fluorescein, tetraethylrhodamine, tetramethylrhodamine isothiocyanate, and phycoerythrin.

[0040] In some embodiments, the enzymes described above include at least one of horseradish peroxidase, alkaline phosphatase, and β-galactosidase.

[0041] In some embodiments, the chemiluminescent agent includes at least one of acridine ester, ruthenium terpyridine, and luminol and its derivatives.

[0042] In some embodiments, the bioluminescent agent includes at least one of amygdalin and biofluorescent protein.

[0043] In some embodiments, the aforementioned micron and nanoparticles include at least one of colloidal gold, colloidal selenium, gold nanoparticles, and quantum dots.

[0044] In one specific example, the tracer mentioned above includes at least one of polyruthenium and polymeric horseradish peroxidase.

[0045] In some embodiments, the first heavy chain variable region is H-CDR1 with an amino acid sequence as shown in SEQ ID NO:1, and the first light chain variable region is L-CDR1 with an amino acid sequence as shown in SEQ ID NO:2, or the first heavy chain variable region is H-CDR2 with an amino acid sequence as shown in SEQ ID NO:13, and the first light chain variable region is L-CDR2 with an amino acid sequence as shown in SEQ ID NO:14.

[0046] Specifically, the amino acid sequence shown in SEQ ID NO:1 is: QVQLQQSGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIGNKGNNHATYYAESVK GRFTVSRDDSKSRVYLQMNSLRVEDTGTYYCTTRFAYWGQGTLVTVSA; the amino acid sequence shown in SEQ ID NO:2 is: DIVMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDY SLTISSLEYEDMGIYYCLQYDEFPRTFGGGTKLEIK; the amino acid sequence shown in SEQ ID NO:13 is: QVQLQESGPGLVKPSQSLSLTCSVTDYSLTSGYYWNWIRQFPGNKIEWMGYIRFDGNNRYNPSIKNRISIT REKSKNQFFLRISSVTPEDTATYYCARGDDAYYRYDGGYFAMDFWGQGTSVTVSS; The amino acid sequence shown in SEQ ID NO:14 is: DILMTQSSSYLSVSLGGRVILTCKASDHINNWIAWYQQKPGNAPRLLISGTTSIETGVPSRFSGSGSGKEYT LSITSIQTEDVATYYCQQYWKSPYTFGGGTKLEIK. The above-mentioned light chain variable region and heavy chain variable region are connected by disulfide bonds.

[0047] It is understood that in other embodiments, the first heavy chain variable region is not limited to the polypeptide with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:13, but may also be other polypeptides containing the above-mentioned heavy chain complementarity-determining region; the first light chain variable region is not limited to the polypeptide with an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:14, but may also be other polypeptides containing the above-mentioned light chain complementarity-determining region.

[0048] Optionally, the nucleotide sequence encoding the first heavy chain variable region is as shown in SEQ ID NO:7, such as CAAGTGCAGCTGCAGCAGAGCGGCGGCGGCCTGGTTCAGCCTGGCGGATCTATGAAACTGAGCTGTGCTGCTTCTGGATTTACCTTCAGCGACGCCTGGATGGATTGGGTCAGACAGAGCCCCGAGAAGGGACTGGAATGGGTGGCCGAGATCGGCAATAAGGGCAACAACCACGCCACATACTACGCCGAGAGCGTGAAGGGCAGATTCACCGTGTCCCGGGACGACAGCAAGAGCCGGGTGTACCTGCAGATGAACAGCCTGAGAGTGGAAGATACAGGCACATATTACTGCACCACCAGATTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCGCC; or the nucleotide sequence encoding the first heavy chain variable region is as shown in SEQ ID NO:17, such as SEQ ID The nucleotide sequence shown in NO:17 is CAAGTGCAGCTGCAGGAGAGCGGCCCTGGCCTGGTGAAGCCTTCTCAGAGCCTGAGCCTGACCTGTAGCGTGACCGATTACAGCCTGACATCTGGATATTACTGGAATTGGATCCGGCAGTTTCCAGGCAACAAG ATCGAATGGATGGGCTACATCAGATTCGATGGCAACAACCGGTACAACCCCAGCATCAAGAATAGAATCAGCATCACCCGGGAAAAAAGCAAGAACCAGTTCTTCCTGAGAATTTCTAGCGTGACCCCTGAGGACACCGCTACATACTACTGCGCCAGAGGCGACGACGCCTACTACAGATACGACGGCGGATACTTCGCCATGGACTTCTGGGGCCAGGGCACCAGCGTCACAGTGTCCTCC.

[0049] Optionally, the nucleotide sequence encoding the nucleic acid of the first light chain variable region is as shown in SEQ ID NO:11, such as GACATCGTGATGACACAGAGCCCTAGCTCCATGTACGCCTCCCTGGGCGAGAGGGTGACAATCACATGCAAGGCCAGCCAGGACATCAATAGCTACCTGAGCTGGTTTCAGCAGAAGCCTGGCAAGTCCCCCAAGACCCTGATCTACAGGGCCAACAGGCTGGTGGATGGCGTGCCTAGCAGGTTCTCCGGCAGCGGCTCCGGCCAGGATTACAGCCTGACAATCTCCAGCCTGGAGTACGAGGACATGGGCATCTACTACTGCCTGCAGTACGATGAGTTCCCTAGAACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG; or the nucleotide sequence encoding the nucleic acid of the first light chain variable region is as shown in SEQ ID NO:20, such as SEQ ID The nucleotide sequence shown in NO:20 is: GATATCCTGATGACCCAGAGCAGCTCTTATCTGAGCGTGTCCCTGGGCGGCAGAGTGATCCTCACCTGTAAAGCCAGCGACCACATCAACAACTGGATCGCCTGGTACCAGCAAAAGCCTGGAAATGCCCCTAGACTGCTGATCAGCGGCACC ACCAGCATCGAGACAGGCGTCCCCAGCCGGTTCAGCGGATCTGGCAGCGGCAAGGAATACACCCTGTCTATCACCAGCATTCAGACCGAGGACGTGGCTACATACTACTGCCAGCAGTACTGGAAGTCCCCATACACATTCGGCGGCGGAACAAAGCTGGAAATCAAG.

