Homocysteine detection kit and preparation method thereof

Abstract

The invention relates to a homocysteine detection kit and a preparation method thereof. The homocysteine detection kit comprises a nitrocellulose membrane coated by an S-adenosylmethionine recombinant antigen, fluorescent microspheres labeled by an SAH (S-adenosylmethionine) antibody and S-adenosylhomocysteine recombinant hydrolase. The kit comprises a reagent strip, a gun head and a sample diluent, the reagent strip comprises a bottom plate, a nitrocellulose membrane, a combination pad, a sample pad and absorbent paper, the sample pad is connected with the combination pad, and the two ends of the nitrocellulose membrane are connected with the combination pad and the absorbent paper respectively and then bonded with the bottom plate. The homocysteine detection kit is convenient to measure, and the detection reagent is a solid reagent, can be directly used and is convenient to store; therefore, the detection speed is high, the sensitivity is high, the accuracy is high, the stability is good, the linear range is wide, the storage time is long, the storage condition does not need refrigeration, and the product can be stored at the room temperature of 4-30 DEG C for more than one and a half year.

Description

Technical Field

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

[0002] Homocysteine ​​is an intermediate product in the process of methionine metabolism. About 70% of homocysteine ​​in plasma is bound to albumin, which is a 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.

[0003] Currently, methods for determining homocysteine ​​include enzymatic method, chemiluminescent immunoassay, high performance liquid chromatography (HPLC), etc., and the stability of existing homocysteine ​​test kits is relatively low.

[0004] Chinese patent document CN 109212232 A discloses a convenient and stable homocysteine ​​detection kit and detection method; the kit includes reagent R1, reagent R2 and HCY liquid calibration solution, the reagent R1 contains buffer, reducing agent, serine, coenzyme preservative, surfactant, the reagent R2 contains buffer, surfactant, lactate dehydrogenase, stabilizer, antioxidant, preservative, cystathionine β synthase, cystathionine β lyase, and the HCY liquid calibration solution contains stabilizer, preservative, homocysteine. This technical solution suppresses the attenuation of the enzyme under various interference factors in many aspects by adding antioxidants to the kit, and effectively improves the stability of the kit under special environment and during shelf life.

[0005] Chinese patent document CN 105181970A discloses a stable homocysteine ​​detection kit, including reagent 1 and reagent 2, wherein the components of reagent 1 are: HCY reducing agent: 1-200mmol / L, serine: 1.0-100mmol / L, NADH: 0.5-0.8g / L, LDH: 50-200KU / L; the components of reagent 2 are: stabilizer: 0.1-10g / L, cystathionine β-synthase: 1-100KU / L, cystathionine β-degrading enzyme: 1-100KU / L. The stabilizer described in reagent 2 is a mixture of soluble starch and glutathione, which can greatly improve the stabilization effect of the homocysteine ​​detection reagent when used together as a stabilizer, and can be stored at 4°C for at least 1 year.

[0006] Chinese patent document CN 115754299A discloses 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 ​​antibody and basic buffer 1; the reagent R2 comprises recombinant S-adenosyl-L-homocysteine ​​hydrolase 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 and basic buffer 4; and further comprises a calibrator, the calibrator comprising HCY antigen and a calibrator diluent, the calibrator diluent comprising a pH regulator, inorganic salt ions, 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 results.

[0007] However, the decay of cystathionine β-lyase in the above kit is still relatively fast, resulting in poor stability of the kit for cystathionine cycle enzyme method under shelf life. The above reagents are all liquid reagents, stored at low temperature (2-8°C), and have a shelf life of 1 year. The reagent storage conditions are relatively high, which will inevitably increase transportation and storage costs, and the poor stability of the reagents will lead to inaccurate test results.

[0008] Therefore, it is necessary to provide a homocysteine ​​detection kit and a preparation method thereof, wherein the kit has a long shelf life, stable reagents and accurate detection results. Summary of the invention

[0009] The technical problem to be solved by the present invention is to provide a homocysteine ​​detection kit, which allows samples to produce a more efficient reaction and obtain more accurate detection results.

