Monoclonal antibody capable of specifically recognizing HCY as well as preparation method and application of monoclonal antibody

Abstract

The invention provides a monoclonal antibody capable of specifically recognizing HCY. A light chain CDR1 of the monoclonal antibody is as shown in SEQ ID NO: 1, a CDR2 of the monoclonal antibody is as shown in SEQ ID NO: 2, and a CDR3 of the monoclonal antibody is as shown in SEQ ID NO: 3; the heavy chain CDR1 is shown as SEQ ID NO: 4, the CDR2 is shown as SEQ ID NO: 5, and the CDR3 is shown as SEQ ID NO: 6. A light chain variable region of the monoclonal antibody for specifically recognizing HCY is shown as SEQ ID NO: 7; the heavy chain variable region is shown as SEQ ID NO: 8. A light chain of the monoclonal antibody for specifically recognizing HCY is shown as SEQ ID NO: 9, and a heavy chain of the monoclonal antibody is shown as SEQ ID NO: 10. The antibody can directly recognize and detect an HCY target object.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody specifically recognizing HCY and a preparation method and application thereof, belonging to the technical field of monoclonal antibodies. Background Art

[0002] Homocysteine (HCY), commonly referred to as homocysteine, is a sulfur-containing amino acid and an intermediate in methionine metabolism. Produced during the metabolism of methionine (an essential amino acid), HCY is converted back to methionine with the help of vitamin B12 and folic acid. With the help of vitamin B6, HCY is converted to cysteine, ultimately producing glutathione or other sulfur-containing compounds. Its levels in the human body are closely associated with various diseases, particularly cardiovascular, cerebrovascular, and neurological disorders.

[0003] Elevated HCY levels can have toxic effects on blood vessels and organs, damaging the vascular endothelium, promoting atherosclerosis, and increasing the risk of coronary heart disease and myocardial infarction. They are closely associated with cerebrovascular diseases such as stroke and cerebral infarction, as well as neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Elevated HCY levels in pregnant women may increase the risk of miscarriage, premature birth, and neural tube defects in the fetus. They are also associated with conditions such as diabetes, chronic kidney disease, and osteoporosis. HCY testing can assess the risk of cardiovascular and cerebrovascular diseases, assist in the diagnosis of metabolic diseases such as vitamin B12 and folic acid deficiencies, monitor the effectiveness of treatment in patients with elevated HCY levels, and facilitate early screening and intervention in healthy individuals.

[0004] Methods for detecting HCY include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), immunofluorescence, and electrochemical methods. Currently, the most widely used immunoassay is the immunoassay. However, among these immunoassays, the current mainstream method requires conversion of Hcy to SAH followed by detection using SAH antibodies. Antibodies that directly bind to Hcy are not yet available. This is likely due to Hcy's small molecular weight (135 Da), its lack of immunogenicity, and its small epitope size after conjugation to a carrier protein, making it difficult to elicit an effective immune response. Furthermore, it is prone to cross-reactivity with other sulfur-containing compounds, such as cysteine. The sensitivity and specificity of detection significantly depend on the specificity and affinity of the SAH antibody. SAH, with a molecular weight only slightly larger than Hcy (384 Da), is formed by the condensation of the hydroxyl group of adenosine with the sulfhydryl group of Hcy. In its overall molecular structure, the Hcy portion is much smaller than the adenosine portion. The preparation of SAH-specific antibodies also faces the challenges of small epitopes, making it difficult to generate an effective immune response, and they are also prone to cross-interference with analogs such as adenosine and cysteine. Therefore, screening and developing highly specific and high-affinity monoclonal antibodies against Hcy or SAH is key to improving and refining Hcy immunoassays. Summary of the Invention

[0005] The present invention provides a monoclonal antibody that specifically recognizes HCY, and a preparation method and application thereof, which can effectively solve the above problems.

[0006] A monoclonal antibody that specifically recognizes HCY, wherein the light chain CDR1 is shown in SEQ ID NO: 1, the CDR2 is shown in SEQ ID NO: 2, and the CDR3 is shown in SEQ ID NO: 3; and the heavy chain CDR1 is shown in SEQ ID NO: 4, the CDR2 is shown in SEQ ID NO: 5, and the CDR3 is shown in SEQ ID NO: 6.

[0007] In some embodiments, the monoclonal antibody that specifically recognizes HCY has a light chain variable region as shown in SEQ ID NO: 7; and a heavy chain variable region as shown in SEQ ID NO: 8.

[0008] In some embodiments, the monoclonal antibody that specifically recognizes HCY has a light chain as shown in SEQ ID NO: 9, and a heavy chain as shown in SEQ ID NO: 10.

