Synthesis of human homocysteine hapten and preparation method and application of human homocysteine hapten antibody

Abstract

The invention relates to synthesis of a human homocysteine hapten and a preparation method and application of an antibody of the human homocysteine hapten. The synthesis of the human homocysteine hapten comprises the following steps: S1-1, preparing a BSA solution, an EDC solution, an NHS solution and a homocysteine HCY solution; s1-2, adding an NHS solution and an EDC solution into the BSA solution, uniformly mixing, and oscillating and incubating at 37 DEG C to obtain a mixed solution I; s1-3, preparing a PBS (Phosphate Buffer Solution), and dialyzing the mixed solution at 4 DEG C; s1-4, quantifying and fixing the volume of the dialyzed mixed solution I, adding a homocysteine HCY solution, uniformly mixing, and oscillating and incubating at 37 DEG C to obtain a mixed solution II; and S1-5, dialyzing the mixed solution II at 4 DEG C, and obtaining the human homocysteine hapten after dialysis is finished. And preparing an antibody from the human homocysteine hapten and applying the antibody to preparation of a corresponding detection kit. The kit has high sensitivity and high specificity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a synthesis of human homocysteine ​​hapten and a preparation method and application of an antibody thereof. Background Art

[0002] Homocysteine ​​is an intermediate product in the metabolism of methionine. About 70% of homocysteine ​​in plasma is bound to albumin and is in the bound form. The remaining free form mainly exists in the form of disulfide homocysteine ​​and homocysteine-cysteine ​​compounds bound by disulfide bonds. Only a small amount of reduced homocysteine ​​exists in plasma. The determination of HCY has important clinical significance in the determination of cardiovascular diseases, uremia, atherosclerosis, diabetes, Alzheimer's disease and other diseases. It is mainly used in clinical practice for the auxiliary diagnosis of hyperhomocysteinemia and the evaluation of cardiovascular disease risks.

[0003] Chemiluminescent immunoassay (CLIA) has received extensive attention in different fields such as life sciences and clinical diagnosis due to its high sensitivity, good specificity, wide range of applications, simple equipment, and wide linear range. Traditional immunoassays always require a long incubation time, which in turn causes the entire analysis to take several hours to complete, so the output and scope of application are largely limited. Researchers have designed different methods to shorten the analysis time by improving mass transport and reaction kinetics. Immunoassays have expanded the scope of application of CLIA.

[0004] Chinese patent document CN111533802A discloses an artificial complete S-adenosylhomocysteine ​​antigen, a preparation method and an application thereof, comprising the following steps: mixing S-adenosylhomocysteine ​​with di-tert-butyl dicarbonate to obtain a first intermediate, wherein the di-tert-butyl dicarbonate is used to protect the antigenic epitope in S-adenosylhomocysteine; adding a carrier protein to the first intermediate to obtain a second intermediate after reaction; and removing the di-tert-butyl dicarbonate in the second intermediate to obtain the artificial complete S-adenosylhomocysteine ​​antigen.

[0005] At present, homocysteine ​​testing is increasingly used in clinical practice. The raw materials for immunoassay testing on the market are generally imported, expensive, and have long and unstable supply cycles. At the same time, the commonly used methods for clinical testing of homocysteine ​​are high-performance liquid chromatography (HPLC), enzyme cycle method (ECA), etc. The HPLC method has complicated operating steps, a long detection cycle, high requirements for instruments and equipment, and is difficult to popularize. The ECA method is rarely used in China, there are few literature reports, and the detection time is long. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for synthesizing human homocysteine ​​hapten. The prepared human homocysteine ​​hapten can be used as a raw material for homocysteine ​​diagnosis, and has high sensitivity, high specificity, simplicity and rapidity.