[0010] In order to solve the above technical solution, the technical solution of the present invention is: the homocysteine ​​detection kit comprises a nitrocellulose membrane coated with S-adenosylmethionine recombinant antigen, fluorescent microspheres labeled with SAH antibodies and S-adenosylhomocysteine ​​recombinant hydrolase.

[0011] By adopting the above technical scheme, the homocysteine ​​detection kit is convenient for determination through a nitrocellulose membrane coated with a recombinant S-adenosylmethionine antigen, fluorescent microspheres labeled with SAH antibodies and S-adenosylhomocysteine ​​recombinant hydrolase, and the detection reagent is a solid reagent, which can be used directly and is easy to store; therefore, the detection speed is fast, the sensitivity is high, the accuracy is high, the stability is good, the linear range is wide, the storage time is long, and the storage conditions do not require refrigeration, and can be stored at room temperature of 4°C to 30°C for more than one and a half years.

[0012] Preferably, the kit comprises a reagent strip, a gun tip and a sample diluent, wherein the reagent strip comprises a base plate, a nitrocellulose membrane, a conjugation pad, a sample pad and absorbent paper, wherein the sample pad is connected to the conjugation pad, and the two ends of the nitrocellulose membrane are respectively connected to the conjugation pad and the absorbent paper, and then bonded to the base plate.

[0013] Preferably, the nitrocellulose membrane is coated with S-adenosylmethionine (SAH) recombinant antigen and goat anti-chicken IgY.

[0014] Preferably, the conjugate pad is coated with fluorescent microspheres of SAH monoclonal antibody; the fluorescent microspheres are prepared from rare earth europium fluorescent microspheres; the fluorescent microspheres of SAH monoclonal antibody are fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody, and the conjugate pad is also coated with fluorescent microspheres 2 labeled with chicken IgY.

[0015] Preferably, the sample diluent comprises citrate buffer, dithiothreitol and ProClin 300, pH=7.4±1.

[0016] Preferably, the gun tip is embedded with S-adenosylhomocysteine ​​recombinant hydrolase.

[0017] Preferably, the concentration of the citrate buffer in the sample diluent is 50 mM, the concentration of dithiothreitol is 1.5 mg / mL; and the concentration of ProClin 300 is 0.1%.

[0018] The technical problem to be solved by the present invention is to provide a method for preparing a homocysteine ​​detection kit, the preparation method has better process and the obtained kit detection result has higher accuracy.

[0019] In order to solve the above technical solution, the technical solution of the present invention is: a method for preparing the homocysteine ​​detection kit, specifically comprising the following steps:

[0020] S1 Preparation of sample diluent: Add citrate with a final concentration of 50 mM, dithiothreitol DTT with a final concentration of 1.5 mg / mL, and ProClin 300 with a final concentration of 0.1% into a container, mix well, make up to volume, and adjust the pH to 7.4 ± 1 after mixing to obtain a sample diluent;

[0021] S2: preparing the conjugate pad: preparing fluorescent microsphere 1 labeled with mouse SAH monoclonal antibody and fluorescent microsphere 2 labeled with chicken IgY respectively, diluting the fluorescent microsphere 1 labeled with mouse SAH monoclonal antibody and the fluorescent microsphere 2 labeled with chicken IgY with buffer, and spraying them onto the conjugate pad using a gold sprayer at a spray volume of 1-2 ul / cm;

[0022] S3 Preparation of nitrocellulose membrane: Dilute S-adenosylmethionine (SAH) recombinant antigen and goat anti-chicken IgY with PB buffer, and then streak them onto the nitrocellulose membrane with a streaker, with a coating amount of 0.5-1ul / cm;

[0023] S4 Preparation of custom pipette tips: After diluting the recombinant S-adenosylhomocysteine ​​hydrolase with the preservation solution, take 5ul and apply it to the pipette tip, put it into the dryer, and drain it for 10-12h.

[0024] S5: Prepare the reagent strip: assemble the reagent plate by sticking the binding pad, nitrocellulose membrane, sample pad and absorbent paper on the bottom plate, and then cut them into the reagent strip according to the size;

[0025] S6 Preparation of the kit: Package the sample diluent, pipette tip, and reagent strip to make a homocysteine ​​detection kit.