[0009] A method for preparing the monoclonal antibody that specifically recognizes HCY is to immunize an animal with the complete HCY antigen R5 as an immunogen, and then screen and prepare the antibody; Wherein, the structural formula of the HCY complete antigen R5 is: .

[0010] In some embodiments, the carrier protein is any one of hemocyanin, ovalbumin, or serum albumin.

[0011] In some embodiments, the method for preparing the HCY complete antigen R5 comprises the following steps: S1, using di-tert-butyl dicarbonate to protect the amino group of Hcy to form R1; S2, linking the thiol group of R1 to the amino group of 2,4,5-triamino-6-chloropyrimidine using a first amine-thiol crosslinker to form R2; S3, under alkaline conditions, R2 reacts with amine-PEG-thiol to form R3; S4, using a second amine-thiol crosslinker to couple R3 to a carrier protein, and under acidic conditions, removing the di-tert-butyl dicarbonate protecting group to obtain the homocysteine complete antigen R5.

[0012] In some embodiments, the amine-PEG-thiol has a PEG degree of polymerization of 2-24.

[0013] In some embodiments, the counter-detectors of the screen are SAH-Bio and Cys-Bio.

[0014] An HCY detection reagent comprises the monoclonal antibody that specifically recognizes HCY.

[0015] An HCY detection kit comprises the monoclonal antibody that specifically recognizes HCY.

[0016] The beneficial effects of the present invention are: The present invention prepares HCY complete antigen R5 as an immunogen to immunize mice, and uses coupled HCY-Bio, SAH-Bio, and Cys-Bio for forward and reverse screening to reduce steric hindrance and more efficiently screen to obtain HCY-specific monoclonal antibody 19A6. The antibody is applied to immune reagent detection, which can reduce the step of converting SAHHase to SAH and directly identify and detect the HCY target. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a diagram showing the expression and purification results of the 19A6 antibody. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0020] An embodiment of the present invention provides a monoclonal antibody that specifically recognizes HCY, wherein the light chain CDR1 is shown in SEQ ID NO: 1, the CDR2 is shown in SEQ ID NO: 2, and the CDR3 is shown in SEQ ID NO: 3; the heavy chain CDR1 is shown in SEQ ID NO: 4, the CDR2 is shown in SEQ ID NO: 5, and the CDR3 is shown in SEQ ID NO: 6.

[0021] In some embodiments, the monoclonal antibody that specifically recognizes HCY has a light chain variable region as shown in SEQ ID NO: 7; and a heavy chain variable region as shown in SEQ ID NO: 8.

[0022] In some embodiments, the monoclonal antibody that specifically recognizes HCY has a light chain as shown in SEQ ID NO: 9, and a heavy chain as shown in SEQ ID NO: 10.

[0023] The embodiment of the present invention provides a method for preparing the monoclonal antibody that specifically recognizes HCY, which is prepared by immunizing an animal with the complete HCY antigen R5 as an immunogen and then screening; Wherein, the structural formula of the HCY complete antigen R5 is: .

[0024] In some embodiments, the carrier protein is any one of hemocyanin, ovalbumin, or serum albumin.

[0025] In some embodiments, the method for preparing the HCY complete antigen R5 comprises the following steps: S1, using di-tert-butyl dicarbonate to protect the amino group of Hcy to form R1; S2, linking the thiol group of R1 to the amino group of 2,4,5-triamino-6-chloropyrimidine using a first amine-thiol crosslinker to form R2; S3, under alkaline conditions, R2 reacts with amine-PEG-thiol to form R3; S4, using a second amine-thiol crosslinker to couple R3 to a carrier protein, and under acidic conditions, removing the di-tert-butyl dicarbonate protecting group to obtain the homocysteine complete antigen R5.

[0026] In some embodiments, the amine-PEG-thiol has a PEG degree of polymerization of 2-24.

[0027] In some embodiments, the counter-detectors of the screen are SAH-Bio and Cys-Bio.

[0028] An embodiment of the present invention provides an HCY detection reagent, comprising the monoclonal antibody that specifically recognizes HCY.

[0029] An embodiment of the present invention provides an HCY detection kit, comprising the monoclonal antibody that specifically recognizes HCY.