[0050] It is understood that, based on the degeneracy of codons, in other embodiments, the nucleic acid encoding the variable region is not limited to the above, but may also be other nucleic acids capable of encoding the first heavy chain variable region as shown in SEQ ID NO:1 or SEQ ID NO:13; the nucleic acid encoding the first light chain variable region is not limited to the above, but may also be other nucleic acids capable of encoding the first light chain variable region as shown in SEQ ID NO:2 or SEQ ID NO:14.

[0051] In one optional specific example, the first binding region is composed of a first heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:13 and a first light chain variable region with an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:14, wherein the first light chain variable region and the first heavy chain variable region are connected by disulfide bonds. In this case, the first heavy chain variable region and the first light chain variable region form an FV region with all antigen binding sites through non-covalent bonding, thus possessing antigen binding capability.

[0052] In some embodiments, the second binding region includes a second heavy chain variable region and a second light chain variable region. The second heavy chain variable region is H-CDR3 with an amino acid sequence as shown in SEQ ID NO:3, and the second light chain variable region is L-CDR3 with an amino acid sequence as shown in SEQ ID NO:4, or the second heavy chain variable region is H-CDR4 with an amino acid sequence as shown in SEQ ID NO:15, and the second light chain variable region is L-CDR4 with an amino acid sequence as shown in SEQ ID NO:16.

[0053] Specifically, the amino acid sequence shown in SEQ ID NO:3 is: EVQLEEFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTIYNQKFEGKATL TVDKSSSTAYMELRSLTSEDTAVYYCARRVLSLWYFDVWGAGTTVTVSS; the amino acid sequence shown in SEQ ID NO:4 is: DIVLTQDQASLSVSVGEIVTITCRASENIYSNLVWYQQKQGKSPQVLVYAATNLADGVPSRFSGSGSGTQY SLKINSLQSEDFGSYYCQNFWVTPWTFGGGTKLEIK; the amino acid sequence shown in SEQ ID NO:15 is: EVLLQQSGPELVKPGASVKLVCKASGYTFTDYNMDWVKQSHGKSIEWIGDINPNNGDSFYNQKFKGKAT The amino acid sequence shown in SEQ ID NO:16 is: DVVMTQTPLSLPVSLGEQASISCRSSQSLLYSNGNTYLHWYIQKPGQSPNLLIYKVSNRFSGVPERFSGSG SGTDFTLKLSRVEAEDLGVYFCSQTTHVPYTFGGGTKLEIK. The second light chain variable region and the second heavy chain variable region are connected by disulfide bonds.

[0054] It is understood that, in other embodiments, the second heavy chain variable region is not limited to the polypeptide with an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:15, but may also be other polypeptides containing the above-mentioned heavy chain complementarity-determining region; the second light chain variable region is not limited to the polypeptide with an amino acid sequence as shown in SEQ ID NO:4 or SEQ ID NO:16, but may also be other polypeptides containing the above-mentioned light chain complementarity-determining region.

[0055] Optionally, the nucleotide sequence encoding the second heavy chain variable region is as shown in SEQ ID NO:9, where the nucleotide sequence is: GAGGTCCAGCTGGAAGAGTTCGGCGCCGAGCTGGTGAAACCTGGCGCCAGCGTGAAGATCAGCTGCAAGGCCTCTGGCTACACCTTTACCGACTACAATATGGACTGGGTTAAGCAGAGCCACGGCAAGTCCCTGGAATGGATCGGCGATCAACCCCAACTACGACAGCACAATCTACAACCAGAAGTTCGAGGGAAAAGCTACACTCACAGTGGATAAGAGCAGCTCTACCGCCTACATGGAACTGAGAAGCCTGACCAGCGAGGACACCGCCGTGTACTACTGTGCCAGACGGGTGCTGAGCCTGTGGTATTTCGACGTGTGGGGAGCTGGCACCACAGTGACCGTGTCCTCT; or the nucleotide sequence encoding the second heavy chain variable region is as shown in SEQ ID NO:18, where SEQ ID NO:18 is the most common nucleotide sequence. The sequence of the nucleotide shown in NO:18 is: GAGGTGCTGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGCGCCAGCGTGAAGCTGGTTTGTAAAGCCTCTGGATATACATTCACCGACTACAACATGGATTGGGTCAAGCAGAGCCACGGCAAAAGCATCGAGTGGATCGGCGATATCAACCCCAACAACGGCGACAGCTTTT ACAATCAAAAGTTCAAGGGCAAGGCTACACTGACCGTGGAAAAGTCCTCCACCACCGCCTACATGGACCTGCGGAGCCTGACAAGCGAGGAAACCGCCGTGTACTACTGCGCCAGACACGAGGGCTACTACAGCAGAATCTACTTCTACACCATGGAATACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC.