[0026] Preferably, the specific steps of preparing the fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody and the fluorescent microspheres 2 labeled with chicken IgY in step S2 are:

[0027] S21 Preparation of fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody: After the fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, mouse SAH monoclonal antibody is added and reacted at 37°C for 3 hours; then a blocking agent is added for blocking, centrifugation, and storage to obtain fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody for standby use;

[0028] S22 Preparation of fluorescent microspheres 2 labeled with chicken IgY: The fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, and chicken IgY is added. The reaction is carried out at 37°C for 3 hours. Then, a blocking agent is added for blocking, centrifugation, and storage to obtain fluorescent microspheres 2 labeled with chicken IgY for use.

[0029] Preferably, the ratio of the fluorescent microspheres to the antibody in the mouse SAH monoclonal antibody in step S21 is 1:2-1:10; the ratio of the fluorescent microspheres to the antibody in the chicken IgY in step S22 is 1:2-1:10. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the structure of the reagent strip of the homocysteine ​​detection kit of the present invention;

[0031] Among them, 1-bottom plate; 2-sample pad; 3-binding pad; 4-nitrocellulose membrane; 5-absorbent paper. DETAILED DESCRIPTION

[0032] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0033] Embodiment: The homocysteine ​​detection kit comprises a nitrocellulose membrane coated with a recombinant S-adenosylmethionine antigen, fluorescent microspheres labeled with SAH antibodies, and a recombinant S-adenosylhomocysteine ​​hydrolase. Figure 1 As shown, the kit includes a reagent strip, a gun tip and a sample diluent, wherein the reagent strip includes a base plate 1, a nitrocellulose membrane 4, a conjugation pad 3, a sample pad 2 and an absorbent paper 5, wherein the sample pad 2 is connected to the conjugation pad 3, and the two ends of the nitrocellulose membrane 4 are respectively connected to the conjugation pad 3 and the absorbent paper 5, and then bonded to the base plate 1; the nitrocellulose membrane 4 is coated with S-adenosylmethionine (SAH) recombinant antigen and goat anti-chicken IgY; the conjugation pad 3 is coated with fluorescent microspheres of SAH monoclonal antibody; the fluorescent microspheres are prepared from rare earth europium fluorescent microspheres; the fluorescent microspheres of SAH monoclonal antibody are fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody, and the conjugation pad 3 is also coated with fluorescent microspheres 2 labeled with chicken IgY; the sample diluent includes citrate buffer, dithiothreitol and ProClin 300, pH=7.4±1; the gun tip is embedded with S-adenosylhomocysteine ​​recombinant hydrolase; the concentration of the citrate buffer in the sample diluent is 50 mM, the concentration of dithiothreitol is 1.5 mg / mL; the concentration of ProClin 300 is 0.1%.

[0034] The preparation method of the homocysteine ​​detection kit specifically comprises the following steps:

[0035] S1 Preparation of sample diluent: Add citrate with a final concentration of 50 mM, dithiothreitol DTT with a final concentration of 1.5 mg / mL, and ProClin 300 with a final concentration of 0.1% into a container, mix well, make up to volume, and adjust the pH to 7.4 ± 1 after mixing to obtain a sample diluent;

[0036] S2: preparing conjugate pad 3: preparing fluorescent microsphere 1 labeled with mouse SAH monoclonal antibody and fluorescent microsphere 2 labeled with chicken IgY respectively, diluting fluorescent microsphere 1 labeled with mouse SAH monoclonal antibody and fluorescent microsphere 2 labeled with chicken IgY with buffer, and spraying them onto conjugate pad 3 using a gold sprayer, with a spray volume of 1-2 ul / cm;

[0037] The formula of the buffer solution includes tris, whose final concentration is 50 mM; sucrose, whose final concentration is 20%; trehalose, whose final concentration is 5%; Tween 20, whose final concentration is 5%; and ProClin 300, whose final concentration is 0.1%. The above raw materials are added into a container and mixed evenly, and the volume is fixed. After mixing, the pH is adjusted to 7.4±1 to obtain a buffer solution.