[0007] In order to solve the above technical solution, the technical solution of the present invention is: a method for synthesizing the human homocysteine ​​hapten, the specific steps are:

[0008] S1-1: prepare BSA solution, EDC solution, NHS solution and homocysteine ​​HCY solution;

[0009] S1-2: Add NHS solution and EDC solution to BSA solution, mix well and incubate at 37°C with shaking to obtain mixed solution 1;

[0010] S1-3: Prepare a PBS solution, and dialyze the mixed solution obtained in step S1-2 at 4°C;

[0011] S1-4: quantitatively adjust the volume of the dialyzed mixed solution 1, add the homocysteine ​​HCY solution prepared in step S1, mix well, and incubate at 37° C. with shaking to obtain a mixed solution 2;

[0012] S1-5: The mixed solution obtained in step S1-4 is dialyzed at 4°C. After the dialysis is completed, human homocysteine ​​hapten is obtained.

[0013] Preferably, in step S1-1, a PBS solution with a concentration of 0.05 to 0.15 M and a pH of 6.0 is used to prepare a BSA solution with a concentration of 20 to 30 mg / mL; pure water is used to prepare EDC with a concentration of 100 mg / mL; pure water is used to prepare NHS with a concentration of 100 mg / mL; and DMSO is used to prepare a homocysteine ​​HCY solution with a concentration of 100 mg / mL.

[0014] Preferably, in step S1-2, 1 mL of BSA solution is measured, and 0.1 mL of NHS solution and 0.1 mL of EDC solution are added, mixed and incubated at 37°C for 15 min to obtain a mixed solution 1; in step S1-4, a certain amount of the mixed solution after dialyzation in step S1-3 is adjusted to a BSA solution concentration of 16 mg / mL, and then 1 mL of BSA solution with a concentration of 16 mg / mL is taken and added to 0.1 mL of homocysteine ​​HCY solution (100 mg / mL) prepared in step S1-1, mixed and incubated at 37°C for 2 h.

[0015] Preferably, in both step S1-3 and step S1-5, a PBS solution with a concentration of 10 mM and a pH of 7.4 is used for dialysis, the dialysis time is 1 hour, and the dialysis ratio is ≥500.

[0016] The technical problem that the present invention also solves is to provide a human homocysteine ​​antibody prepared using human homocysteine ​​hapten, which can be used for the detection of homocysteine ​​and has high sensitivity, high specificity, simplicity and rapidity.

[0017] In order to solve the above technical solution, the technical solution of the present invention is: the human homocysteine ​​antibody comprises a heavy chain and a light chain, wherein the sequence of the heavy chain is shown as SEQ ID NO.1, and the sequence of the light chain is shown as SEQ ID NO.2;

[0018] Wherein, SEQ ID NO.1 is:

[0019] TCTVSGFSFINYGVHWIRQSPGQGLEWLGVIWNSGKTDYNAAFISRLSLTKDN

[0020] SKSQVFFLMNSLQPDDTAIYYCARNPYAPFDYWGQGTTITVSSAKTTAPSVYP

[0021] IAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTL

[0022] SSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLG

[0023] GPSVFIFPPKIKDVLMLSLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT

[0024] QTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS

[0025] VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDLYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS;

[0026] SEQ ID NO.2 is:

[0027] LSVTPGDRVSLSCRASQNINNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGS

[0028] GSGTDFTLSLNSVETEDFGMYFCQQSNSWWTTFGGGTKVEIKADAAPTVSIFPP

[0029] SSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP.

[0030] The technical problem that the present invention also solves is to provide a method for preparing human homocysteine ​​antibodies using human homocysteine ​​hapten, which can be used for the detection of homocysteine ​​and has high sensitivity, high specificity, and is simple and quick.

[0031] In order to solve the above technical solution, the technical solution of the present invention is: the method for preparing human homocysteine ​​antibody using human homocysteine ​​hapten, the specific steps are:

[0032] S2-1 immunization: Human homocysteine ​​hapten was emulsified with Freund's complete adjuvant, mixed in proportion, and then immunized into 4-6 week old female Balb / c mice;

[0033] S2-2 cell fusion: Aseptically prepare a suspension of mouse spleen cells that meet the immune standard, then mix the mouse spleen cell suspension with mouse myeloma cells sp2 / 0 in proportion, centrifuge, add serum-free DMEM solution, mix and centrifuge, add serum, blow up the cells, add thymus cells mixed with 10xHAT, and mix again; then evenly distribute them into multiple 96-well plates with feeder cells, and culture them in an incubator at a temperature of 37°C and filled with 5% CO2;

