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CN-121978357-A - Stable oxidized low-density lipoprotein cholesterol detection kit and preparation method thereof

CN121978357ACN 121978357 ACN121978357 ACN 121978357ACN-121978357-A

Abstract

The invention relates to the technical field of detection reagent preparation, and discloses a stable oxidized low-density lipoprotein cholesterol detection kit and a preparation method thereof, wherein the kit is composed of a component R1 and a component R2, the component R1 at least comprises a buffer solution, a sensitizer, a surfactant, a stabilizer, an anti-interference agent and a preservative, the component R2 at least comprises an ion buffer solution, a stabilizer, a blocking agent, an antibody-carboxyl microsphere conjugate and a preservative, and the antibody-carboxyl microsphere conjugate is an oxidized low-density lipoprotein antibody and carboxyl microsphere conjugate. The formula of the oxidized low-density lipoprotein reagent composition, reagent R1 and reagent R2 disclosed by the invention not only effectively reduces the production cost, but also can be used on a full-automatic biochemical analyzer, has high automation degree, has higher accuracy, repeatability and stability compared with similar products, and achieves unexpected technical effects.

Inventors

  • SU MINGXI
  • Si Dalei
  • YU MAN

Assignees

  • 柏定生物工程(北京)有限公司

Dates

Publication Date
20260505
Application Date
20260304

Claims (10)

  1. 1. The stable oxidized low-density lipoprotein cholesterol detection kit is composed of a component R1 and a component R2, and is characterized in that the component R1 at least comprises a buffer solution, a sensitizer, a surfactant, a stabilizer, an anti-interference agent and a preservative, and the component R2 at least comprises an ion buffer solution, a stabilizer, a blocking agent, an antibody-carboxyl microsphere conjugate and a preservative; The antibody-carboxyl microsphere conjugate is a conjugate of an oxidized low density lipoprotein antibody and a carboxyl microsphere.
  2. 2. The stabilized oxidized low-density lipoprotein cholesterol assay kit according to claim 1, wherein the buffer in component R1 is at least one of Tris-HCl buffer, glycine-NaOH solution, sodium glutamate solution, borate buffer, carbonate-bicarbonate buffer.
  3. 3. The stabilized oxidized low-density lipoprotein cholesterol assay kit according to claim 1, wherein said sensitizer in component R1 is at least one of polydextrose sulfate, polyethylene glycol, heparin sodium, dextran sulfate, polyvinylpyrrolidone.
  4. 4. The stabilized oxidized low-density lipoprotein cholesterol assay kit according to claim 1, wherein the surfactant in component R1 is at least one of poloxamer 188, triton X-100, tween-80, tween-20, and sodium deoxycholate.
  5. 5. The stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, wherein the stabilizer is a 10-15mmol/L NaCl solution containing 0.50% -2.0% sodium octoate, 0.05% -0.10% bovine serum albumin, 0.20% -3.00% galactose, in parts by mass in the component R1.
  6. 6. The stable oxidized low-density lipoprotein cholesterol detection kit according to claim 1, wherein the preservative is an HPO solution containing gentamicin, and the mass ratio of gentamicin to HPO is (0.01-0.05).
  7. 7. The stabilized oxidized low-density lipoprotein cholesterol test kit according to claim 1, wherein the stabilizer in the component R2 is prepared from galactose and lactoferrin in a mass ratio of (0.1-1): 0.1-0.2.
  8. 8. The stable oxidized low-density lipoprotein cholesterol assay kit according to claim 1, wherein in said component R2, the preservative is a kanamycin-containing HPO solution, and the mass ratio of kanamycin to HPO is 0.05 (0.05-0.1).
  9. 9. The stable oxidized low density lipoprotein cholesterol assay kit of claim 1 wherein in component R2, the antibody-carboxyl microsphere conjugate is prepared by: A1, cleaning and re-suspending a carboxyl modified polystyrene microsphere suspension to obtain a microsphere suspension, diluting an anti-human oxLDL monoclonal antibody with a buffer solution to obtain an antibody solution, mixing the microsphere suspension and the antibody solution, and incubating the mixed solution at room temperature; a2, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide solution into the mixed solution of the A1, and carrying out light-shielding reaction for 1-2 hours at room temperature; a3, directly adding a fetal bovine serum albumin solution into the reaction system after the reaction is finished, and incubating the system for 1-2 hours; a4, transferring the reaction solution of the A3 to a centrifuge tube, adding a coupling cleaning buffer solution, centrifuging, discarding the supernatant, repeatedly adding the coupling cleaning buffer solution and centrifuging, and cleaning for 2-3 times to obtain the antibody-carboxyl microsphere conjugate.
  10. 10. The method for preparing a stabilized oxidized low-density lipoprotein cholesterol detection kit according to any one of claims 1 to 9, which comprises the steps of: s1, sequentially adding a buffer solution, a stabilizer and a sensitizer into deionized water, stirring until the materials are dissolved, adding a surfactant and a preservative, adjusting the pH value of a system to 8-9 after the deionized water is subjected to constant volume, filtering, sterilizing and preserving in a dark place to obtain an R1 component; S2, preparing an R2 component according to a formula, filtering and sterilizing the system, pre-cooling at 0-4 ℃ to obtain an R2 basic storage solution, taking 10-20 mL, adding an antibody-carboxyl microsphere conjugate into the basic storage solution, and performing ultrasonic treatment under ice bath to obtain a heavy suspension, wherein the heavy suspension is diluted by using the R2 basic storage solution to ensure that the concentration of the antibody-microsphere conjugate in a final R2 reagent is 0.10-0.30%, so as to obtain the R2 component; S3, sub-packaging the R1 component and the R2 component into double reagent bottles according to a preset volume ratio to obtain the stable oxidized low-density lipoprotein cholesterol detection kit.

