Homocysteine detection kit and preparation method thereof

Abstract

The invention provides a homocysteine detection kit and a preparation method thereof, and belongs to the field of in-vitro diagnostic reagents, the homocysteine detection kit provided by the invention comprises a first reagent, a second reagent, a third reagent and a calibrator, the first reagent is an AMPSO buffer solution containing TCEP, adenosine, an antioxidant, a macromolecular coagulant and a preservative; the second reagent is an HEPES buffer solution containing a recombinant S-adenosine-L-homocysteine hydrolase, an S-adenosine-L-homocysteine coupled bovine serum albumin compound, a protein substance, a carbohydrate substance, glycerol, a surfactant and a preservative; the third reagent is a latex particle preservation solution marked with a mouse anti-human SAH antibody; the calibrator is a plurality of homocysteine solutions or homocysteine dry powder with different concentration levels and the concentration of 0-100 [mu] mol / L. The kit provided by the invention has the advantages of convenience and simplicity in use and high stability.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and in particular to a homocysteine ​​detection kit and a preparation method thereof. Background Art

[0002] Homocysteine ​​(Hcy) is a sulfur-containing amino acid and an important intermediate product produced in the metabolism of methionine and cysteine. Homocysteine ​​can be synthesized into methionine under the action of N5-CH3-FH4 methyltransferase. Under normal circumstances, homocysteine ​​can be decomposed and metabolized in the body, and the concentration is maintained at a low level. However, in daily life, primary and secondary reasons can affect the metabolism of blood homocysteine, leading to the accumulation and increase of homocysteine ​​concentration, referred to as high blood homocysteine. It will greatly increase the risk of coronary heart disease, peripheral vascular disease and cerebrovascular disease.

[0003] At present, the methods used to detect Hcy mainly include high-performance liquid chromatography, enzyme cycle method, enzyme-linked immunosorbent assay and chemiluminescence immunoassay. High-performance liquid chromatography (HPLC) requires derivatization treatment, which is relatively complicated to operate. It can only detect a single sample, has low efficiency, cannot meet market demand, and can only be used for scientific research. The enzyme cycle method is simple to operate and is currently a commonly used method for detecting Hcy on a fully automatic biochemical analysis platform. The fee is low, but this method mainly uses enzymes, and multiple enzymes are used together. The storage conditions and adaptive buffer environment of each enzyme are different, resulting in the stability of enzyme Hcy reagents has always been a pain point in the market. The enzyme-linked immunosorbent assay is a manual method, which is extremely inconvenient and time-consuming for clinical use. The luminescence immunoassay has accurate results, simple operation, and stable reagents, but the test takes a long time, the fee is expensive, and the luminescence has four reagent components, while the currently commonly used fully automatic biochemical analysis platform can only realize the automatic liquid collection and mixing of two reagent components. The stability of the reagents in the luminescence immunoassay after pre-mixing is significantly reduced, and the accuracy of the results of the machine detection is reduced. Therefore, the existing luminescence immunoassay cannot be popularized on the fully automatic biochemical analysis platform. Due to the limitations of these methods, there are disadvantages such as multiple reagent components (3 or more), complex operations, inability to perform automated analysis, or slow testing speed of automated analysis equipment. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to detect Hcy more sensitively, quickly and accurately.

[0005] In order to solve the above problems, the present invention provides a homocysteine ​​detection kit in a first aspect, the kit comprising a first reagent, a second reagent, a third reagent and a calibrator, wherein:

[0006] The first reagent is an AMPSO buffer containing TCEP, adenosine, antioxidants, macromolecular coagulants, and preservatives;

[0007] The second reagent is a HEPES buffer containing recombinant S-adenosyl-L-homocysteine ​​hydrolase, S-adenosylhomocysteine ​​coupled bovine serum albumin complex, protein substances, sugar substances, glycerol, surfactants and preservatives;

[0008] The third reagent is a latex particle preservation solution containing labeled mouse anti-human SAH antibodies;

[0009] The calibrator is homocysteine ​​solution or homocysteine ​​powder at several different concentration levels ranging from 0 to 100 μmol / L.