[0056] Optionally, the nucleotide sequence encoding the second light chain variable region is as shown in SEQ ID NO:10, and the nucleotide sequence shown in SEQ ID NO:10 is: GACATCGTGCTGACCCAGGACCAGGCCAGCCTGAGCGTGAGCGTGGGCGAGATCGTGACAATCACATGCAGGGCCTCCGAGAATATCTACAGCAATCTGGTGTGGTACCAGCAGAAGCAGGGCAAGTCCCCTCAGGTGCTGGTGTACGCCGCCACAAATCTGGCCGACGGCGTGCCCAGCAGATTCTCCGGCAGCGGCAGCGGCACCCAGTACTCCCTGAAGATCAATTCCCTGCAGTCCGAGGACTTTGGCAGCTACTACTGCCAGAACTTTTGGGTGACCCCTTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG; or the nucleotide sequence encoding the second light chain variable region is as shown in SEQ ID NO:19, and the nucleotide sequence encoding the second light chain variable region is as shown in SEQ ID NO:19. The nucleotide sequence shown in NO:19 is: GATGTGGTGATGACCCAGACCCCTGTCCCTGCCTGTGTCCCTGGGCGAGCAGGCCAGCATCAGCTGTAGAAGCAGCCAAAGCCTCCTGTACAGCAACGGCAACACCTACCTGCACTGGTATATCCAGAAACCTGGCCAGAGCCCCAACCTGCTGATCTA CAAGGTGTCTAATAGATTCAGCGGCGTGCCCGAGCGGTTTAGCGGCTCCGGCTCTGGCACAGACTTCACCCTGAAGCTGTCTAGAGTGGAAGCCGAGGACCTGGGAGTCTACTTCTGCAGCCAGACAACACACGTGCCATACACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG.

[0057] It is understood that, based on the degeneracy of codons, in other embodiments, the nucleic acid encoding the variable region is not limited to the above, but may also be other nucleic acids capable of encoding the second heavy chain variable region as shown in SEQ ID NO:3 or SEQ ID NO:15; the nucleic acid encoding the second light chain variable region is not limited to the above, but may also be other nucleic acids capable of encoding the light chain variable region as shown in SEQ ID NO:4 or SEQ ID NO:16.

[0058] In some embodiments, the first binding region includes a first heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 (H-CDR1), and a first light chain variable region having an amino acid sequence as shown in SEQ ID NO:2 (L-CDR1); and

[0059] The second binding region includes a second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:3 (H-CDR3) and a second light chain variable region having an amino acid sequence as shown in SEQ ID NO:4 (L-CDR3).

[0060] In other embodiments, the first binding region includes a first heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 13 (H-CDR2), and a first light chain variable region having an amino acid sequence as shown in SEQ ID NO: 14 (L-CDR2); and

[0061] The second binding region includes a second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:15 (H-CDR4) and a second light chain variable region having an amino acid sequence as shown in SEQ ID NO:16 (L-CDR4).

[0062] In some embodiments, the bispecific antibody further includes a heavy chain constant region and a light chain constant region. The heavy chain constant region is connected to a first heavy chain variable region and a second heavy chain variable region, respectively, and the light chain constant region is connected to a first light chain variable region and a second light chain variable region, respectively. The heavy chain constant region is also connected to the light chain constant region to form a Y-shaped monomer structure at both ends.

[0063] In one optional specific example, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:5; the amino acid sequence of the light chain constant region is shown in SEQ ID NO:6; it is understood that in other embodiments, the amino acid sequences of the heavy chain constant region and the light chain constant region are not limited to the above, and may also be other polypeptide fragments that can serve as constant regions.

[0064] Specifically, such as SEQ ID The amino acid sequence shown in NO:5 is: AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVD DVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK; such as SEQ The amino acid sequence shown in IDNO:6 is: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS TLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0065] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain constant region is shown in SEQ ID NO:8.The nucleotide sequence shown in SEQ ID NO:8 is GCCAAGACCACACCACCTAGCGTGTACCCTCTCGCCCCTGGCTCTGCCGCTCAAACAAACAGCATGGTGACCCTGGGATGTCTGGTGAAGGGCTACTTCCCTGAGCCAGTGACCGTGACATGGAACAGCGGCAGCCTGAGCTCCGGCGTGCATACCTTTCCCGCCGTGCTGCAGAGCGACCTGTACACACTGTCTTCTAGCGTGACAGTGCCTAGCAGCACCTGGCCTAGCGAAACCGTCACATGCAACGTGGCCCACCCCGCCAGCAGCACCAAGGTTGATAAGAAGATCGTGCCAAGAGATTGCGGCTGCAAGCCCTGCATCTGCACCGTCCCCGAGGTGTCCTCCGTGTTCATCTTCCCTCCCAAGCCTAAAGACGTACTGACCATCACCCTGACCCCCAAGGTGACCTGCGTGGTGGTCGACATCAGCAAGGACGACCCCGAGGTGCAGTTCAGCTGGTTCGTGGACGATGTGGAAGTGCACACAGCCCAGACCCAGCCCAGAGAGGAACAGTTTAACAGCACATTTCGGAGCGTGTCTGAGCTGCCTATCATGCACCAGGACTGGCTGAACGGCAAGGAGTTCAAGTGCCGGGTGAACAGCGCCGCCTTCCCTGCTCCTATCGAGAAAACCATCTCTAAGACAAAGGGAAGACCTAAGGCCCCTCAGGTGTACACCATCCCTCCTCCAAAGGAACAGATGGCCAAGGATAAGGTGTCCCTGACATGTATGATCACCGACTTCTTCCCGGAAGATATCACTGTGGAATGGCAGTGGAACGGCCAGCCTGCCGAGAACTACAAGAACACCCAACCTATTATGGACACAGACGGCAGCTACTTCGTGTACAGCAAACTGAATGTGCAGAAATCCAACTGGGAGGCCGGAAATACCTTCACCTGTAGCGTTCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGAGCCTGTCCCACTCTCCCGGCAAG。,