[0034] S2-3 ELISA screening of positive hybridoma cells: After 7 days of culture in the incubator, the HAT medium was replaced with HT medium. One day later, 100 μL of supernatant protein was taken for ELISA screening;

[0035] S2-4 subcloning: The positive clones screened out were subcloned using the gradient dilution method until a homozygous positive cell line was screened out;

[0036] S2-5 Ascites preparation: Logarithmic growth phase cells were washed and suspended in serum-free medium and counted for about 10 61 mL of the suspended cells was intraperitoneally injected into mice sensitized with paraffin oil in advance; the ascites was collected 7 days later; the ascites was centrifuged at 4000 rpm for 10 min at 4°C; the ascites in the middle was carefully aspirated and collected in a centrifuge tube and stored at 4°C or -20°C;

[0037] Purification of S2-6 monoclonal antibody: The antibody was purified from ascites using HiTrap rProtein A FF affinity chromatography; the purified antibody was stored at -20°C;

[0038] S2-7 Obtained antibody sequence: The HCY antibody sequence was obtained by cell line sequencing.

[0039] Preferably, in the step S2-1, human homocysteine ​​hapten and Freund's complete adjuvant are mixed and emulsified in a ratio of 1:1, and then 4-6 week old female Balb / c mice are immunized, wherein the specific steps of immunizing mice are: subcutaneous injection is performed on the abdomen of the mice, 5 points per mouse, and the dose is 100 μg / mouse; boost immunization is performed once every 14 days, and the human homocysteine ​​hapten is emulsified with Freund's incomplete adjuvant, and the dose is 50 μg / mouse; 7 days after the third boost immunization, the polyantibody titer against the immunogen in the mouse serum is detected by indirect ELISA (wavelength 450nm), and the mouse with the highest titer is immunized by tail vein injection, and the antigen is mixed with physiological saline, and the dose is 50 μg / mouse.

[0040] Preferably, the specific steps of step S2-2 are:

[0041] S2-21: First prepare the mouse spleen cell suspension that has reached the immune standard, then mix the mouse spleen cell suspension with mouse myeloma cells at a ratio of 5:1, and then centrifuge at 1200 rpm for 5 minutes; discard the supernatant;

[0042] S2-22: Place the centrifuge tube in a 37°C water bath, slowly add 1 mL of PEG1500 within 1 minute, and stir the cells; after standing in warm water for 1 minute, add 10 mL of serum-free DMEM solution, mix well, and centrifuge at 1000 rpm for 5 minutes; discard the supernatant;

[0043] S2-23: Add 10 mL of serum to the centrifuge tube, carefully pipette the cells up, and add 5 mL of thymocytes mixed with 10xHAT and mix well;

[0044] S2-24: The solution was then evenly distributed into 10 96-well plates containing feeder cells and cultured in an incubator at 37°C filled with 5% CO2.

[0045] Preferably, the step S2-6 further comprises using SDS-PAGE gel to identify the purity of the purified antibody, and then using OD value / 1.4 to determine the antibody concentration.

[0046] The technical problem that the present invention also solves is to provide an application of a human homocysteine ​​hapten and an antibody thereof, which can be used for the detection of homocysteine ​​and has high sensitivity, high specificity, simplicity and rapidity.

[0047] In order to solve the above technical solution, the technical solution of the present invention is: application of the human homocysteine ​​hapten and the antibody thereof in a human homocysteine ​​detection kit.

[0048] Compared with the prior art, the present invention has the beneficial effects that the human homocysteine ​​hapten and the antibody prepared by the present invention can be used as raw materials for homocysteine ​​diagnosis, and the obtained raw materials are applied to chemiluminescence detection technology and can be used for the detection of homocysteine, which has the advantages of high sensitivity, high specificity, simplicity and speed, etc. DETAILED DESCRIPTION

[0049] In order to deepen the understanding of the present invention, the present invention will be further described in detail with reference to the embodiments below. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0050] Embodiment: The method for synthesizing the human homocysteine ​​hapten comprises the following specific steps:

[0051] S1-1: preparing BSA solution, EDC solution, NHS solution and homocysteine ​​HCY solution; in the step S1-1, a PBS solution with a concentration of 0.05-0.15M (a PBS solution with a concentration of 0.1M in this embodiment) and a pH of 6.0 is used to prepare the BSA solution into a 20-30mg / mL BSA solution (20mg / mL BSA in this embodiment); EDC is prepared into an EDC solution with a concentration of 100mg / mL using pure water; NHS is prepared into an NHS solution with a concentration of 100mg / mL using pure water; and a homocysteine ​​HCY solution with a concentration of 100mg / mL is prepared using DMSO;

[0052] S1-2: adding NHS solution and EDC solution to the BSA solution, mixing and incubating at 37° C. with shaking to obtain a mixed solution 1; measuring 1 mL of the BSA solution in step S1-2, adding 0.1 mL of the NHS solution and 0.1 mL of the EDC solution, mixing and incubating at 37° C. with shaking for 15 min to obtain a mixed solution 1;

[0053] S1-3: Prepare a PBS solution, and dialyze the mixed solution obtained in step S1-2 at 4°C (3°C to 5°C is also acceptable); use a PBS solution with a concentration of 10 mM and a pH of 7.4 for 1 hour, and a dialysis ratio of ≥500;

[0054] S1-4: the dialyzed mixed solution 1 is quantitatively adjusted to a fixed volume, the homocysteine ​​HCY solution prepared in step S1 is added, and the mixture is shaken and incubated at 37°C (36°C to 38°C is also acceptable) after mixing, to obtain a mixed solution 2; in the step S1-4, the dialyzed mixed solution in step S1-3 is adjusted to a certain volume so that the concentration of the BSA solution is 16 mg / mL, and then 1 mL of the BSA solution with a concentration of 16 mg / mL is taken and added to 0.1 mL of the homocysteine ​​HCY solution (100 mg / mL) prepared in step S1-1, and the mixture is shaken and incubated at 37°C for 2 h after mixing;

[0055] S1-5: The mixed solution obtained in step S1-4 is dialyzed at 4°C (3°C to 5°C is also acceptable) using a 10 mM PBS solution with a pH of 7.4. The dialysis time is 1 h, and the dialysis ratio is ≥500. After the dialysis is completed, human homocysteine ​​hapten is obtained.

[0056] The human homocysteine ​​antibody comprises a heavy chain and a light chain, wherein the sequence of the heavy chain is shown in SEQ ID NO.1, and the sequence of the light chain is shown in SEQ ID NO.2;

[0057] SEQ ID NO.1 is:

[0058] TCTVSGFSFINYGVHWIRQSPGQGLEWLGVIWNSGKTDYNAAFISRLSLTKDN

[0059] SKSQVFFLMNSLQPDDTAIYYCARNPYAPFDYWGQGTTITVSSAKTTAPSVYP

[0060] IAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTL

[0061] SSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLG

[0062] GPSVFIFPPKIKDVLMLSLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT

[0063] QTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS

[0064] VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDLYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS;

[0065] SEQ ID NO.2 is:

[0066] LSVTPGDRVSLSCRASQNINNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGS

[0067] GSGTDFTLSLNSVETEDFGMYFCQQSNSWWTTFGGGTKVEIKADAAPTVSIFPP

[0068] SSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP.

[0069] The method for preparing human homocysteine ​​antibody by using human homocysteine ​​hapten comprises the following specific steps:

[0070] S2-1 immunization: Human homocysteine ​​hapten was emulsified with Freund's complete adjuvant (Sigma), mixed in proportion, and then immunized into 4-6 week old female Balb / c mice;

[0071] In the step S2-1, human homocysteine ​​hapten and Freund's complete adjuvant are mixed and emulsified at a ratio of 1:1, and then female Balb / c mice aged 4-6 weeks are immunized, wherein the specific steps of immunizing the mice are: subcutaneous injection is performed on the abdomen of the mice, 5 points per mouse, and the dose is 100 μg / mouse; boost immunization is performed once every 14 days, and the human homocysteine ​​hapten is emulsified with Freund's incomplete adjuvant (Sigma) at a dose of 50 μg / mouse; 7 days after the third boost immunization, the polyantibody titer against the immunogen in the mouse serum is detected by indirect ELISA (wavelength 450nm), and the mouse with the highest titer is immunized by tail vein injection, and the antigen is mixed with physiological saline at a dose of 50 μg / mouse;

