Homocysteine ​​detection kit

Abstract

The present invention relates to a homocysteine ​​detection kit, comprising reagent R1, reagent R2, reagent R3 and reagent R4, wherein the reagent R1 comprises magnetic particles coated with S-adenosyl-L-homocysteine ​​antibodies and a basic buffer solution one; the reagent R2 comprises recombinant S-adenosyl-L-homocysteine ​​hydrolase and a basic buffer solution two; the reagent R3 comprises S-adenosyl-L-cysteine ​​conjugates and a basic buffer solution three labeled with a detectable marker; the reagent R4 comprises dithiothreitol and a basic buffer solution four; and further comprises a calibration product, the calibration product comprising an HCY antigen and a calibration product diluent, the calibration product diluent comprising a pH regulator, an inorganic salt ion, a surfactant and a stabilizer, and the stabilizer comprises bovine serum albumin. The homocysteine ​​detection kit has good stability, is easy to use and store, and can improve the accuracy of the test result.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro detection, and particularly to a homocysteine ​​detection kit. Background Art

[0002] Homocysteine ​​(HCY) is a sulfur-containing amino acid generated by the demethylation of methionine in cells. When circulating extracorporeally, most homocysteine ​​exists in an oxidized form bound to plasma proteins, that is, it is bound to albumin in plasma through a disulfide bond to form protein-HCY. Only a small amount of reduced homocysteine ​​and disulfide homocysteine ​​(HCY-SS-HCY) exist in the blood. Total homocysteine ​​refers to all types of homocysteine ​​(free HCY and albumin-bound) in serum or plasma.

[0003] Extremely elevated total homocysteine ​​concentrations occur in homocystinuria, a rare inherited defect in the enzyme that metabolizes homocysteine. Symptoms of homocystinuria include intellectual disability, early atherosclerosis, and arteriovenous thrombosis. In addition, some less severe inherited defects can cause moderate elevations in total homocysteine ​​levels. Studies investigating the relationship between elevated homocysteine ​​levels and cardiovascular disease (CVD) have shown that homocysteine ​​is an important marker in risk assessment. In the presence of known coronary artery disease (CAD), homocysteine ​​has been shown to be an independent marker for CAD-related mortality. In patients at intermediate risk, elevated homocysteine ​​levels correlate with the degree of coronary artery calcification. Elevated homocysteine ​​levels in these patients are an independent risk factor for coronary heart disease (CHD).

[0004] At present, HCY is mostly detected by biochemical enzyme cycle method. However, this method has shortcomings: low sensitivity, easy to be interfered, and unable to meet the requirements of accurate detection of large number of clinical samples.

[0005] Chemiluminescent immunoassay is a non-radioactive immunoassay technology that has developed rapidly in the world in recent years. The detection principle is based on luminescent substances. This method has high sensitivity and a wide detection range, and is an important development direction of immunological detection.

[0006] Chinese patent document (CN 114813712 A) discloses a homocysteine ​​detection kit and a homocysteine ​​detection method, wherein the homocysteine ​​detection kit comprises a first reagent, a second reagent, a third reagent and a fourth reagent, wherein the first reagent is a magnetic particle working solution coated with SAH antibody, the second reagent is an enzyme-labeled working solution of a SAH derivative labeled with alkaline phosphatase, the third reagent is a reducing agent buffer, the reducing agent is at least one of DTT, TCEP and β-mercaptoethanol, and the fourth reagent is a buffer of SAHH and Ado. However, the stability of the homocysteine ​​detection kit in this technical solution is still not high.

[0007] Chinese patent document (CN 109001462 A) discloses a homocysteine ​​detection kit, including reagent R1 and reagent R2, wherein the components of reagent R1 are serine 0.5-20mmol / L, HCY reducing agent 0.5-10mmol / L, NADH 0.5-1.8g / L, LDH 10-200KU / L, and buffer 20-100mmol / L; the components of reagent R2 are cystathionine β-synthase 0.5-10KU / L, cystathionine β-degradase 10-50KU / L, and buffer 20-100mmol / L. The sensitivity and stability of the homocysteine ​​detection kit in this technical solution are still not high.