[0030] The reagents used in the embodiments of the present invention are as follows: The extraction kit for the target plasmid is a high-purity plasmid mini-extraction kit (DP107), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the 293F cells are from Xiamen University; the amplification primers are synthesized by Guangzhou Qingke Biotechnology Co., Ltd.; the Escherichia coli DH5a competent strain is purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the LB liquid culture medium reagent is purchased from Sigma; the molecular amplification reagent and cloning ligation kit are purchased from Takara; the 96-well plate for PCR is purchased from Axygen; the fresh culture medium is OPM 293 CD05 culture medium, purchased from Shanghai Aopuma Biotechnology Co., Ltd.; the feed culture medium is OPM 293ProFeed culture medium, purchased from Shanghai Aopuma Biotechnology Co., Ltd.; the Opti-MEM culture medium is purchased from Thermo Fisher Scientific (China) Co., Ltd.; the nickel column, protein G The column was purchased from Huiyan Biotechnology Co., Ltd.; the lymphocyte separation fluid was purchased from Dayu; the SA was purchased from Tiandirenhe Biotechnology Co., Ltd.; the NHS-PEG4-Biotin was purchased from Sigma; the goat anti-mouse IgG-HRP secondary antibody was purchased from Sigma; the Elisa plate and cell plate were purchased from Guangzhou Jiete Biofiltration Co., Ltd.; the eluent and preservation solution reagents were purchased from Sinopharm Group; the analysis methods included SDS-PAGE protein gel electrophoresis, Elisa detection and agarose gel electrophoresis.

[0031] Example 1 Preparation of S1 complete antigen R5 Dissolve 1 eq of Hcy in 0.1 mol / L sodium bicarbonate and stir in an ice-water bath for 15 minutes. Once completely dissolved, add 1.2 eq of di-tert-butyl dicarbonate in DMF and stir in an ice-water bath for 40 minutes. Then, heat to 25°C and react for 18 hours. After the reaction is complete, extract with dichloromethane three times. Separate the aqueous phase and freeze-dry to recover the product, the first intermediate R1 Boc-Hcy.

[0032] Dissolve 5 eq of R1, 1 eq of 2,4,5-triamino-6-chloropyrimidine, and 5 eq of BMPS in DMF. Allow to react at room temperature for 4 h. After the reaction, purify by column chromatography. The product is R2.

[0033] Dissolve 1 eq of R2, 1.5 eq of triethylamine, and 1.5 eq of amino-PEG6-thiol in DMF. Heat at 50°C for 16 h. After the reaction, purify by column chromatography. The product is R3.

[0034] Dissolve 10 mg of R3 in 10 ml of DMF, add 5 mg of silica gel, and stir for 30 minutes. Then, add 50 μL of 10% trifluoroacetic acid dropwise at 30°C. After reacting for 5 minutes, immediately add 2 ml of 0.1 mol / L sodium bicarbonate solution and stir in an ice-water bath for 5 minutes. The product is purified by column chromatography and freeze-dried to obtain hapten R4.

[0035] Dissolve 10 mg of R3 and 4 mg of EMCS in 1 ml of DMF and slowly add dropwise to a 2 mg / ml KLH protein solution at a pH of 7.0-7.2. Add EDTA to a final concentration of 5 mM and stir at room temperature for 4 hours. After the reaction, dialyze into 20 mmol / L phosphate buffer at pH 7.4. This is the complete antigen R5.

[0036] S2 immunized mice The homemade HCY complete antigen R5 was used as the immunogen. After dissolution, it was evenly emulsified with an equal volume of Freund's complete adjuvant (Sigma). 6-8 week old SPF grade Balb / c mice were taken and injected subcutaneously at multiple points with 200 μg / mouse. After an interval of 2 weeks, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points with 100 μg / mouse. The immunization was boosted twice and the shock was performed by intraperitoneal injection 3 days before fusion.

[0037] S3 Preparation of forward and reverse screening samples 1) Dissolve HCY / SAH / Cys in 10 mmol / l Bicine Buffer (pH 8.5) to prepare a solution. 2) 10mM NHS-dPEG4-Biotin Formulation: Weigh 0.56mg NHS-dPEG4-Biotin (molecular weight 587) and dissolve in 95µl ultrapure water. Prepare immediately before use and do not store. Use immediately. NHS-PEG4-Biotin is highly deliquescent, so allow to equilibrate to room temperature before opening. 3) Add the prepared biotin labeling reagent solution to the protein solution, with a molar ratio of protein to biotin labeling reagent of 1:5; 4) After thorough mixing, incubate in a water bath shaker (25°C) for 2-4 hours; 5) Open the cap on the chromatography column and discard the filling solution; 6) Open the cap of the column outlet and elute with PBS several times, collecting approximately 10 ml of eluate from each column to allow the gel to equilibrate; 7) Use a pipette to draw 500 μl of the labeled protein solution into the chromatography column and discard the flow-through; 8) Place a 2 ml sample tube at the outlet of the chromatography column, add 1 ml of PBS to the chromatography column, and collect the biotinylated protein that flows out.