[0072] S2-2 cell fusion: Aseptically prepare a suspension of mouse spleen cells that meet the immune standard, then mix the mouse spleen cell suspension with mouse myeloma cells sp2 / 0 in proportion, centrifuge, add serum-free DMEM solution, mix and centrifuge, add serum, blow up the cells, add thymus cells mixed with 10xHAT, and mix again; then evenly distribute them into multiple 96-well plates with feeder cells, and culture them in an incubator at a temperature of 37°C and filled with 5% CO2;

[0073] The specific steps of step S2-2 are:

[0074] S2-21: First prepare the mouse spleen cell suspension that has reached the immune standard, then mix the mouse spleen cell suspension with mouse myeloma cells at a ratio of 5:1, and then centrifuge at 1200 rpm for 5 minutes; discard the supernatant;

[0075] S2-22: Place the centrifuge tube in a 37°C water bath, slowly add 1 mL of PEG1500 (Sigma) within 1 minute, and stir the cells; after standing in warm water for 1 minute, add 10 mL of serum-free DMEM solution (Sigma), mix well, and centrifuge at 1000 rpm for 5 minutes; discard the supernatant;

[0076] S2-23: Add 10 mL of serum to the centrifuge tube, carefully blow up the cells, and add 5 mL of thymocytes mixed with 10xHAT (Sigma) and mix well;

[0077] S2-24: Then evenly dispense into 10 96-well plates with feeder cells and culture in an incubator at 37°C filled with 5% CO2;

[0078] S2-3 ELISA screening of positive hybridoma cells: After 7 days of culture in the incubator, the HAT medium was replaced with HT medium. One day later, 100 μL of supernatant protein was taken for ELISA screening;

[0079] S2-4 subcloning: The positive clones screened out were subcloned using the gradient dilution method until a homozygous positive cell line was screened out;

[0080] S2-5 Ascites preparation: Logarithmic growth phase cells were washed and suspended in serum-free medium and counted for about 10 6 1 mL of the suspended cells was intraperitoneally injected into mice sensitized with paraffin oil in advance; the ascites was collected 7 days later; the ascites was centrifuged at 4000 rpm for 10 min at 4°C; the ascites in the middle was carefully aspirated and collected in a centrifuge tube and stored at 4°C or -20°C;

[0081] Purification of S2-6 monoclonal antibody: The antibody was purified from ascites using HiTrap rProtein AFF (GE) affinity chromatography; the purified antibody was stored at -20°C;

[0082] The step S2-6 also includes using SDS-PAGE gel to identify the purity of the purified antibody, and then using OD value / 1.4 to determine the antibody concentration;

[0083] S2-7 Obtained antibody sequence: The HCY antibody sequence was obtained by cell line sequencing.

[0084] The human homocysteine ​​hapten and the antibody thereof are used in a human homocysteine ​​detection kit.

[0085] (1) Preparation of a human homocysteine ​​detection kit, specifically:

[0086] The human homocysteine ​​detection kit comprises a magnetic bead-SA coating (reagent R1), an acridinium ester-HCY antibody marker (reagent R3), an HCY-BSA-Biotin marker (reagent R2), a sample processing solution 1 and a sample processing solution 2; the HCY antibody concentration in the acridinium ester-HCY antibody marker is 5ug / L, BSA, a phosphate buffer solution with a pH value of 5.5, etc.; the HCY-BSA Biotin marker has an HCY-BSA concentration of 30ug / L, a Biotin concentration of 3ug / L, a phosphate buffer solution with a pH value of 7.4, etc.; the magnetic bead-SA coating is 1mg / ml; the sample processing solution 1 contains 1g / L of DTT (reagent R4) and a pH value of 5.5; the sample processing solution 2 contains 40g / L of sodium hydroxide, etc.

[0087] (2) Preparation of HCY detection kit: Prepare reagent R1, reagent R2, reagent R3 and reagent R4 according to Table 1 below.