[0008] Therefore, a chemiluminescent immunoassay kit suitable for HCY detection with high sensitivity, high accuracy and high stability is still a problem that needs to be solved. Summary of the invention

[0009] The purpose of the present invention is to provide a homocysteine ​​detection kit with high stability, high sensitivity and high accuracy.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is that the homocysteine ​​detection kit comprises reagent R1, reagent R2, reagent R3 and reagent R4, wherein the reagent R1 comprises magnetic particles coated with S-adenosine-L-homocysteine ​​antibody and basic buffer one; the reagent R2 comprises recombinant S-adenosine-L-homocysteine ​​hydrolase (SAHH) and basic buffer two; the reagent R3 comprises S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker and basic buffer three; and the reagent R4 comprises dithiothreitol (DTT) and basic buffer four.

[0011] By adopting the above technical solution, the defoaming agent propylene glycol is contained in the reagent R1 to prevent bubbles from appearing when mixing with the magnetic particles and affecting the sample addition, thereby improving the stability of the detection system; in addition, the homocysteine ​​detection kit is used for HCY detection with high sensitivity and accuracy.

[0012] Preferably, the basic buffer 1, basic buffer 2, basic buffer 3 and basic buffer 4 all include a pH regulator, inorganic salt ions and a surfactant, and the pH value is a mixture of one or more of phosphate buffer, citric acid buffer, MES buffer, Tris buffer and HEPES buffer; the inorganic salt ions are sodium chloride and / or potassium chloride; the surfactant is a mixture of one or more of Tween, SPAN, Triton and EMULGEN series surfactants. The amount of the pH regulator can be selected by yourself; the inorganic salt ion refers to a type of reagent that maintains the ion concentration of the solution in a relatively stable equilibrium state. For the type of inorganic salt ions, the present invention is selected from one or more of sodium chloride and potassium chloride; for the amount of inorganic salt ions, it can be selected by yourself; the pH regulator refers to a reagent that keeps the pH value of the solution relatively stable; the surfactant refers to a substance that can significantly change the interface state of its solution system by adding a small amount, which has fixed hydrophilic and lipophilic groups and can be arranged in a directional manner on the surface of the solution; the amount of the surfactant can be selected by yourself.

[0013] Preferably, the basic buffer 1, basic buffer 2 and basic buffer 3 further include a stabilizer, the stabilizer includes bovine serum albumin, and the content of the bovine serum albumin is 10-20 g / L; the basic buffer 1 also includes a defoamer, and the defoamer is glycerol. The defoamer glycerol is contained in the reagent R1 to prevent bubbles from appearing when mixing with the magnetic particles and affecting the sample addition, thereby improving the stability of the detection system; in addition, the homocysteine ​​detection kit has high stability, high sensitivity and high accuracy.

[0014] Preferably, the amount of the magnetic particles coated with S-adenosine-L-homocysteine ​​antibody in the reagent R1 is 0.01-2.5 mg / mL; the S-adenosine-L-homocysteine ​​antibody is an immunoglobulin molecule and a fragment thereof that specifically binds to S-adenosine-L-homocysteine.

[0015] Preferably, the concentration of the recombinant S-adenosyl-L-homocysteine ​​hydrolase (SAHH) in the reagent R2 is 0.1-50 μg / mL.

[0016] Preferably, the detectable marker-labeled S-adenosyl-L-cysteine ​​conjugate in the reagent R3 refers to an acridinium ester-labeled S-adenosyl-L-cysteine ​​conjugate with a concentration of 0.2 to 2 μg / mL. The "detectable marker" is an "acridine chemiluminescent substance"; "acridine chemiluminescent substance" refers to acridinium esters and their derivatives, acridinium sulfonamide chemiluminescent substances and lucigenin, which are suitable for chemiluminescent immunoassay systems. In an alkaline H2O2 solution, when the molecule is attacked by hydrogen peroxide ions, unstable ethylene dioxide is generated, which decomposes into CO2 and electronically excited N-methylacridone, which emits photons when it returns to the ground state. In the present invention, the "detectable marker" is an acridinium ester derivative.

[0017] Preferably, the concentration of dithiothreitol (DTT) in the reagent R4 is 0.01-100 mg / mL.