[0066] Optionally, the nucleotide sequence encoding the light chain constant region of the nucleic acid is as shown in SEQ ID NO:12, and the nucleotide sequence shown in SEQ ID NO:12 is AGGGCCGACGCCGCCCCAACAGTGTCCATCTTCCCCCCTAGCTCCGAGCAGCTGACCAGCGGAGGAGCCTCCGTGGTGTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGATCGATGGCTCTGAGCGGCAGAACGGCGTGCTGAATAGCTGGACAGACCAGGATAGCAAGGACTCCACCTATTCTATGTCTAGCACCCTGACACTGACCAAGGATGAGTACGAGCGGCACAATTCCTATACATGCGAGGCCACCCACAAGACATCCACCTCTCCCATCGTGAAGTCTTTTAACAGAAATGAGTGT.

[0067] It is understood that, based on the degeneracy of codons, in other embodiments, the nucleic acid encoding the constant region is not limited to the above, but may also be other nucleic acids that can encode the heavy chain constant region of the amino acid sequence as shown in SEQ ID NO:5; the nucleic acid encoding the light chain constant region is not limited to the above, but may also be other nucleic acids that can encode the light chain constant region of the amino acid sequence as shown in SEQ ID NO:6.

[0068] In addition, one embodiment of this application also provides the above-mentioned bispecific nucleic acid.

[0069] In some embodiments, the nucleic acid comprises a nucleic acid fragment encoding a first heavy chain variable region of a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:1 and a nucleic acid fragment encoding a first light chain variable region of a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:2; and a nucleic acid fragment encoding a second heavy chain variable region of a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:3 and a nucleic acid fragment encoding a second light chain variable region of a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:4. Further, the nucleic acid also includes a nucleic acid fragment encoding a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:5 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:6.

[0070] Optionally, the nucleotide sequence of the nucleic acid encoding the first heavy chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:1 is shown in SEQ ID NO:7. The nucleotide sequence of the nucleic acid encoding the first light chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:2 is shown in SEQ ID NO:11. The nucleotide sequence of the nucleic acid encoding the second heavy chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:3 is shown in SEQ ID NO:9. The nucleotide sequence of the nucleic acid encoding the second light chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:4 is shown in SEQ ID NO:10. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequences of the nucleic acid fragments encoding the heavy chain variable regions with amino acid sequences as shown in SEQ ID NO:1 and SEQ ID NO:2 and the light chain variable regions with amino acid sequences as shown in SEQ ID NO:3 and SEQ ID NO:4 are not limited to the above, and may be other sequences.

[0071] It should be noted that, in the above nucleic acids, the nucleic acid fragments encoding the first heavy chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:1 and the nucleic acid fragments encoding the first light chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:2 may exist in different reagent systems (e.g., the two nucleic acid fragments are located on different expression vectors), or they may exist simultaneously in the same reagent system (e.g., the two nucleic acid fragments are located on the same expression vector). Similarly, in the above nucleic acids, the nucleic acid fragments encoding the second heavy chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:3 and the nucleic acid fragments encoding the second light chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:4 may exist in different reagent systems (e.g., the two nucleic acid fragments are located on different expression vectors), or they may exist simultaneously in the same reagent system (e.g., the two nucleic acid fragments are located on the same expression vector).

[0072] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain constant region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:5 is shown in SEQ ID NO:8. The nucleotide sequence of the nucleic acid encoding the light chain constant region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:6 is shown in SEQ ID NO:12. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequences of the nucleic acid fragments encoding the heavy chain constant region and light chain constant region with the amino acid sequences as shown in SEQ ID NO:5 and SEQ ID NO:6 are not limited to the above, and may be other sequences.

[0073] In other embodiments, the nucleic acid comprises a nucleic acid fragment encoding a first heavy chain variable region of a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:13 and a nucleic acid fragment encoding a first light chain variable region of a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:14; and a nucleic acid fragment encoding a second heavy chain variable region of a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:15 and a nucleic acid fragment encoding a second light chain variable region of a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:16. Further, the nucleic acid also includes a nucleic acid fragment encoding a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO:5 and a light chain constant region with an amino acid sequence as shown in SEQ ID NO:6.

[0074] Optionally, the nucleotide sequence of the nucleic acid encoding the first heavy chain variable region of the bispecific antibody with the amino acid sequence shown in SEQ ID NO:13 is shown in SEQ ID NO:17. The nucleotide sequence of the nucleic acid encoding the first light chain variable region of the bispecific antibody with the amino acid sequence shown in SEQ ID NO:14 is shown in SEQ ID NO:20. The nucleotide sequence of the nucleic acid encoding the second heavy chain variable region of the bispecific antibody with the amino acid sequence shown in SEQ ID NO:15 is shown in SEQ ID NO:18. The nucleotide sequence of the nucleic acid encoding the second light chain variable region of the bispecific antibody with the amino acid sequence shown in SEQ ID NO:16 is shown in SEQ ID NO:19. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequences of the nucleic acid fragments encoding the heavy chain variable regions with amino acid sequences shown in SEQ ID NO:13 and SEQ ID NO:15 and the light chain variable regions with amino acid sequences shown in SEQ ID NO:14 and SEQ ID NO:16 are not limited to the above, and may be other sequences.