[0038] S4 cell fusion and subclone screening Spleens from immunized mice were harvested and ground to isolate single splenocytes. Splenocytes were then fused with myeloma cells using an electrofusion instrument. After stabilization, the cells were plated in culture medium onto 96-well plates. After one week, the medium was changed and the supernatant was analyzed by ELISA. The cells were coated with SA (Tian Di Ren He) and HCY-Bio was added as a detection source. Goat anti-mouse IgG-HRP (Sigma) was used as an enzyme-labeled secondary antibody to evaluate the cell supernatant. Positive wells were further tested for SAH-Bio and Cys-Bio by ELISA. SA (Tian Di Ren He) was coated with SA (Tian Di Ren He) and SAH-Bio and Cys-Bio were added as counter-detection sources. Goat anti-mouse IgG-HRP (Sigma) was used as an enzyme-labeled secondary antibody to evaluate the cell supernatant. Wells positive for HCY-Bio but negative for SAH-Bio / Cys-Bio were selected and subcloned by limiting dilution. After one week of culture, ELISA analysis was repeated 3-4 times until all wells were positive and single colonies were present. The specific hybridoma cell line 19A6 was then expanded.

[0039] Table 1 Fusion pores detected by forward screening and reverse screening ELISA

[0040] Table 2 Elisa detection results of 19A6 anti-complex antibody activity

[0041] As shown in Table 2, the obtained 19A6 antibody is an HCY-specific antibody.

[0042] Ascites preparation of S5 positive cell lines Single colony cells were expanded and cultured, and then injected into mice that had been pre-immunized with IFA to prepare ascites. The ascites was collected to obtain the complex antibody, which was then affinity purified using a protein G column.

[0043] S6 gene retrieval The 19A6 hybridoma cell line was expanded, mRNA was extracted, and cDNA products were obtained by reverse transcription. The products were subjected to A addition reaction with Taq DNA polymerase and inserted into the pMD-19T vector. The cells were transformed into DH5α competent cells, and 10 plaques each of heavy chain and light chain gene clones were collected and sent to a gene sequencing company for sequencing.

[0044] Sequence analysis of the S7 antibody gene The gene sequences obtained from the above sequencing were placed in the IMGT antibody database for analysis, and snapgene software was used to analyze and determine the correct heavy chain and light chain variable region genes.

[0045] Construction of S8 recombinant antibody expression plasmid Recombinant antibody expression vectors were constructed using pTT5, which already contained a signal peptide and constant region. Based on the antibody variable region gene sequencing results from pMD-19T, specific primer pairs for the light and heavy chains were designed for homologous recombination. The light and heavy chain gene fragments were amplified by PCR. Homologous primers were designed for the pTT5 vector, which already contained a signal peptide and constant region. After PCR amplification, the vector fragments were recovered by electrophoresis. The gene and vector fragments were homologously ligated and transformed into DH5α competent cells. After positive colonies were confirmed by PCR, they were sequenced. Normal colonies were selected for expansion and culture, and the vector plasmids containing the ligated heavy and light chain variable regions were isolated and isolated. These plasmids are referred to as pTT5-19A6 H chain and pTT5-19A6 L chain.

[0046] The primer sequences are as follows: Primer name Primer sequence (5' to 3') 19A6-H-FgggtgcccggatccaccggcGAGGTGCAGCTGCAGG (SEQ ID NO: 11) 19A6-H-RgatgggcccttggtgctagcTGCAGAGACAGTGACCAG (SEQ ID NO: 2) 19A6-L-FgggtgcccggatccaccggcGACATTGTGATGACCCAGTCT (SEQ ID NO: 13) 19A6-L-RgatggtgcagccaccgtacgAGCCCGTTTGATTTCCAG (SEQ ID NO: 4) PTT5-H-Fgctagcaccaagggcccatc (SEQ ID NO: 15) PTT5-H-Rgccggtggatccgggcaccc (SEQ ID NO: 16) PTT5-L-Fcgtacggtggctgcaccatc (SEQ ID NO: 17) PTT5-L-Rgccggtggatccgggcaccc (SEQ ID NO: 18) Note: The lowercase sequence is the homologous connecting part, H is the heavy chain, and L is the light chain S8 recombinant antibody expression The target plasmid obtained in the above steps was transfected into 293F cells by PEI transfection method. After successful transfection, the cells were cultured in an incubator for 24 h, and then fresh OPM-293 CD05 culture medium with the same volume as the culture medium was added. When the cells grew to 4×10 6 When the density reached 1 / mL, 1% by volume of OPM-293 ProFeed feed medium was added every day, and the cells were returned to the incubator at 37°C, 8% CO2, and 120 rpm for further 96 h to obtain a culture fluid of 293F cells expressing the recombinant anti-complex antibody 19A6.