[0018] Preferably, the S-adenosyl-L-homocysteine ​​antibody in the reagent R1 includes chimeric antibodies, humanized antibodies, fully human antibodies, CDR-grafted antibodies, antibody constructs, and recombinant or synthetic antibodies.

[0019] Preferably, the concentration of the recombinant S-adenosyl-L-homocysteine ​​hydrolase (SAHH) in the reagent R2 is 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, or 50 μg / mL.

[0020] Preferably, the detectable marker in the S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker in the reagent R3 is an acridinium ester derivative; the S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker is an acridinium ester-labeled S-adenosine-L-cysteine ​​conjugate, and its usage concentration is 0.2 μg / mL or 0.25 μg / mL or 0.5 μg / mL or 1 μg / mL or 2 μg / mL.

[0021] Preferably, the concentration of dithiothreitol (DTT) in the reagent R4 is 0.01 mg / mL or 0.1 mg / mL or 0.5 mg / mL or 1 mg / mL or 10 mg / mL or 100 mg / mL.

[0022] Preferably, when the pH value is phosphate buffer, its concentration is 0.1-10 g / L; when the inorganic salt ion is sodium chloride, its concentration is 2-30 g / L; when the surfactant is Tween 20 or Triton 100, its concentration is 0.05-20 mL / L.

[0023] Preferably, the homocysteine ​​detection kit further comprises a calibrator, the calibrator comprises an HCY antigen and a calibrator diluent, the calibrator diluent comprises a pH regulator, an inorganic salt ion, a surfactant, a stabilizer and bovine serum albumin; the content of the bovine serum albumin is 10 to 100 g / L. Adding bovine serum albumin in the above concentration range to the calibrator diluent can not only improve the stability of the calibrator, but also improve the specificity of the calibration curve, thereby improving the accuracy of the test results.

[0024] Preferably, the content of bovine serum albumin is 10-70 g / L or 20-60 g / L or 30-50 g / L. After a large number of experiments, it is found that adding bovine serum albumin in the above concentration range to the calibration diluent can not only improve the stability of the main calibrator, but also improve the specificity of the calibration curve, thereby improving the accuracy of the test results.

[0025] Preferably, the concentrations of HCY antigens in the master calibrators are: Cal A: 0-0.1 μmol / L; Cal B: 1.0-1.5 μmol / L; Cal C: 4.0-5.0 μmol / L; Cal D: 7.5-8.5 μmol / L; Cal E: 20.0-30.0 μmol / L.

[0026] Compared with the prior art, the present invention has the following beneficial effects: the homocysteine ​​test kit contains a defoaming agent in the reagent R1 to prevent bubbles from appearing when mixing with magnetic particles and affecting sample addition, thereby improving the stability of the detection system; bovine serum albumin is added to the calibration diluent, which has good stability, is easy to use and store, and can improve the accuracy of the detection results. In addition, combined with the method of the present invention, the detection sensitivity and accuracy can be further improved. DETAILED DESCRIPTION