[0088] Table 1 Formulas for preparing reagent R1, reagent R2, reagent R3 and reagent R4

[0089]

[0090] (3) The preparation formula of the calibration dilution solution is shown in Table 2.

[0091] Table 2 Preparation formula of calibrator dilution solution

[0092] Reagent name Calibrator diluent Trehalose 50g / L BSA 40g / L Mannitol 100g / L Tween-20 3mL / L PC-300 1g / L Purified water Make up to 1L

[0093] (3) The HCY detection kit in the embodiment was compared with the kit of Abbott Laboratories, and the measurement results are shown in Table 3 below.

[0094] Table 3. Comparison of test results with Abbott's test kit

[0095]

[0096] As can be seen from Table 3 above, the test results of the test kit prepared with self-produced raw materials are consistent with those of the control manufacturer (Abbott).

[0097] For ordinary technicians in this field, the embodiments are only illustrative descriptions of the present invention. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned methods. As long as various non-substantial improvements are made using the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. A method for synthesizing human homocysteine ​​hapten, characterized in that: The specific steps are: S1-1: prepare BSA solution, EDC solution, NHS solution and homocysteine ​​HCY solution; S1-2: Add NHS solution and EDC solution to BSA solution, mix well and incubate at 37°C with shaking to obtain mixed solution 1; S1-3: preparing a PBS solution, and dialyzing the mixed solution obtained in step S1-2; S1-4: quantitatively adjust the volume of the dialyzed mixed solution 1, add the homocysteine ​​HCY solution prepared in step S1, mix well, and incubate with shaking to obtain a mixed solution 2; S1-5: dialyze the mixed solution 2 obtained in step S1-4. After the dialysis is completed, human homocysteine ​​hapten is obtained.

2. The method for synthesizing human homocysteine ​​hapten according to claim 1, characterized in that: In the step S1-1, a PBS solution with a concentration of 0.05 to 0.15 M and a pH of 6.0 is used to prepare a BSA solution with a concentration of 20 to 30 mg / mL; EDC is prepared with pure water to prepare an EDC solution with a concentration of 100 mg / mL; NHS is prepared with pure water to prepare an NHS solution with a concentration of 100 mg / mL; and DMSO is prepared to prepare a homocysteine ​​HCY solution with a concentration of 100 mg / mL.

3. The method for synthesizing human homocysteine ​​hapten according to claim 1, characterized in that: In the step S1-2, 1 mL of BSA solution was measured, and 0.1 mL of NHS solution and 0.1 mL of EDC solution were added, mixed, and incubated at 37° C. for 15 min to obtain a mixed solution 1; in the step S1-4, a certain amount of the mixed solution after dialyzed in step S1-3 was adjusted to a BSA solution concentration of 16 mg / ml, and then 1 mL of BSA solution with a concentration of 16 mg / mL was taken and added to 0.1 mL of homocysteine ​​HCY solution prepared in step S1-1, mixed, and incubated at 37° C. for 2 h.

4. The method for synthesizing human homocysteine ​​hapten according to claim 1, characterized in that: In both step S1-3 and step S1-5, dialysis is performed using a PBS solution with a concentration of 10 mM and a pH of 7.

4. The dialysis time is 1 hour, and the dialysis ratio is ≥500.

5. A human homocysteine ​​antibody prepared using human homocysteine ​​hapten, characterized in that: The human homocysteine ​​antibody comprises a heavy chain and a light chain, wherein the sequence of the heavy chain is shown as SEQ ID NO.1, and the sequence of the light chain is shown as SEQ ID NO.2; Wherein, SEQ ID NO.1 is: TCTVSGFSFINYGVHWIRQSPGQGLEWLGVIWNSGKTDYNAAFISRLSLTKDNSKSQVFFLMNSLQPDDTAIYYCARNPYAPFDYWGQGTTITVSSAKTTA PSVYPIAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPPKIKDVLMLSLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN KDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDLYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS; SEQ ID NO.2 is: LSVTPGDRVSLSCRASQNINNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSLNSVETEDFGMYFCQQSNSWWTTFGGGTKVEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP.