[0047] S9 recombinant antibody purification The 293F cell culture medium obtained in the above step was centrifuged at 9000 rpm for 15 minutes. The supernatant was collected and filtered through a 0.22 μm filter membrane. Protein G was used for protein purification. Before use, the Protein G column was equilibrated with 5 column volumes of equilibration buffer containing 0.02 M PB and 0.15 M NaCl, pH 7.4. The supernatant was passed through the column and then washed with 5 column volumes of equilibration buffer containing 0.02 M PB and 0.15 M NaCl, pH 7.4. The column was then eluted with 5 column volumes of eluent containing 0.1 M Glycine-HCl, pH 2.7. The eluent was immediately neutralized with 1.0 M Tris-HCl, pH 9.0. Finally, the purified 19A6 antibody was dialyzed into PBS, pH 8.0, to obtain the recombinant anti-complex antibody 19A6.

[0048] The results of 19A6 antibody expression and purification are as follows Figure 1 shown.

[0049] After testing, the sequence information of HCY-specific antibody 19A6 is as follows: L-CDR1: KASQDVSTAVA (SEQ ID NO: 1) L-CDR2: SASYRYT (SEQ ID NO: 2) L-CDR3: QQHYSTPWT (SEQ ID NO: 3) H-CDR1: SYTMH (SEQ ID NO: 4) H-CDR2: YINPSSGYTEYNQKFKD (SEQ ID NO: 5) H-CDR3: GAYYGSRFAY (SEQ ID NO: 6) Light chain variable region VL: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKRA (SEQ ID NO: 7) Heavy chain variable region VH: EVQLQESAAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWIGYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLSSLTSEDSAVYYCARGAYYGSRFAYWGQGTLVTVSA (SEQ ID NO: 8) Light chain: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKRART VAAPSVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQID NO: 9) Heavy chain: EVQLQESAAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWIGYINPSSGYTEYNQKFKDKTTLTADKSSSTAYMQLSSSLTSEDSAVYYCARGAYYGSRFAYWGQG TLVTVSAASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQID NO: 10) The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically recognizes HCY, characterized in that: Its light chain CDR1 is shown in SEQ ID NO: 1, CDR2 is shown in SEQ ID NO: 2, and CDR3 is shown in SEQ ID NO: 3; its heavy chain CDR1 is shown in SEQ ID NO: 4, CDR2 is shown in SEQ ID NO: 5, and CDR3 is shown in SEQ ID NO:

6.

2. The monoclonal antibody that specifically recognizes HCY according to claim 1, wherein The light chain variable region is shown in SEQ ID NO: 7; the heavy chain variable region is shown in SEQ ID NO:

8.

3. The monoclonal antibody that specifically recognizes HCY according to claim 1, wherein Its light chain is shown in SEQ ID NO: 9, and its heavy chain is shown in SEQ ID NO:

10.

4. A method for preparing a monoclonal antibody that specifically recognizes HCY according to any one of claims 1 to 3, characterized in that: It was prepared by immunizing animals with HCY complete antigen R5 as immunogen and then screening; Wherein, the structural formula of the HCY complete antigen R5 is: 。 5. The preparation method according to claim 4, characterized in that The carrier protein is any one of hemocyanin, ovalbumin or serum albumin.

6. The preparation method according to claim 4, characterized in that The preparation method of the HCY complete antigen R5 comprises the following steps: S1, using di-tert-butyl dicarbonate to protect the amino group of Hcy to form R1; S2, linking the thiol group of R1 to the amino group of 2,4,5-triamino-6-chloropyrimidine using a first amine-thiol crosslinker to form R2; S3, under alkaline conditions, R2 reacts with amine-PEG-thiol to form R3; S4, using a second amine-thiol crosslinker to couple R3 to a carrier protein, and under acidic conditions, removing the di-tert-butyl dicarbonate protecting group to obtain the homocysteine complete antigen R5.

7. The preparation method according to claim 4, characterized in that The PEG degree of polymerization of the amine-PEG-thiol is 2-24.

8. The preparation method according to claim 4, characterized in that Counter assays for the screen were SAH-Bio and Cys-Bio.

9. An HCY detection reagent, characterized in that The invention comprises the monoclonal antibody specifically recognizing HCY according to any one of claims 1 to 3.

10. An HCY detection kit, characterized in that The invention comprises the monoclonal antibody specifically recognizing HCY according to any one of claims 1 to 3.

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