[0027] Embodiment: The homocysteine ​​detection kit (abbreviated as: HCY detection kit) comprises reagent R1, reagent R2, reagent R3 and reagent R4, wherein the reagent R1 comprises magnetic particles coated with S-adenosyl-L-homocysteine ​​antibody and basic buffer 1; the reagent R2 comprises recombinant S-adenosyl-L-homocysteine ​​hydrolase (SAHH) and basic buffer 2; the reagent R3 comprises S-adenosyl-L-cysteine ​​conjugate labeled with a detectable marker and basic buffer 3; the reagent R4 comprises dithiothreitol (DTT) and basic buffer 4; the reagent R1 comprises The defoaming agent glycerol prevents bubbles from appearing when mixing with magnetic particles and affecting sample addition, thereby improving the stability of the detection system; the basic buffer solution 1, basic buffer solution 2, basic buffer solution 3 and basic buffer solution 4 all include a pH regulator, inorganic salt ions and a surfactant, and the pH value is a mixture of one or more of phosphate buffer, citric acid buffer, MES buffer, Tris buffer and HEPES buffer; the inorganic salt ions are sodium chloride and / or potassium chloride; the surfactant is a mixture of one or more of Tween, SPAN, Triton and EMULGEN series surfactants. The amount of pH regulator can be selected by yourself; inorganic salt ions refer to a type of reagent that maintains the ion concentration of the solution in a relatively stable equilibrium state.The type of inorganic salt ions is selected from one or more of sodium chloride and potassium chloride; the dosage of inorganic salt ions can be selected by oneself; pH regulator refers to a reagent that keeps the pH value of the solution relatively stable; surfactant refers to a substance that can significantly change the interface state of its solution system by adding a small amount, which has fixed hydrophilic and lipophilic groups and can be oriented on the surface of the solution; the dosage of surfactant can be selected by oneself; the basic buffer solution 1, basic buffer solution 2 and basic buffer solution 3 also include a stabilizer, which includes bovine serum albumin, and the content of bovine serum albumin is 10-20g / L; the basic buffer solution 1 also includes a defoamer, which is glycerol; the defoamer glycerol is contained in the reagent R1 to prevent bubbles from appearing when mixing with the magnetic particles and affecting the sample addition, thereby improving the stability of the detection system; The dosage of the magnetic particles coated with S-adenosine-L-homocysteine ​​antibody in the reagent R1 is 0.01-2.5 mg / mL; the S-adenosine-L-homocysteine ​​antibody is an immunoglobulin molecule and its fragments that specifically bind to S-adenosine-L-homocysteine; the concentration of the recombinant S-adenosine-L-homocysteine ​​hydrolase (SAHH) in the reagent R2 is 0.1-50 μg / mL; the S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker in the reagent R3 refers to the S-adenosine-L-cysteine ​​conjugate labeled with an acridinium ester, and the dosage concentration is 0.2-2 μg / mL; the "detectable marker" is an "acridine chemiluminescent substance"; the "acridine chemiluminescent substance" refers to acridinium esters and their derivatives, acridinium sulfonamide chemiluminescent substances and lucigenin, which are suitable for chemiluminescent immunoassay systems.In an alkaline H2O2 solution, when the molecule is attacked by hydrogen peroxide ions, unstable ethylene dioxide is generated, which decomposes into CO2 and N-methylacridone in an electronically excited state, and emits photons when it returns to the ground state; the "detectable marker" in the present invention is an acridinium ester derivative; the concentration of dithiothreitol (DTT) in the reagent R4 is 0.01-100 mg / mL; the S-adenosyl-L-homocysteine ​​antibody in the reagent R1 includes chimeric antibodies, humanized antibodies, fully human antibodies, and CDR-transplanted antibodies and antibody constructs and antibody recombinant or synthetic antibodies; the concentration of recombinant S-adenosine-L-homocysteine ​​hydrolase (SAHH) in the reagent R2 is 0.1 μg / mL or 1 μg / mL or 5 μg / mL or 10 μg / mL or 50 μg / mL; the detectable marker in the S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker in the reagent R3 is an acridinium ester derivative; the S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker is an acridinium ester-labeled S-adenosine-L-cysteine ​​conjugate; The cystine conjugate has a dosage concentration of 0.2 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, or 2 μg / mL; the concentration of dithiothreitol (DTT) in the reagent R4 is 0.01 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, or 100 mg / mL; when the pH value is phosphate buffer, its concentration is 0.1 to 10 g / L; when the inorganic salt ion is sodium chloride, its concentration is 2 to 30 g / L. L; when the surfactant is Tween 20 or Triton 100, its concentration is 0.05-20 mL / L; the homocysteine ​​detection kit also includes a calibrator, which includes an HCY antigen and a calibrator diluent, and the calibrator diluent includes a pH regulator, an inorganic salt ion, a surfactant, a stabilizer and bovine serum albumin; the content of the bovine serum albumin is 10-100 g / L; the content of the bovine serum albumin is 10-70 g / L or 20-60 g / L or 30-50 g / L. After a large number of experiments, it was found that adding bovine serum albumin in the above concentration range to the calibration diluent can not only improve the stability of the calibration, but also improve the specificity of the calibration curve, thereby improving the accuracy of the test results; the concentrations of HCY antigens in the main calibrators are: Cal A: 0-0.1 μmol / L; Cal B: 1.0-1.5 μmol / L; Cal C: 4.0-5.0 μmol / L; Cal D: 7.5-8.5 μmol / L; Cal E: 20.0-30.0 μmol / L.