[0075] It should be noted that, in the above nucleic acids, the nucleic acid fragments encoding the first heavy chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:13 and the nucleic acid fragments encoding the first light chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:14 may exist in different reagent systems (e.g., the two nucleic acid fragments are located on different expression vectors), or they may exist simultaneously in the same reagent system (e.g., the two nucleic acid fragments are located on the same expression vector). Similarly, in the above nucleic acids, the nucleic acid fragments encoding the second heavy chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:15 and the nucleic acid fragments encoding the second light chain variable region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:16 may exist in different reagent systems (e.g., the two nucleic acid fragments are located on different expression vectors), or they may exist simultaneously in the same reagent system (e.g., the two nucleic acid fragments are located on the same expression vector).

[0076] Optionally, the nucleotide sequence of the nucleic acid encoding the heavy chain constant region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:5 is shown in SEQ ID NO:8. The nucleotide sequence of the nucleic acid encoding the light chain constant region of the bispecific antibody with the amino acid sequence as shown in SEQ ID NO:6 is shown in SEQ ID NO:12. It is understood that, based on codon degeneracy, in other embodiments, the nucleotide sequences of the nucleic acid fragments encoding the heavy chain constant region and light chain constant region with the amino acid sequences as shown in SEQ ID NO:5 and SEQ ID NO:6 are not limited to the above, and may be other sequences.

[0077] In some embodiments, the nucleic acid of any of the above embodiments further includes transcription elements, such as promoters and terminators. It is understood that in some embodiments, the nucleic acid of any of the above embodiments may not include transcription elements. In this case, during use, the nucleic acid can be inserted into a vector having the corresponding transcription elements for expression.

[0078] Furthermore, one embodiment of this application also provides a carrier containing the nucleic acid of any of the above embodiments. The nucleic acid fragments encoding the first heavy chain variable region as shown in SEQ ID NO:1, the first light chain variable region as shown in SEQ ID NO:2, the second heavy chain variable region as shown in SEQ ID NO:3, the second light chain variable region as shown in SEQ ID NO:4, the heavy chain constant region as shown in SEQ ID NO:5, and the light chain constant region as shown in SEQ ID NO:6 are all included in this embodiment.

[0079] In other embodiments, the vector contains the nucleic acid of any of the above embodiments. The nucleic acid fragment encoding the first heavy chain variable region as shown in SEQ ID NO:13, the nucleic acid fragment encoding the first light chain variable region as shown in SEQ ID NO:14, the nucleic acid fragment encoding the second heavy chain variable region as shown in SEQ ID NO:15, the nucleic acid fragment encoding the second light chain variable region as shown in SEQ ID NO:16, the nucleic acid fragment encoding the heavy chain constant region as shown in SEQ ID NO:5, and the nucleic acid fragment encoding the light chain constant region as shown in SEQ ID NO:6.

[0080] In one embodiment, the vector is a pCDNA3.1 vector. Of course, in other embodiments, the vector is not limited to the pCDNA3.1 vector, but can be other vectors.

[0081] Furthermore, one embodiment of this application also provides a host cell transformed with a vector comprising any of the above embodiments. The recombinant expression vector comprises the nucleic acid fragment encoding the first heavy chain variable region as shown in SEQ ID NO:1 and SEQ ID NO:13, the nucleic acid fragment encoding the first light chain variable region as shown in SEQ ID NO:2 and SEQ ID NO:14, the nucleic acid fragment encoding the second heavy chain variable region as shown in SEQ ID NO:3 and SEQ ID NO:15, and the nucleic acid fragment encoding the second light chain variable region as shown in SEQ ID NO:4 and SEQ ID NO:16.

[0082] In another embodiment, the host cells are transformed with a recombinant expression vector containing a nucleic acid fragment encoding the heavy chain variable region of the bispecific antibody and a recombinant expression vector containing a nucleic acid fragment encoding the light chain variable region of the bispecific antibody. Placing the nucleic acid fragment encoding the first light chain variable region of the first binding region of the bispecific antibody and the nucleic acid fragment encoding the first heavy chain variable region of the first binding region of the bispecific antibody on different expression vectors and co-transfecting them into the host cells facilitates control over the amount of light chain and heavy chain variable regions of the first binding region of the bispecific antibody produced.

[0083] In one embodiment, the host cell is HEK293. Of course, in other embodiments, the host cell is not limited to HEK293 and can be other cells.

[0084] This application also provides the application of the above-mentioned bispecific antibody, the above-mentioned nucleic acid, the above-mentioned vector, and the above-mentioned host cell in the preparation of detection reagents.

[0085] In some embodiments, the above-described immunoassay reagent is used to perform at least one of the following detection methods: enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunogold immunoassay, immunochemiluminescence assay, immunoturbidimetric assay, immunoblotting, and dot blot assay.

[0086] In some embodiments, the above-mentioned bispecific antibody, nucleic acid, vector, and host cell are used in the preparation of alpha-fetoprotein detection reagents.

[0087] In addition, one embodiment of this application provides a detection reagent comprising the bispecific antibody of any of the above embodiments.

[0088] One embodiment of this application also provides a detection kit comprising the bispecific antibody of any of the above embodiments, for detecting alpha-fetoprotein and improving the accuracy of immunodiagnosis. Optionally, the bispecific antibody can be used as a capture antibody or as a labeling antibody.

[0089] In some embodiments, the detection kit includes a first binding region and a second binding region. The first binding region includes a first light chain variable region and a first heavy chain variable region. The first heavy chain variable region of the first binding region includes a complementarity-determining region with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:13, and the first light chain variable region of the first binding region includes a complementarity-determining region with an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:14. The second binding region includes a second light chain variable region and a second heavy chain variable region. The second heavy chain variable region of the second binding region includes a complementarity-determining region with an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:15, and the second light chain variable region of the second binding region includes a complementarity-determining region with an amino acid sequence as shown in SEQ ID NO:4 or SEQ ID NO:16.