[0010] The homocysteine ​​detection kit provided by the present invention is a latex enhanced immunoturbidimetry kit. Different forms of homocysteine ​​in a detection sample can be reduced to free homocysteine ​​(L-Hcy) under the action of TCEP, and L-Hcy can be converted into S-adenosyl-L-homocysteine ​​hydrolase in a second reagent. Meanwhile, the converted S-adenosyl-L-homocysteine ​​competes with an S-adenosylhomocysteine-coupled bovine serum albumin complex to generate turbidity, thereby realizing quantitative detection of homocysteine ​​in the detection sample.

[0011] Preferably, in the first reagent, the antioxidant is selected from any one or more of sodium pyrosulfite, sodium sulfite, and sodium bisulfite;

[0012] The macromolecular coagulant is selected from any one or more of dextran, polyethylene glycol, and polyvinyl pyrrolidone;

[0013] The preservative is selected from any one or more of sodium azide, thimerosal, gentamicin and proclin.

[0014] Preferably, in the second reagent, the protein substance is selected from any one or more of bovine serum albumin, casein, and ovalbumin;

[0015] The sugar substance is selected from any one or more of sucrose, glucose, trehalose and sorbitol;

[0016] The surfactant is selected from any one or more of Tween-20, Tween-80-Triton X-100, Triton X-405, and Brij35;

[0017] The preservative is selected from any one or more of sodium azide, thimerosal, gentamicin and proclin.

[0018] Preferably, in the first reagent, the concentration of TCEP is 0.5-10 mM, the concentration of adenosine is 10-100 mg / L, the mass fraction of the antioxidant is 0.5-5%, the mass fraction of the macromolecular coagulant is 1-10%, the mass fraction of the preservative is 0.01-0.1%, and the pH of the first reagent is 8.0-10.0.

[0019] Preferably, in the second reagent, the concentration of recombinant S-adenosyl-L-homocysteine ​​hydrolase is 0.1-10 U / mL, the concentration of S-adenosylhomocysteine ​​coupled bovine serum albumin complex is 0.5-10 mg / L, the mass fraction of protein substances is 0.5-5%, the mass fraction of carbohydrate substances is 1-10%, the mass fraction of glycerol is 2-20%, the mass fraction of surfactants is 0.1-2%, the mass fraction of preservatives is 0.01-0.1%, and the pH of the second reagent is 7.0-8.3.

[0020] Preferably, the latex particles labeled with mouse anti-human SAH antibodies are P0113 latex microspheres, and the pH of the third reagent is 7.0-9.0.

[0021] Furthermore, the second aspect of the present invention provides a method for preparing the homocysteine ​​detection kit according to the first aspect, comprising the following steps:

[0022] S1: prepare the first reagent by taking AMPSO buffer, TCEP, adenosine, antioxidant, macromolecular coagulant and preservative;

[0023] S2: prepare the second reagent by taking recombinant S-adenosyl-L-homocysteine ​​hydrolase, HEPES buffer, S-adenosyl-homocysteine ​​coupled bovine serum albumin complex, protein substances, sugar substances, glycerol, surfactants and preservatives;

[0024] S3: prepare the third reagent;

[0025] S4: Prepare calibrators.

[0026] Preferably, in step S2, the S-adenosylhomocysteine-bovine serum albumin complex is prepared by the following steps:

[0027] S21: Weigh bovine serum albumin and dissolve it in HEPES buffer to obtain solution A;

[0028] S22: dissolving EDC in HEPES buffer to obtain solution B, mixing solution B with solution A and reacting them sufficiently to obtain solution C;

[0029] S23: weighing S-adenosylhomocysteine ​​and dissolving it in HEPES buffer to obtain solution D, mixing solution D with solution C and reacting them sufficiently to obtain solution E;

[0030] S24: removing EDC and S-adenosylhomocysteine ​​from solution E to obtain an S-adenosylhomocysteine-coupled bovine serum albumin complex.