[0028] Specific embodiment 1: The homocysteine ​​detection kit comprises reagent R1, reagent R2, reagent R3 and reagent R4; and also comprises calibrators 1 to calibrators 5; the preparation formulas of reagent R1, reagent R2, reagent R3 and reagent R4 are specifically shown in Table 1;

[0029] The specific formula of the reagent R1 for preparing 1L is: magnetic microparticles coated with S-adenosyl-L-homocysteine ​​antibody at a concentration of 0.5 mg / mL, Tris at a concentration of 6.06 g / L, glycerol as a defoaming agent at a concentration of 10 mL / L, bovine serum albumin at a concentration of 10 g / L, PC-300 at a concentration of 1 g / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0030] The specific formula of the reagent R2 for preparing 1L is: recombinant S-adenosyl-L-homocysteine ​​hydrolase with a concentration of 0.1 mg / mL, MES with a concentration of 50 mmol / L, bovine serum albumin with a concentration of 10 g / L, PC-300 with a concentration of 1 g / L, Tween 20 with a concentration of 0.5 mL / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0031] The specific formula of the reagent R3 for preparing 1L is: acridinium ester labeled S-adenosyl-L-cysteine ​​conjugate with a concentration of 0.2 μg / mL, disodium hydrogen phosphate with a concentration of 17.9 g / L, sodium dihydrogen phosphate with a concentration of 7.8 g / L, bovine serum albumin with a concentration of 10 g / L, PC-300 with a concentration of 1 g / L, Tween 20 with a concentration of 0.5 mL / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0032] The specific formula for preparing 1L of the reagent R4 is: dithiothreitol with a concentration of 1 mg / mL, disodium hydrogen phosphate with a concentration of 17.9 g / L, sodium dihydrogen phosphate with a concentration of 7.8 g / L, PC-300 with a concentration of 1 g / L, and the rest is purified water, and the volume is adjusted to 1L with purified water.

[0033] Table 1 Preparation formula of reagent R1, reagent R2, reagent R3 and reagent R4 in specific embodiments

[0034]

[0035] Prepare calibration diluent 1. The specific formula for preparing 1 L of calibration diluent 1 is as follows: trehalose with a concentration of 50 g / L, bovine serum albumin with a concentration of 10 g / L, mannitol with a concentration of 100 g / L, Tween-20 with a concentration of 3 mL / L, PC-300 with a concentration of 1 g / L, and the rest is purified water. Make up to 1 L with purified water.

[0036] Then, calibrator diluent 1 was used to prepare master calibrators 1 to 5. The concentrations of HCY antigen added to calibrator diluent 1 were: CalA: 0.1 μmol / L; CalB: 1.15 μmol / L; CalC: 4.35 μmol / L; CalD: 7.52 μmol / L; CalE: 29.13 μmol / L, respectively, to obtain master calibrators 1 to 5.

[0037] Specific embodiment 2: The homocysteine ​​detection kit comprises reagent R1, reagent R2, reagent R3 and reagent R4; and also comprises calibrators 6 to 10.

[0038] The specific formula of the reagent R1 for preparing 1L is: magnetic microparticles coated with S-adenosyl-L-homocysteine ​​antibody at a concentration of 0.5 mg / mL, Tris at a concentration of 6.06 g / L, glycerol as a defoaming agent at a concentration of 10 mL / L, bovine serum albumin at a concentration of 10 g / L, PC-300 at a concentration of 1 g / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0039] The specific formula of the reagent R2 for preparing 1L is: recombinant S-adenosyl-L-homocysteine ​​hydrolase with a concentration of 0.1 mg / mL, MES with a concentration of 50 mmol / L, bovine serum albumin with a concentration of 10 g / L, PC-300 with a concentration of 1 g / L, Tween 20 with a concentration of 0.5 mL / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0040] The specific formula of the reagent R3 for preparing 1L is: acridinium ester labeled S-adenosyl-L-cysteine ​​conjugate with a concentration of 0.2 μg / mL, disodium hydrogen phosphate with a concentration of 17.9 g / L, sodium dihydrogen phosphate with a concentration of 7.8 g / L, bovine serum albumin with a concentration of 10 g / L, PC-300 with a concentration of 1 g / L, Tween 20 with a concentration of 0.5 mL / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0041] The specific formula for preparing 1L of the reagent R4 is: dithiothreitol with a concentration of 1 mg / mL, disodium hydrogen phosphate with a concentration of 17.9 g / L, sodium dihydrogen phosphate with a concentration of 7.8 g / L, PC-300 with a concentration of 1 g / L, and the rest is purified water, and the volume is adjusted to 1L with purified water.