[0090] In one embodiment, the above-described detection kit further includes at least one of a solid support and a buffer solution. Optionally, the solid support is a magnetic bead. The buffer solution includes phosphate buffer and citric acid solution, used as a sample dilution buffer and an elution buffer, respectively.

[0091] The above-mentioned test kit includes the aforementioned bispecific antibody, which has high sensitivity, good affinity and specificity, and high batch-to-batch consistency, and can be used for the development of immunodiagnostic products.

[0092] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, the embodiments do not include components other than unavoidable impurities. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature or books, or methods recommended by the manufacturer.

[0093] Example 1

[0094] (1) Preparation of bispecific antibody expression vector

[0095] In this case, the first binding region of the bispecific antibody can specifically bind to the human CA199 antigen, and its heavy chain variable region sequence is shown in SEQ ID NO:1, and its light chain variable region sequence is shown in SEQ ID NO:2; the second binding region can specifically bind to polymeric horseradish peroxidase (poly-HRP), and its heavy chain variable region sequence is shown in SEQ ID NO:3, and its light chain variable region sequence is shown in SEQ ID NO:4; the heavy chain constant region sequence of the antibody is shown in SEQ ID NO:5, and the light chain constant region sequence is shown in SEQ ID NO:6.

[0096] The expression vector construction scheme is as follows: Expression vector 1 is a synthetically produced gene fragment consisting of the first binding region heavy chain variable region gene (SEQ ID NO:7), the heavy chain constant region gene (SEQ ID NO:8), the second binding region heavy chain variable region gene (SEQ ID NO:9), and the second binding region light chain variable region gene (SEQ ID NO:10), ligated into the mammalian cell expression vector pCDNA3.1; Expression vector 2 is a gene fragment consisting of the first binding region light chain variable region gene (SEQ ID NO:11) and the light chain constant region gene (SEQ ID NO:12), ligated into the mammalian cell expression vector pCDNA3.1. The synthesis of the above gene fragments and the ligation of the vectors were completed by Jiangsu Saisofe Biotechnology Co., Ltd. After the expression vectors were constructed, plasmids were extracted in large quantities using an endotoxin-free plasmid large-scale extraction kit (Nanjing Novizan Biotechnology Co., Ltd., DC202).

[0097] (2) Expression of bispecific antibodies

[0098] Suspension Expi293F cells (Thermo Fisher Scientific Inc., A14527CN) were cultured in OPM-293CD05 medium (Shanghai Aopumai Biotechnology Co., Ltd., 81075-001) at 37℃, 110 rpm, and 6% CO2. 24 hours before transfection, cells were seeded into 1000mL breathable cap culture flasks (784011, Wuxi Naisi Biotechnology Co., Ltd.) with fresh OPM-293CD05 medium (240mL volume) at a cell density of 2×10⁶ cells / mL. 6 Cells / mL. On the day of transfection, preheat 16 mL of OPM-293CD05 medium at 37°C, then add 120 μg of expression vector 1 and 120 μg of expression vector 2, gently mix, and then add 480 μg of PEI transfection reagent (Polysciences, 23966, 1 mg / mL). After inverting and mixing, let stand at room temperature for 20 min, and then slowly add the resulting DNA and PEI complex into the cultured cells, which is counted as day 1. On days 2 and 4 of culture, glucose (Sangon Biotech (Shanghai) Co., Ltd., A610219-0500, 2M) and glutamine solution (Thermo Fisher Scientific, 25030149, 200mM) to a final concentration of 50 mM and 4 mM, respectively, are added simultaneously. After 6-7 days of culture, the cell expression supernatant is collected for protein purification.

[0099] (3) Purification of bispecific antibodies

[0100] First, the rProtein A affinity chromatography column (GLK-gel proteinA, Wuxi Galek Chromatography Technology Co., Ltd.) was connected to an AKTA pure150L (Cytiva) instrument. The column was equilibrated with 5CV equilibration buffer (PBS, pH 7.4). Then, cell culture medium was flow-throughd through the rProtein A affinity chromatography packing material to allow antibody binding. After loading, equilibration was continued until the UV baseline stabilized. The antibody on the column was eluted with 20mM citric acid (pH 3.0), and the elution peak was collected. The pH of the eluent was adjusted to 7.4 with Tris-HCl (pH 9.0). Then, the antibody buffer was replaced with PBS at pH 7.4 using a 14kDa dialysis bag (Beijing Jingke Hongda Biotechnology Co., Ltd., 300059534). The purified antibody concentration was determined using a UV5Nano ultra-micro UV-Vis spectrophotometer (Mettler Toledo Technologies (China) Co., Ltd.), and the molecular weight and purity of the antibody were analyzed by SDS-PAGE. Figure 1Analysis showed that the target molecular weights of the recombinant bispecific antibody expressed by reducing SDS-PAGE were 75 kDa and 25 kDa, respectively, while the target band of the non-reducing SDS-PAGE was approximately 200 kDa, which is consistent with the theoretical molecular weight.

[0101] (4) The bispecific antibody was reacted with polymeric horseradish peroxidase (poly-HRP) in HRP enzyme stabilizer for 2 h to obtain a complex.

[0102] (5) Use HRP enzyme stabilizer to prepare a reaction solution of 0.5 μg / mL for the complex.

[0103] Example 2

[0104] (1) Preparation of bispecific antibody expression vector

[0105] The first binding region of the bispecific antibody can specifically bind to human AFP antigen, and its heavy chain variable region sequence is shown in SEQ ID NO:13, and its light chain variable region sequence is shown in SEQ ID NO:14; the second binding region can specifically bind to polyruthenium, and its heavy chain variable region sequence is shown in SEQ ID NO:15, and its light chain variable region sequence is shown in SEQ ID NO:16; the heavy chain constant region sequence of the antibody is shown in SEQ ID NO:5, and the light chain constant region sequence is shown in SEQ ID NO:6.