6. A method for preparing human homocysteine ​​antibodies using human homocysteine ​​hapten, characterized in that: The specific steps are: S2-1 immunization: Human homocysteine ​​hapten was emulsified with Freund's complete adjuvant, mixed in proportion, and then immunized into 4-6 week old female Balb / c mice; S2-2 cell fusion: Aseptically prepare a suspension of mouse spleen cells that meet the immune standard, then mix the mouse spleen cell suspension with mouse myeloma cells sp2 / 0 in proportion, centrifuge, add serum-free DMEM solution, mix and centrifuge, add serum, blow up the cells, add thymus cells mixed with 10xHAT, and mix again; then evenly distribute them into multiple 96-well plates with feeder cells, and culture them in an incubator at a temperature of 37°C and filled with 5% CO2; S2-3 ELISA screening of positive hybridoma cells: After 7 days of culture in the incubator, the HAT medium was replaced with HT medium. One day later, 100 μL of supernatant protein was taken for ELISA screening; S2-4 subcloning: The positive clones screened out were subcloned using the gradient dilution method until a homozygous positive cell line was screened out; S2-5 Ascites preparation: Logarithmic growth phase cells were washed and suspended in serum-free medium and counted for 10 6 1 mL of the suspended cells was intraperitoneally injected into mice sensitized with paraffin oil in advance; the ascites was collected 7 days later; the ascites was centrifuged at 4000 rpm for 10 min at 4°C; the ascites in the middle was aspirated and collected in a centrifuge tube and stored at 4°C or -20°C; Purification of S2-6 monoclonal antibody: The antibody was purified from ascites using HiTrap rProtein AFF affinity chromatography; the purified antibody was stored at -20°C; S2-7 Obtained antibody sequence: The HCY antibody sequence was obtained by cell line sequencing.

7. The method for preparing human homocysteine ​​antibody using human homocysteine ​​hapten according to claim 6, characterized in that: In the step S2-1, human homocysteine ​​hapten and Freund's complete adjuvant are mixed and emulsified in a ratio of 1:1, and then 4-6 week old female Balb / c mice are immunized, wherein the specific steps of immunizing the mice are: subcutaneous injection is performed on the abdomen of the mice, 5 points per mouse, and the dose is 100 μg / mouse; boost immunization is performed once every 14 days, and the human homocysteine ​​hapten is emulsified with Freund's incomplete adjuvant, and the dose is 50 μg / mouse; 7 days after the third boost immunization, the polyantibody titer against the immunogen in the mouse serum is detected by indirect ELISA, and the mouse with the highest titer is immunized by tail vein injection, and the antigen is mixed with physiological saline, and the dose is 50 μg / mouse.

8. The method for preparing human homocysteine ​​antibody using human homocysteine ​​hapten according to claim 6, characterized in that: The specific steps of step S2-2 are: S2-21: First prepare the mouse spleen cell suspension that has reached the immune standard, then mix the mouse spleen cell suspension with mouse myeloma cells at a ratio of 5:1, and then centrifuge at 1200 rpm for 5 minutes; discard the supernatant; S2-22: Place the centrifuge tube in a 37°C water bath, slowly add 1 mL of PEG1500 within 1 minute, and stir the cells; after standing in warm water for 1 minute, add 10 mL of serum-free DMEM solution, mix well, and centrifuge at 1000 rpm for 5 minutes; discard the supernatant; S2-23: Add 10 mL of serum to the centrifuge tube, pipette the cells up, and add 5 mL of thymocytes mixed with 10xHAT and mix well; S2-24: The solution was then evenly distributed into 10 96-well plates containing feeder cells and cultured in an incubator at 37°C filled with 5% CO2.

9. The method for preparing human homocysteine ​​antibody using human homocysteine ​​hapten according to claim 7, characterized in that: The step S2-6 also includes using SDS-PAGE gel to identify the purity of the purified antibody, and then using OD value / 1.4 to determine the antibody concentration.

10. A use of a human homocysteine ​​hapten and an antibody thereof, characterized in that: The human homocysteine ​​hapten and the antibody thereof are used in a human homocysteine ​​detection kit.

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