[0038] The specific steps of preparing the fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody and the fluorescent microspheres 2 labeled with chicken IgY in step S2 are as follows:

[0039] S21 Preparation of fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody: After the fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, mouse SAH monoclonal antibody is added and reacted at 37°C for 3 hours; then a blocking agent is added for blocking, centrifugation, and storage to obtain fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody for standby use;

[0040] The ratio of the fluorescent microspheres to the antibody in the mouse SAH monoclonal antibody in step S21 is 1:2-1:10, and in this embodiment, the ratio of the fluorescent microspheres to the antibody in the mouse SAH monoclonal antibody is 1:4; the ratio of the fluorescent microspheres to the antibody in the chicken IgY in step S22 is 1:2-1:10, and in this embodiment, the ratio of the fluorescent microspheres to the antibody in the chicken IgY is 1:10;

[0041] S22 Preparation of fluorescent microspheres 2 labeled with chicken IgY: After the fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, chicken IgY is added and reacted at 37°C for 3 hours, followed by addition of a blocking agent, centrifugation, and storage to obtain fluorescent microspheres 2 labeled with chicken IgY for later use;

[0042] The blocking agent in step S21 and step S22 is: BSA, casein, CE210, glycine, etc. In this embodiment, BSA is used as the blocking agent;

[0043] S3 Preparation of nitrocellulose membrane 4: Dilute S-adenosylmethionine (SAH) recombinant antigen and goat anti-chicken IgY with PB diluent, and then streak them onto nitrocellulose membrane 4 with a streak meter, with a coating amount of 0.5-1 ul / cm;

[0044] S4 Preparation of custom pipette tips: Dilute the recombinant S-adenosylhomocysteine ​​hydrolase with the preservation solution, apply 5ul to the pipette tip, place it in a vacuum dryer, and drain for 10-12h;

[0045] The formula of the preservation solution is: tris, with a final concentration of 50 mM; mannitol, with a final concentration of 1%; trehalose, with a final concentration of 5%; PVA-10, with a final concentration of 0.05%; glycerol, with a final concentration of 1%; ProClin 300, with a final concentration of 0.1%; add the above solutions into a container and mix evenly, make up to volume, and adjust the pH to 7.4±1 after mixing to obtain the preservation solution;

[0046] S5: Prepare the reagent strip: assemble the reagent plate by pasting the binding pad 3, nitrocellulose membrane 4, sample pad 2 and absorbent paper 5 on the bottom plate 1, and then cut and assemble into the reagent strip according to the size;

[0047] S6 Preparation of the kit: Package the sample diluent, pipette tip, and reagent strip to make a homocysteine ​​detection kit.

[0048] The kit of the present invention and the kit prepared by the conventional process were tested by using the same sample, and the test results are shown in Table 1.

[0049] Table 1 Results of comparative tests of the kits obtained by conventional process and the preparation method of the present invention

[0050] Sample concentration Conventional process method T / C Preparation method kit T / C of the present invention 0μmol / / L 0.6241 0.8857 1.1μmol / / L 0.6085 0.8354 4.9μmol / / L 0.4547 0.6045 10.5μmol / / L 0.3557 0.3054 31.2μmol / / L 0.1857 0.0798 45.9μmol / / L 0.0158 0.0025

[0051] From the comparative test results in Table 1, it can be seen that the T / C measurement value of the kit prepared by the preparation method of the present invention is obviously better than that of the conventional process, the fold difference is better, and the accuracy is higher.

[0052] The kit prepared by the present invention was subjected to a storage stability test, and the test results are shown in Table 2.

[0053] Table 2 Storage stability test

[0054]

[0055]

[0056] It can be seen from the test results in Table 2 that the test kit prepared by the present invention is stable within 24 months when stored at room temperature (10-30° C.), and the test results are still accurate.

[0057] For ordinary technicians in this field, the specific 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 concepts and technical solutions of the present invention, or the concepts and technical solutions 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 homocysteine ​​detection kit, characterized in that: The homocysteine ​​detection kit comprises a nitrocellulose membrane coated with a recombinant S-adenosylmethionine antigen, fluorescent microspheres labeled with SAH antibodies, and a recombinant S-adenosylhomocysteine ​​hydrolase.