[0042] Prepare calibration diluent 2. The specific formula for preparing 1 L of calibration diluent 2 is: trehalose with a concentration of 50 g / L, bovine serum albumin with a concentration of 40 g / L, mannitol with a concentration of 100 g / L, Tween-20 with a concentration of 3 mL / L, PC-300 with a concentration of 1 g / L, and the rest is purified water. Make up to 1 L with purified water.

[0043] Then, calibrator diluent 2 was used to prepare master calibrators 6 to 10. The concentrations of HCY antigen added to calibrator diluent 2 were: CalA: 0.1 μmol / L; CalB: 1.15 μmol / L; CalC: 4.35 μmol / L; CalD: 7.52 μmol / L; CalE: 29.13 μmol / L, respectively, to obtain master calibrators 6 to 10.

[0044] Specific embodiment 3: The homocysteine ​​detection kit comprises reagent R1, reagent R2, reagent R3 and reagent R4; and also comprises calibrators 11 to 15.

[0045] The specific formula of the reagent R1 for preparing 1L is: magnetic microparticles coated with S-adenosyl-L-homocysteine ​​antibody at a concentration of 0.5 mg / mL, Tris at a concentration of 6.06 g / L, glycerol as a defoaming agent at a concentration of 10 mL / L, bovine serum albumin at a concentration of 10 g / L, PC-300 at a concentration of 1 g / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0046] The specific formula of the reagent R2 for preparing 1L is: recombinant S-adenosyl-L-homocysteine ​​hydrolase with a concentration of 0.1 mg / mL, MES with a concentration of 50 mmol / L, bovine serum albumin with a concentration of 10 g / L, PC-300 with a concentration of 1 g / L, Tween 20 with a concentration of 0.5 mL / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0047] The specific formula of the reagent R3 for preparing 1L is: acridinium ester labeled S-adenosyl-L-cysteine ​​conjugate with a concentration of 0.2 μg / mL, disodium hydrogen phosphate with a concentration of 17.9 g / L, sodium dihydrogen phosphate with a concentration of 7.8 g / L, bovine serum albumin with a concentration of 10 g / L, PC-300 with a concentration of 1 g / L, Tween 20 with a concentration of 0.5 mL / L, and the rest is purified water, and the volume is fixed to 1L with purified water;

[0048] The specific formula for preparing 1L of the reagent R4 is: dithiothreitol with a concentration of 1 mg / mL, disodium hydrogen phosphate with a concentration of 17.9 g / L, sodium dihydrogen phosphate with a concentration of 7.8 g / L, PC-300 with a concentration of 1 g / L, and the rest is purified water, and the volume is adjusted to 1L with purified water.

[0049] Prepare calibration diluent 3. The specific formula for preparing 1 L of calibration diluent 3 is as follows: trehalose with a concentration of 50 g / L, bovine serum albumin with a concentration of 40 g / L, mannitol with a concentration of 100 g / L, Tween-20 with a concentration of 3 mL / L, PC-300 with a concentration of 1 g / L, and the rest is purified water. Make up to 1 L with purified water.

[0050] Then, calibrator diluent 3 was used to prepare master calibrators 11 to 15. The concentrations of HCY antigen added to calibrator diluent 3 were: CalA: 0.1 μmol / L; CalB: 1.15 μmol / L; CalC: 4.35 μmol / L; CalD: 7.52 μmol / L; CalE: 29.13 μmol / L, respectively, to obtain master calibrators 11 to 15.