Description

Stable oxidized low-density lipoprotein cholesterol detection kit and preparation method thereof Technical Field The invention relates to the technical field of detection reagent preparation, in particular to a stable oxidized low-density lipoprotein cholesterol detection kit and a preparation method thereof. Background Oxidized low density lipoprotein (oxLDL) is the product of the oxidation modification of polyunsaturated fatty acids in low density lipoprotein. In the cardiovascular disease risk assessment field, oxidized low density lipoprotein is used as a key pathogenicity modified lipoprotein, and the accurate detection of the oxidized low density lipoprotein has important clinical significance. However, the existing detection technology has a plurality of defects, which limit the wide application and the reliability of results. The traditional ELISA method has the defects of complicated operation steps, time consumption, serious dependence on manual operation, difficulty in realizing high-throughput automatic detection, poor reagent stability, large batch-to-batch variation and incapability of meeting the requirements of quick, efficient and standardized modern clinical laboratories. Although detection technology based on latex-enhanced immunoturbidimetry is developed subsequently, automated analysis is realized initially, but the core performance is still not ideal. The existing method has the defects of general insufficient reagent stability, easy inactivation of the antibody in the storage process, easy non-specific aggregation or sedimentation of latex microspheres, short reagent validity period and detection signal drift. Meanwhile, the anti-interference capability of the method is weak, and common conditions such as high fat, hemolysis, jaundice and the like in serum of a patient are very easy to generate background interference on turbidity detection, so that the false increase or decrease is caused. More importantly, the existing method has the defects of insufficient optimization of the coupling process of the antibody and the microsphere, low coupling efficiency and insufficient exposure of active sites, so that the detection sensitivity is limited, a low-concentration sample is difficult to accurately measure, and a wide linear range between the sensitivity and the upper detection limit is difficult to obtain. In addition, the design of a core reaction system of the existing kit is not perfect enough, and a synergistic effect cannot be formed by a buffer environment, a polymerization promotion condition and a sealing scheme, so that the precision and the accuracy of detection are difficult to simultaneously meet the clinical high-end requirements. Therefore, developing an oxidized low-density lipoprotein detection kit which is high in stability, high in sensitivity, strong in interference resistance and suitable for a full-automatic platform becomes a technical problem to be solved urgently. Disclosure of Invention Technical problem to be solved Aiming at the defects of the prior art, the invention provides a stable oxidized low-density lipoprotein cholesterol detection kit and a preparation method thereof. Technical proposal In order to achieve the above purpose, the present invention provides the following technical solutions: A stable oxidized low-density lipoprotein cholesterol detection kit is composed of a component R1 and a component R2, wherein the component R1 at least comprises a buffer solution, a sensitizer, a surfactant, a stabilizer, an anti-interference agent and a preservative, and the component R2 at least comprises an ion buffer solution, a stabilizer, a blocking agent, an antibody-carboxyl microsphere conjugate and a preservative; The antibody-carboxyl microsphere conjugate is a conjugate of an oxidized low density lipoprotein antibody and a carboxyl microsphere. Further, in the component R1, the buffer solution is at least one of Tris-HCl buffer solution, glycine-NaOH solution, sodium glutamate solution, borate buffer solution and carbonate-bicarbonate buffer solution. Further preferably, the buffer solution is a sodium glutamate solution with the concentration of 50-150mmol/L. In the above preferred technical scheme, the buffer solution is sodium glutamate solution, and the pH value of the reaction system is maintained in a stable range of 8.0-9.0, which is favorable for antigen-antibody combination and also favorable for maintaining the activity of each component. Further, in the component R1, the sensitizer is at least one of polydextrose sulfate, polyethylene glycol, heparin sodium, dextran sulfate and polyvinylpyrrolidone. Further preferably, in the component R1, the sensitizer is polydextrose sulfate. In the above preferred technical scheme, dextran sulfate is selected as a sensitizer, which is a polymer with a great amount of negative charges, can neutralize the negative charges of oxLDL itself, and can reduce partial negative charges on the surface of the antibod