[0106] The expression vectors were constructed as follows: Expression vector 1 consisted of artificially synthesized gene fragments in the following order: the heavy chain variable region gene of the first binding region (SEQ ID NO:17), the heavy chain constant region gene (SEQ ID NO:8), the heavy chain variable region gene of the second binding region (SEQ ID NO:18), and the light chain variable region gene of the second binding region (SEQ ID NO:19), which were ligated into the mammalian cell expression vector pCDNA3.1. Expression vector 2 consisted of the light chain variable region gene of the first binding region (SEQ ID NO:20) and the light chain constant region gene (SEQ ID NO:12), which were ligated into the mammalian cell expression vector pCDNA3.1. The synthesis of the above gene fragments and the ligation of the vectors were completed by Jiangsu Saisofe Biotechnology Co., Ltd. After construction, the expression vectors were used for large-scale plasmid extraction using an endotoxin-free plasmid extraction kit (Nanjing Novizan Biotechnology Co., Ltd., DC202).

[0107] (2) Expression of bispecific antibodies

[0108] Suspension Expi293F cells (Thermo Fisher Scientific Inc., A14527CN) were cultured in OPM-293CD05 medium (Shanghai Aopumai Biotechnology Co., Ltd., 81075-001) at 37℃, 110 rpm, and 6% CO2. 24 hours before transfection, cells were seeded into 1000mL breathable cap culture flasks (784011, Wuxi Naisi Biotechnology Co., Ltd.) with fresh OPM-293CD05 medium (240mL volume) at a cell density of 2×10⁶ cells / mL. 6 Cells / mL. On the day of transfection, preheat 16 mL of OPM-293CD05 medium at 37°C, then add 120 μg of expression vector 1 and 120 μg of expression vector 2, gently mix, and then add 480 μg of PEI transfection reagent (Polysciences, 23966, 1 mg / mL). After inverting and mixing, let stand at room temperature for 20 min, and then slowly add the resulting DNA and PEI complex into the cultured cells, which is counted as day 1. On days 2 and 4 of culture, glucose (Sangon Biotech (Shanghai) Co., Ltd., A610219-0500, 2M) and glutamine solution (Thermo Fisher Scientific, 25030149, 200mM) to a final concentration of 50 mM and 4 mM, respectively, are added simultaneously. After 6-7 days of culture, the cell expression supernatant is collected for protein purification.

[0109] (3) Purification of bispecific antibodies

[0110] First, the rProtein A affinity chromatography column (GLK-gel proteinA, Wuxi Galek Chromatography Technology Co., Ltd.) was connected to an AKTA pure150L (Cytiva) instrument. The column was equilibrated with 5CV equilibration buffer (PBS, pH 7.4). Then, cell culture medium was flow-throughd through the rProtein A affinity chromatography packing material to allow antibody binding. After loading, equilibration was continued until the UV baseline stabilized. The antibody on the column was eluted with 20mM citric acid (pH 3.0), and the elution peak was collected. The pH of the eluent was adjusted to 7.4 with Tris-HCl (pH 9.0). Then, the antibody buffer was replaced with PBS at pH 7.4 using a 14kDa dialysis bag (Beijing Jingke Hongda Biotechnology Co., Ltd., 300059534). The purified antibody concentration was determined using a UV5Nano ultra-micro UV-Vis spectrophotometer (Mettler Toledo Technologies (China) Co., Ltd.), and the molecular weight and purity of the antibody were analyzed by SDS-PAGE. Figure 2Analysis showed that the target molecular weights of the recombinant bispecific antibody expressed by reducing SDS-PAGE were 75 kDa and 25 kDa, respectively, while the target band of the non-reducing SDS-PAGE was approximately 200 kDa, which is consistent with the theoretical molecular weight.

[0111] (4) The bispecific antibody was reacted with polyruthenium in a stabilizer for 2 hours to obtain a complex.

[0112] (5) The complex was prepared to a concentration of 0.5 μg / mL using a stabilizer as the R2 reaction solution.

[0113] Comparative Example 1

[0114] (1) Weigh 25 mg HRP and dissolve it in 1.25% glutaraldehyde solution, and let it stand overnight at room temperature.

[0115] (2) The enzyme solution after the reaction was eluted with physiological saline using a Sephadex G-25 column. The flow rate was controlled at 1 mL / 1 min, and the brown eluent was collected. If the volume was greater than 5 mL, it was concentrated to 5 mL with PEG. The solution was then placed in a 25 mL beaker and stirred slowly.

[0116] (3) Dilute 12.5 mg of the CA199 secondary antibody to be labeled with physiological saline to 5 mL, and add it dropwise to the enzyme solution while stirring.

[0117] (4) Use 0.25 mL of 1M pH 9.5 carbonate buffer to adjust the pH to 9.0-9.5 and stir for 3 hours.

[0118] (5) Add 0.25 mL of 0.2 M lysine, mix well, and let stand at room temperature for 2 hours.

[0119] (6) Add an equal volume of saturated ammonium sulfate dropwise while stirring, and let stand at 4°C for 1 hour.

[0120] (7) Centrifuge at 3000 rpm for half an hour and discard the supernatant. Wash the precipitate twice with semi-saturated ammonium sulfate, and finally dissolve the precipitate in a small amount of 0.15M pH7.4 PBS.