[0031] Preferably, the mass ratio of bovine serum albumin in step S21 to S-adenosylhomocysteine ​​in step S23 is 5:1.

[0032] Beneficial effects of the present invention: The homocysteine ​​detection kit prepared by the preparation method of the homocysteine ​​detection kit provided by the present invention includes three detection reagents and a set of calibrators. Compared with the existing homocysteine ​​detection kit, it does not need to prepare sample diluent separately, nor does it need to mix reagents in advance before detection, and has the advantages of being simple and convenient to use. At the same time, the homocysteine ​​detection kit provided by the present invention can be stably stored at 4°C for more than 14 months, which fully meets the requirements of the biochemical platform for reagent stability. In summary, the homocysteine ​​detection kit provided by the present invention has the advantages of being simple and convenient to use and having high stability. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0034] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0035] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0036] As described in the background art, the current Hcy detection method has the disadvantages of low accuracy and sensitivity, complicated operation process, high cost, low automation, etc. Based on this, the specific embodiment of the present invention provides a homocysteine ​​detection kit by latex enhanced immunoturbidimetry.

[0037] The homocysteine ​​detection kit provided in the specific embodiment of the present invention is developed based on a biochemical platform and comprises three detection reagents, namely a first reagent, a second reagent and a third reagent, and a calibrator, wherein:

[0038] The first reagent is an AMPSO buffer containing TCEP, adenosine, an antioxidant, a macromolecular coagulant and a preservative, wherein the concentration of TCEP is 0.5-10 mM, the concentration of adenosine is 10-100 mg / L, the mass fraction of the antioxidant is 0.5-5%, the mass fraction of the macromolecular coagulant is 1-10%, the mass fraction of the preservative is 0.01-0.1%, and the pH of the first reagent is 8.0-10.0;

[0039] The second reagent is a HEPES buffer containing recombinant S-adenosyl-L-homocysteine ​​hydrolase, S-adenosyl-homocysteine ​​coupled bovine serum albumin complex, protein substances, sugar substances, glycerol, surfactants and preservatives, wherein the concentration of recombinant S-adenosyl-L-homocysteine ​​hydrolase is 0.1-10 U / mL, the concentration of S-adenosyl-homocysteine ​​coupled bovine serum albumin complex is 0.5-10 mg / L, the mass fraction of protein substances is 0.5-5%, the mass fraction of sugar substances is 1-10%, the mass fraction of glycerol is 2-20%, the mass fraction of surfactants is 0.1-2%, the mass fraction of preservatives is 0.01-0.1%, and the pH of the second reagent is 7.0-8.3;

[0040] The third reagent is a latex particle preservation solution containing labeled mouse anti-human SAH antibodies;

[0041] The calibrator is homocysteine ​​solution or homocysteine ​​powder at several different concentration levels ranging from 0 to 100 μmol / L.

[0042] In the first reagent of the above embodiment, the antioxidant is selected from any one or more of sodium pyrosulfite, sodium sulfite, and sodium bisulfite;

[0043] The macromolecular coagulant is selected from any one or more of dextran, polyethylene glycol, and polyvinyl pyrrolidone;

[0044] The preservative is selected from any one or more of sodium azide, thimerosal, gentamicin and proclin.

[0045] In the second reagent of the above embodiment, the protein substance is selected from any one or more of bovine serum albumin, casein, and ovalbumin;

[0046] The sugar substance is selected from any one or more of sucrose, glucose, trehalose and sorbitol;

[0047] The surfactant is selected from any one or more of Tween-20, Tween-80-Triton X-100, Triton X-405, and Brij35;

[0048] The preservative is selected from any one or more of sodium azide, thimerosal, gentamicin and proclin.

[0049] In the third reagent of the above embodiment, the latex particles labeled with mouse anti-human SAH antibodies are P0113 latex microspheres, and the pH of the third reagent is 7.0-9.0.