2. The homocysteine ​​detection kit according to claim 1, characterized in that The test kit comprises a reagent strip, a gun tip and a sample diluent. The reagent strip comprises a base plate, a nitrocellulose membrane, a conjugation pad, a sample pad and absorbent paper. The sample pad is connected to the conjugation pad. The two ends of the nitrocellulose membrane are respectively connected to the conjugation pad and the absorbent paper and then bonded to the base plate.

3. The homocysteine ​​detection kit according to claim 2, characterized in that: The nitrocellulose membrane is coated with S-adenosylmethionine recombinant antigen and goat anti-chicken IgY.

4. The homocysteine ​​detection kit according to claim 2, characterized in that The conjugate pad is coated with fluorescent microspheres of SAH monoclonal antibody; the fluorescent microspheres are prepared from rare earth europium fluorescent microspheres; the fluorescent microspheres of SAH monoclonal antibody are fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody, and the conjugate pad is also coated with fluorescent microspheres 2 labeled with chicken IgY.

5. The homocysteine ​​detection kit according to claim 2, characterized in that: The sample diluent includes citrate buffer, dithiothreitol and ProClin 300, pH=7.4±1.

6. The homocysteine ​​detection kit according to claim 2, characterized in that: The gun tip is embedded with S-adenosylhomocysteine ​​recombinant hydrolase.

7. The homocysteine ​​detection kit according to claim 5, characterized in that: The concentration of the citrate buffer in the sample diluent is 50 mM, the concentration of dithiothreitol is 1.5 mg / mL; and the concentration of ProClin 300 is 0.1%.

8. A method for preparing a homocysteine ​​detection kit, characterized in that: The specific steps include: S1 Preparation of sample diluent: Add citrate with a final concentration of 50 mM, dithiothreitol DTT with a final concentration of 1.5 mg / mL, and ProClin 300 with a final concentration of 0.1% into a container, mix well, make up to volume, and adjust the pH to 7.4 ± 1 after mixing to obtain a sample diluent; S2: preparing the conjugate pad: preparing fluorescent microsphere 1 labeled with mouse SAH monoclonal antibody and fluorescent microsphere 2 labeled with chicken IgY respectively, diluting the fluorescent microsphere 1 labeled with mouse SAH monoclonal antibody and the fluorescent microsphere 2 labeled with chicken IgY with buffer, and spraying them onto the conjugate pad using a gold sprayer at a spray volume of 1-2 ul / cm; S3 Preparation of nitrocellulose membrane: Dilute S-adenosylmethionine SAH recombinant antigen and goat anti-chicken IgY with 20mMPB buffer, and then streak them onto nitrocellulose membrane with a streaker, with a coating amount of 0.5-1ul / cm; S4 Preparation of pipette tip: Dilute the recombinant S-adenosylhomocysteine ​​hydrolase with the preservation solution, apply 5ul of the solution to the pipette tip, place it in a vacuum dryer, and drain for 10-12 hours. S5: Prepare the reagent strip: assemble the reagent plate by sticking the binding pad, nitrocellulose membrane, sample pad and absorbent paper on the bottom plate, and then cut them into the reagent strip according to the size; S6 Preparation of the kit: Package the sample diluent, pipette tip, and reagent strip to make a homocysteine ​​detection kit.

9. The method for preparing a homocysteine ​​detection kit according to claim 8, characterized in that: The specific steps of preparing the fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody and the fluorescent microspheres 2 labeled with chicken IgY in step S2 are as follows: S21 Preparation of fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody: After the fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, mouse SAH monoclonal antibody is added and reacted at 37°C for 3 hours; then a blocking agent is added for blocking, centrifugation, and storage to obtain fluorescent microspheres 1 labeled with mouse SAH monoclonal antibody for standby use; S22 Preparation of fluorescent microspheres 2 labeled with chicken IgY: The fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, and chicken IgY is added. The reaction is carried out at 37°C for 3 hours. Then, a blocking agent is added for blocking, centrifugation, and storage to obtain fluorescent microspheres 2 labeled with chicken IgY for use.

10. The method for preparing a homocysteine ​​detection kit according to claim 8, characterized in that: The ratio of the fluorescent microspheres to the antibody in the mouse SAH monoclonal antibody in step S21 is 1:2-1:10; the ratio of the fluorescent microspheres to the antibody in the chicken IgY in step S22 is 1:2-1:10.

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