[0121] (8) Put the above solution into a dialysis bag and dialyze it against 0.15M pH 7.4 PB buffered saline to remove ammonium ions (detected with Naphthyl reagent). Centrifuge at 10,000 rpm for 30 minutes to remove the precipitate. The supernatant is the CA199 antibody labeled with HRP enzyme.

[0122] (9) Use HRP enzyme stabilizer to prepare a reaction solution of 0.5 μg / mL CA199 antibody labeled with enzyme.

[0123] Comparative Example 2

[0124] (1) Dilute 5 mg of the AFP secondary antibody to be labeled with physiological saline to 5 mL, stir and add 9 μL of ruthenium dropwise, and mix at 37 °C for 2 hours.

[0125] (2) The above solution was placed in a dialysis bag and dialyzed against 0.15M pH 7.4 PB buffered saline to remove free ruthenium. The solution was centrifuged at 10,000 rpm for 30 minutes to remove the precipitate. The supernatant was the ruthenium-labeled AFP antibody.

[0126] (3) Use a stabilizer to prepare the ruthenium-standardized AFP antibody to a concentration of 0.5 μg / mL as the R2 reaction solution.

[0127] test:

[0128] 1. Protein chip detection was performed on Example 1 and Comparative Example 1 above.

[0129] (1) Preparation of spotting solution: Take surfactant, protectant, moisturizer and biological buffer, mix them evenly to obtain spotting solution;

[0130] (2) Spotting: The spotting solution prepared in step (1) of CA199 antibody is diluted and then spotted on the chip matrix.

[0131] (3) Blocking treatment: The chip after spotting in step (2) is blocked with blocking solution for 4 hours. The solid carrier is removed and the residual blocking solution is removed by centrifugation to obtain the biochip blocking solution, which is a buffer solution containing blocking protein. The blocking protein is bovine serum albumin or ovalbumin. The buffer is selected from any one of PBS buffer, Tris buffer, HEPS buffer, and MOPS buffer.

[0132] Signal strength test:

[0133] Low-quality control samples were added to the protein chip prepared above to allow for reaction. Then, reaction solutions of the same concentrations as those in Example 1 and Comparative Example 1 were added, and the reaction was repeated. Finally, the chemiluminescent substrate luminol was added, and images were captured using a biochip analyzer (Jiangsu Sanlian Biotechnology Co., Ltd., SLXP-001B). The captured images are shown below. Figure 3 and Figure 4 As shown in Table 1 below, the signal values ​​obtained from the test are shown in Table 1.

[0134] Table 1

[0135] Experimental Example 1 195 14651 14570 14952 14468 13730 Compare with Example 1 168 2319 2504 2237 2504 2237

[0136] 2. Electrochemiluminescence detection was performed on Example 2 and Comparative Example 2.

[0137] The electrochemiluminescence platform was manufactured by Jiangsu Sanlian Biotechnology Co., Ltd., and its model number is SE1200.

[0138] Example 2:

[0139] M: Magnetic beads coated with streptavidin.

[0140] R1: Biotinylated anti-alpha-fetoprotein antibody; Biotinylated anti-alpha-fetoprotein monoclonal antibody (mouse) concentration 4.5 mg / L, phosphate buffer 100 mmol / L, pH 6.0.

[0141] R2: is the bispecific antibody complex reaction solution from Example 2.

[0142] Comparative Example 2:

[0143] Electrochemiluminescence platform (Jiangsu Sanlian Biotechnology Co., Ltd., SE1200)

[0144] M: Magnetic beads coated with streptavidin.

[0145] R1: Biotinylated anti-alpha-fetoprotein antibody; Biotinylated anti-alpha-fetoprotein monoclonal antibody (mouse) concentration 4.5 mg / L, phosphate buffer 100 mmol / L, pH 6.0.

[0146] R2: is the ruthenium-standardized antibody reaction solution of Comparative Example 2.

[0147] The quality control low value was measured 5 times using the above two methods, and the signal values ​​obtained are shown in Table 2 below.

[0148] Table 2

[0149] Experiment Example 2 13504 13624 14109 13756 13954 Compare with Example 2 1687 1654 1721 1696 1630

[0150] The comparison of the images above shows that the spots detected using bispecific antibodies as secondary antibodies are brighter and more uniform. A comparison of the signal values ​​in Tables 1 and 2 reveals that the signals detected by direct HRP or ruthenium labeling in Comparative Examples 1 and 2 are significantly lower than those detected by bispecific antibodies. The signal detected by bispecific antibodies as secondary antibodies is approximately 5 to 6 times higher than the control, indicating a significant improvement in detection sensitivity. In conclusion, the bispecific antibodies proposed in this technical solution, which specifically bind to antigens and tracers, can significantly increase detection sensitivity and signal intensity, and can be used in the development of related immunodiagnostic products.

[0151] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0152] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A bispecific antibody for enhancing the sensitivity of immunoassays, characterized in that, The bispecific antibody includes a first binding region that has the ability to specifically bind to the antigen, wherein the first binding region specifically binds to human alpha-fetoprotein antigen. And a second binding region, which specifically binds polyruthenium; The first binding region includes a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:13, and the first light chain variable region is an amino acid sequence as shown in SEQ ID NO:14; The second binding region includes a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:15, and the second light chain variable region is an amino acid sequence as shown in SEQ ID NO:

16.

2. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody further includes a heavy chain constant region and a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO:5; the amino acid sequence of which is shown in SEQ ID NO:

6.

3. The use of the bispecific antibody according to any one of claims 1 to 2 in the preparation of immunoassay reagents, characterized in that, The immunoassay reagent is used to perform the following detection method: immunochemiluminescence immunoassay.

4. A test kit, characterized in that, Includes the bispecific antibody as described in any one of claims 1 to 2.

Images