[0050] Each component of the homocysteine ​​detection kit provided by the above embodiment of the present invention can be stably stored at 4°C for more than 14 months, which fully meets the requirements of the biochemical platform for reagent stability. There is no need to prepare a separate sample diluent, nor is it necessary to mix the two reagents in advance before use.

[0051] The technical solution of the present invention is further described below through specific examples. It should be noted that, in the following, Hcy is the abbreviation of homocysteine; SAH-BSA is the abbreviation of S-adenosylhomocysteine ​​coupled bovine serum albumin complex;

[0052] Example 1

[0053] Preparation of a homocysteine ​​detection kit

[0054] The final concentrations of the components of the first reagent are: 100 mmol / L AMPSO (pH 9.0), 50.0 g / L polyethylene glycol 6000, 0.5 g / L TCEP, 20 mg / L adenosine, 9.0 g / L sodium chloride, 1 g / L sodium azide;

[0055] The final concentrations of the components of the second reagent are: 50 mmol / L HEPES (pH 7.4), 2 U / mL recombinant S-adenosyl-L-homocysteine ​​hydrolase (SAHHase), 1 mg / L SAH-BSA antigen complex, 2% BSA, 5% trehalose, 10% glycerol, 0.2% Tween 20, 1 g / L sodium azide;

[0056] Among them, the SAH-BSA antigen complex was synthesized in the laboratory and obtained by coupling SAH and BSA through EDC. The specific coupling method is as follows:

[0057] Take a 50mL centrifuge tube, add 10mL of 10mM HEPES (pH7.4), weigh 5mg of BSA and add it to the centrifuge tube, invert and mix until BSA is completely dissolved; take a certain amount of EDC and dissolve it in 10mM HEPES (pH7.4) to a concentration of 10mg / mL, take 20μLEDC solution and add it to the above centrifuge tube, mix quickly and react at room temperature for 30min; take 1mg SAH and add it to 10mL of 10mM HEPES (pH7.4) and heat it to 60℃ until it is completely dissolved, add it to the 50mL centrifuge tube in step 1, mix and react at room temperature for 2h; after the reaction, dialyze the above reaction solution with a dialysis bag for 24h, and change the solution three times in the middle to remove unreacted EDC and SAH small molecules, which is conducive to the stability of SAH-BSA storage.

[0058] The final concentration of each component of the third reagent is: a storage solution containing latex particles labeled with mouse anti-human SAH antibodies. The specific preparation process of latex particles labeled with mouse anti-human SAH antibodies is as follows:

[0059] Take a 50mL centrifuge tube, add 10mL of 10mM HEPES (pH7.4), take 100μL of P0113 latex microspheres and add them to the above centrifuge tube; take a certain amount of EDC and dissolve it with 10mM HEPES (pH7.4) to a concentration of 10mg / mL, take 20μL of EDC solution and add it to the above centrifuge tube, mix it quickly and react at room temperature for 30min; 3. Take 1mg of SAH antibody, add it to the above centrifuge tube, mix it at room temperature for 2h; after the reaction is completed, add 1mg BSA for blocking, and the blocking time is 1h; after the blocking is completed, centrifuge at 15000rpm for 30min, and remove the supernatant; add 10mL of preservation solution to the centrifuge tube, mix it by ultrasonication, and obtain 10mL of the third reagent.

[0060] The calibrators are divided into six concentrations and the preparation compositions are as follows:

[0061] Calibrator 1: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide;

[0062] Calibrator 2: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide, 12.5μmol / L homocysteine;

[0063] Calibrator 3: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide, 25μmol / L homocysteine;

[0064] Calibrator 4: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide, 50μmol / L homocysteine;

[0065] Calibrator 5: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide, 75μmol / L homocysteine;

[0066] Calibrator 6: 50 mmol / L phosphate buffer (pH 7.4), 9 g / L sodium chloride, 20 g / L BSA, 1 g / L sodium azide, 100 μmol / L homocysteine.