[0051] The preparation formulas of the three different calibration dilution solutions in the above-mentioned Specific Implementation 1 to Specific Implementation 3 are shown in Table 2.

[0052] Table 2 Preparation formulas of three different calibrant diluents in Specific Embodiment 1 to Specific Embodiment 3

[0053] Reagent name Calibrator diluent 1 Calibrator diluent 2 Calibrator diluent 3 Trehalose 50g / L 50g / L 50g / L BSA 10g / L 40g / L 100g / L Mannitol 100g / L 100g / L 100g / L Tween-20 3mL / L 3mL / L 3mL / L PC-300 1g / L 1g / L 1g / L Purified water Make up to 1L Make up to 1L Make up to 1L

[0054] (I) Analyze the stability of the homocysteine ​​detection kits of Specific Embodiment 1 to Specific Embodiment 3.

[0055] The master calibrators (master calibrators 1 to 15) with different HCY antigen concentrations prepared with different calibrator diluents (calibrator diluent 1 to calibrator diluent 3) were placed in a 4°C environment, and then the diluted master calibrators 1 to 15 were detected using an HCY detection kit prepared with reagents R1, R2, R3 and R4 prepared in Table 1; then, the master calibrators 1 to 15 with different HCY antigen concentrations prepared with the above-mentioned different calibrator diluents (calibrator diluent 1 to calibrator diluent 3) were placed in a 37°C environment for 7 days, and then detected using an HCY detection kit prepared with reagents R1, R2, R3 and R4 prepared in Table 1, and the obtained calibration curve signal values ​​and calibration curve signal retention rates were as shown in Table 3.

[0056] Table 3 Stability analysis of HCY master calibrator prepared with three different calibrator diluents

[0057]

[0058] It can be concluded from Table 3 above that the stabilities of the master calibrators prepared with calibrator diluent 2 and calibrator diluent 3 are comparable, and their stabilities are significantly better than that of the master calibrator prepared with calibrator diluent 1. In addition, the specificities of the calibration curves obtained with calibrator diluent 2 and calibrator diluent 3 are both high.

[0059] (II) Accuracy analysis

[0060] The three calibrator dilutions (calibrator dilution 1 to calibrator dilution 3) used in the above stability study were used to prepare the main calibrators respectively. The HCY detection kit prepared by reagent R1, reagent R2, reagent R3 and reagent R4 prepared in Table 1 was used to measure 8 random samples. The main calibrator should be measured before the sample measurement, and the results should be calibrated according to the calibration rules. The measured sample results shall be based on the calibration results of the main calibrator.

[0061] In order to compare the accuracy of the test results, the above 8 samples were also tested using Abbott's HCY assay kit (chemiluminescent microparticle immunoassay).

[0062] The components of each reagent in Abbott's HCY assay kit are shown in Table 4 below.

[0063] Table 4 Reagent components in Abbott's HCY assay kit

[0064]

[0065] The HCY detection kits prepared using reagents R1, R2, R3 and R4 prepared in Table 1 were matched with the main calibrators prepared with the above three calibrator diluents (HCY antigen concentrations were: CalA: 0.1 μmol / L; CalB: 1.15 μmol / L; CalC: 4.35 μmol / L; CalD: 7.52 μmol / L; CalE: 29.13 μmol / L), and 8 samples (S1-S8) were measured respectively with Abbott's kit. The measurement results are shown in Table 5 below.

[0066] Table 5 Comparison results of the HCY test kit and the Abbott test kit

[0067]

[0068] It can be seen from Table 5 above that the content of bovine serum albumin in the calibrator diluent will affect the accuracy of the HCY detection kit. The results of the samples measured after calibration with the main calibrator prepared with calibrator diluent 2 are more accurate.

[0069] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention, such as changes in the shape or material of a component, should be included in the scope of protection of the present invention.