[0067] The method of using the kit prepared in this embodiment is as follows: after adding the sample or calibration substance to the first reagent, incubate at 37°C for 1.5 minutes, add the second reagent, incubate at 37°C for 3.5 minutes, add the third reagent, mix well, take the absorbance A1, incubate at 37°C for 5 minutes, read the absorbance A2, and calculate △A=A2-A1; the analysis parameters are: wavelength 600nm, sample or calibration substance volume 8μL, first reagent volume 75μL, second reagent volume 75μL, third reagent volume 50μL.

[0068] Example 2

[0069] Preparation of a homocysteine ​​detection kit

[0070] The final concentrations of the components of the first reagent are: 100mmol / L AMPSO (pH10.0), 10.0g / L dextran, 2.5g / L TCEP, 100mg / L adenosine, 9.0g / L sodium chloride, 0.5g / L sodium azide;

[0071] The final concentrations of the components of the second reagent were: 50 mmol / L HEPES (pH 8.3), 10 U / mL recombinant S-adenosyl-L-homocysteine ​​hydrolase (SAHHase), 10 mg / L SAH-BSA antigen complex, 5% casein, 10% glucose, 20% glycerol, 2% Triton X-100, and 0.1 g / L thimerosal;

[0072] Among them, the SAH-BSA antigen complex was synthesized in the laboratory and obtained by coupling SAH and BSA through EDC. The specific coupling method is as follows:

[0073] Take a 50mL centrifuge tube, add 10mL of 10mM HEPES (pH7.4), weigh 5mg of BSA and add it to the centrifuge tube, invert and mix until BSA is completely dissolved; take a certain amount of EDC and dissolve it in 10mM HEPES (pH7.4) to a concentration of 10mg / mL, take 20μLEDC solution and add it to the above centrifuge tube, mix quickly and react at room temperature for 30min; take 1mg SAH and add it to 10mL of 10mM HEPES (pH7.4) and heat it to 60℃ until it is completely dissolved, add it to the 50mL centrifuge tube in step 1, mix and react at room temperature for 2h; after the reaction, dialyze the above reaction solution with a dialysis bag for 24h, and change the solution three times in the middle to remove unreacted EDC and SAH small molecules, which is conducive to the stability of SAH-BSA storage.

[0074] The final concentration of each component of the third reagent is: a storage solution containing latex particles labeled with mouse anti-human SAH antibodies. The specific preparation process of latex particles labeled with mouse anti-human SAH antibodies is as follows:

[0075] Take a 50mL centrifuge tube, add 10mL of 10mM HEPES (pH7.4), take 100μL of P0113 latex microspheres and add them to the above centrifuge tube; take a certain amount of EDC and dissolve it with 10mM HEPES (pH7.4) to a concentration of 10mg / mL, take 20μL of EDC solution and add it to the above centrifuge tube, mix it quickly and react at room temperature for 30min; 3. Take 1mg of SAH antibody, add it to the above centrifuge tube, mix it at room temperature for 2h; after the reaction is completed, add 1mg BSA for blocking, and the blocking time is 1h; after the blocking is completed, centrifuge at 15000rpm for 30min, and remove the supernatant; add 10mL of preservation solution to the centrifuge tube, mix it by ultrasonication, and obtain 10mL of the third reagent.

[0076] The calibrators are divided into six concentrations and the preparation compositions are as follows:

[0077] Calibrator 1: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide;

[0078] Calibrator 2: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide, 12.5μmol / L homocysteine;

[0079] Calibrator 3: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide, 25μmol / L homocysteine;

[0080] Calibrator 4: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide, 50μmol / L homocysteine;

[0081] Calibrator 5: 50mmol / L phosphate buffer (pH7.4), 9g / L sodium chloride, 20g / L BSA, 1g / L sodium azide, 75μmol / L homocysteine;

[0082] Calibrator 6: 50 mmol / L phosphate buffer (pH 7.4), 9 g / L sodium chloride, 20 g / L BSA, 1 g / L sodium azide, 100 μmol / L homocysteine.