Claims

1. A homocysteine ​​detection kit, characterized in that: The invention comprises reagent R1, reagent R2, reagent R3 and reagent R4, wherein the reagent R1 comprises magnetic particles coated with S-adenosine-L-homocysteine ​​antibody and basic buffer one, and the basic buffer one also comprises a defoamer, which is glycerol; the reagent R2 comprises recombinant S-adenosine-L-homocysteine ​​hydrolase and basic buffer two; the reagent R3 comprises an S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker and basic buffer three; the reagent R4 comprises dithiothreitol and basic buffer four; the basic buffer one, basic buffer two, basic buffer three and basic buffer four all comprise a pH regulator, an inorganic salt ion and a surfactant; The homocysteine ​​detection kit also includes a calibrator, which includes an HCY antigen and a calibrator diluent, and the calibrator diluent includes a pH value regulator, inorganic salt ions, a surfactant, a stabilizer and bovine serum albumin; the content of the bovine serum albumin is 10-70 g / L or 20-60 g / L or 30-50 g / L.

2. The homocysteine ​​detection kit according to claim 1, characterized in that The pH value is a mixture of one or more of phosphate buffer, citric acid buffer, MES buffer, Tris buffer and HEPES buffer; the inorganic salt ions are sodium chloride and / or potassium chloride; the surfactant is one or a mixture of Tween, SPAN, Triton and EMULGEN series surfactants.

3. The homocysteine ​​detection kit according to claim 2, characterized in that: The basic buffer solution 1, basic buffer solution 2 and basic buffer solution 3 further include a stabilizer, and the stabilizer includes bovine serum albumin, and the content of the bovine serum albumin is 10-20 g / L.

4. The homocysteine ​​detection kit according to claim 1, characterized in that The dosage of the magnetic particles coated with S-adenosine-L-homocysteine ​​antibody in the reagent R1 is 0.01-2.5 mg / mL; the S-adenosine-L-homocysteine ​​antibody is an immunoglobulin molecule and a fragment thereof that specifically binds to S-adenosine-L-homocysteine.

5. The homocysteine ​​detection kit according to claim 1, characterized in that: The concentration of the recombinant S-adenosyl-L-homocysteine ​​hydrolase in the reagent R2 is 0.1-50 μg / mL.

6. The homocysteine ​​detection kit according to claim 1, characterized in that: The detectable marker-labeled S-adenosine-L-cysteine ​​conjugate in the reagent R3 refers to an acridinium ester-labeled S-adenosine-L-cysteine ​​conjugate with a concentration of 0.2 to 2 μg / mL.

7. The homocysteine ​​detection kit according to claim 1, characterized in that: The concentration of dithiothreitol in the reagent R4 is 0.01-100 mg / mL.

8. The homocysteine ​​detection kit according to claim 4, characterized in that: The S-adenosyl-L-homocysteine ​​antibody in the reagent R1 includes chimeric antibodies, humanized antibodies, fully human antibodies, CDR-grafted antibodies, antibody constructs, and recombinant or synthetic antibodies.

9. The homocysteine ​​detection kit according to claim 5, characterized in that: The concentration of the recombinant S-adenosyl-L-homocysteine ​​hydrolase in the reagent R2 is 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, or 50 μg / mL.

10. The homocysteine ​​detection kit according to claim 6, characterized in that: The detectable marker in the S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker in the reagent R3 is an acridinium ester derivative; the S-adenosine-L-cysteine ​​conjugate labeled with a detectable marker is an acridinium ester labeled S-adenosine-L-cysteine ​​conjugate, and its usage concentration is 0.2 μg / mL or 0.25 μg / mL or 0.5 μg / mL or 1 μg / mL or 2 μg / mL.

11. The homocysteine ​​detection kit according to claim 7, characterized in that: The concentration of dithiothreitol in the reagent R4 is 0.01 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL or 100 mg / mL.

12. The homocysteine ​​detection kit according to claim 2, characterized in that: When the pH value is phosphate buffer, its concentration is 0.1-10 g / L; when the inorganic salt ion is sodium chloride, its concentration is 2-30 g / L; when the surfactant is Tween 20 or Triton 100, its concentration is 0.05-20 mL / L.

13. The homocysteine ​​detection kit according to claim 1, characterized in that: The concentrations of HCY antigens in the master calibrators are: CalA: 0-0.1 μmol / L; CalB: 1.0-1.5 μmol / L; CalC: 4.0-5.0 μmol / L; CalD: 7.5-8.5 μmol / L; CalE: 20.0-30.0 μmol / L.

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