[0083] The method of using the kit prepared in this embodiment is as follows: after adding the sample or calibration substance to the first reagent, incubate at 37°C for 1.5 minutes, add the second reagent, incubate at 37°C for 3.5 minutes, add the third reagent, mix well, take the absorbance A1, incubate at 37°C for 5 minutes, read the absorbance A2, and calculate △A=A2-A1; the analysis parameters are: wavelength 600nm, sample or calibration substance volume 8μL, first reagent volume 75μL, second reagent volume 75μL, third reagent volume 50μL.

[0084] Example 3

[0085] Accuracy test

[0086] The kit prepared in Example 1 was used to test the homocysteine ​​frozen human serum national standard product with a test batch number of 360047-202001, and the accuracy of the kit prepared in Example 1 was tested. The relevant results are shown in Table 1:

[0087] Table 1

[0088]

[0089] The results showed that the final relative deviation value was less than 10%, indicating that the homocysteine ​​detection kit prepared in Example 1 had high accuracy.

[0090] Example 4

[0091] Repeatability test

[0092] Three samples of low, medium and high were tested and repeated 10 times respectively. The mean, standard deviation and coefficient of variation were calculated. The results are shown in Table 2:

[0093] Table 2

[0094]

[0095] The results showed that the cvs of the three samples with low, medium and high test levels were all less than 5%, indicating that the homocysteine ​​detection kit in Example 1 had good repeatability and high precision.

[0096] Example 5

[0097] On-machine stability test

[0098] Take the test kits of Examples 1 and 2 as quality control level 1 and quality control level 2, and divide the samples of quality control level 1 and quality control level 2 into small packages and freeze them in a -20°C refrigerator in advance for use. Place the first reagent in the position set on the R1 reagent disk of the Hitachi 7180 fully automatic biochemical analyzer, and place the second and third reagents in the position set on the R2 reagent disk of the Hitachi 7180 fully automatic biochemical analyzer. Calibrate on day 0, test quality control 1 and quality control 2 3 times each and take the average value. Without recalibrating on days 1 / 3 / 5 / 7 / 14 / 21 / 31, test quality control 1 and quality control 2 3 times each and take the average value, and calculate the relative deviation of the average value compared with day 0. The results are shown in Table 3:

[0099]

[0100]

[0101] As can be seen from Table 3, the deviation of the test results of the first reagent, the second reagent and the third reagent after opening the instrument is less than 10%, which meets the clinical requirements, indicating that the reagent can be stable for 1 month after opening, and the reagent has good opening stability.

[0102] Example 6

[0103] Real-time stability testing

[0104] The kits of Examples 1 and 2 were taken as quality control level 1 and quality control level 2, and stored at 4° C. on the day of storage, one month, three months, six months, nine months, twelve months, and fourteen months, respectively, and the homocysteine ​​concentration of the same quality control product was tested with the kits. The relative deviations of the test values ​​at one month, three months, six months, nine months, twelve months, and fourteen months of storage and the test values ​​on the day of storage were calculated.

[0105] The stability test results of the homocysteine ​​detection kit of Example 1-2 at 4°C are shown in Table 4 below.

[0106]

[0107] As can be seen from Table 4, the reagent can be stable in real time for 14 months, and the reagent has good real-time stability.

[0108] In summary, the homocysteine ​​detection kit provided by the specific embodiment of the present invention has high accuracy, sensitivity, repeatability and stability after opening the bottle.

[0109] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A homocysteine ​​detection kit, characterized in that, It includes a first reagent, a second reagent, a third reagent and a calibrator, wherein: The first reagent is an AMPSO buffer containing TCEP, adenosine, antioxidants, macromolecular coagulants, and preservatives; The second reagent is a HEPES buffer containing recombinant S-adenosyl-L-homocysteine ​​hydrolase, S-adenosylhomocysteine ​​coupled bovine serum albumin complex, protein substances, sugar substances, glycerol, surfactants and preservatives; The third reagent is a latex particle preservation solution containing labeled mouse anti-human SAH antibodies; The calibrator is homocysteine ​​solution or homocysteine ​​powder at several different concentration levels ranging from 0 to 100 μmol / L.

2. The homocysteine ​​detection kit according to claim 1, characterized in that In the first reagent, the antioxidant is selected from any one or more of sodium metabisulfite, sodium sulfite, and sodium bisulfite; The macromolecular coagulant is selected from any one or more of dextran, polyethylene glycol, and polyvinyl pyrrolidone; The preservative is selected from any one or more of sodium azide, thimerosal, gentamicin and proclin.

3. The homocysteine ​​detection kit according to claim 1, characterized in that In the second reagent, the protein substance is selected from any one or more of bovine serum albumin, casein, and ovalbumin; The sugar substance is selected from any one or more of sucrose, glucose, trehalose and sorbitol; The surfactant is selected from any one or more of Tween-20, Tween-80, Triton X-100, Triton X-405, and Brij35; The preservative is selected from any one or more of sodium azide, thimerosal, gentamicin and proclin.

4. The homocysteine ​​detection kit according to claim 1, characterized in that In the first reagent, the concentration of TCEP is 0.5-10mM, the concentration of adenosine is 10-100mg / L, the mass fraction of antioxidant is 0.5-5%, the mass fraction of macromolecular coagulant is 1-10%, the mass fraction of preservative is 0.01-0.1%, and the pH of the first reagent is 8.0-10.

0.

5. The homocysteine ​​detection kit according to claim 1, characterized in that In the second reagent, the concentration of recombinant S-adenosyl-L-homocysteine ​​hydrolase is 0.1-10 U / mL, the concentration of S-adenosylhomocysteine ​​coupled bovine serum albumin complex is 0.5-10 mg / L, the mass fraction of protein substances is 0.5-5%, the mass fraction of carbohydrate substances is 1-10%, the mass fraction of glycerol is 2-20%, the mass fraction of surfactants is 0.1-2%, the mass fraction of preservatives is 0.01-0.1%, and the pH of the second reagent is 7.0-8.

3.

6. The homocysteine ​​detection kit according to claim 1, characterized in that The latex particle preservation solution contains latex particles labeled with mouse anti-human SAH antibodies, the latex particles labeled with mouse anti-human SAH antibodies are P0113 latex microspheres, and the pH of the third reagent is 7.0-9.

0.

7. A method for preparing the homocysteine ​​detection kit according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1: prepare the first reagent by taking AMPSO buffer, TCEP, adenosine, antioxidant, macromolecular coagulant and preservative; S2: prepare the second reagent by taking recombinant S-adenosyl-L-homocysteine ​​hydrolase, HEPES buffer, S-adenosyl-homocysteine ​​coupled bovine serum albumin complex, protein substances, sugar substances, glycerol, surfactants and preservatives; S3: prepare the third reagent; S4: Prepare calibrators.

8. The preparation method according to claim 7, characterized in that: In step S2, the S-adenosylhomocysteine-bovine serum albumin complex is prepared by the following steps: S21: Weigh bovine serum albumin and dissolve it in HEPES buffer to obtain solution A; S22: dissolving EDC in HEPES buffer to obtain solution B, mixing solution B with solution A and reacting them sufficiently to obtain solution C; S23: weighing S-adenosylhomocysteine ​​and dissolving it in HEPES buffer to obtain solution D, mixing solution D with solution C and reacting them sufficiently to obtain solution E; S24: removing EDC and S-adenosylhomocysteine ​​from solution E to obtain an S-adenosylhomocysteine-coupled bovine serum albumin complex.

9. The preparation method according to claim 8, characterized in that: The mass ratio of bovine serum albumin in step S21 to S-adenosylhomocysteine ​​in step S23 is 5:1.

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