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CN-122017257-A - Detection method and detection kit for oxidized low-density lipoprotein

CN122017257ACN 122017257 ACN122017257 ACN 122017257ACN-122017257-A

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a detection method and a detection kit for oxidized low-density lipoprotein. The invention provides a new application of quaternized chitosan in a kit, in particular to application of quaternized chitosan with different molecular weights in an immunonephelometry detection process of oxidized low-density lipoprotein, which shows that when the quaternized chitosan with a large molecular weight of 100-200 kDa and the quaternized chitosan with a small molecular weight of 30-50 kDa are used as components of the oxidized low-density lipoprotein detection kit, gel particles with different mechanical strengths can be formed by virtue of reaction of the quaternized chitosan with different molecular weights and citrate, the detection of the content of the oxidized low-density lipoprotein can be realized by an immunonephelometry method, and the use of the quaternized chitosan with different molecular weights greatly improves the sensitivity and the linear range of the oxidized low-density lipoprotein detection kit.

Inventors

  • HAN ZHIXUAN
  • WANG JULIANG
  • YANG JINLONG
  • ZHANG BEI
  • ZENG QI

Assignees

  • 济南玖方生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260413

Claims (8)

  1. 1. The application of the quaternary ammonium salt chitosan in the detection of oxidized low-density lipoprotein.
  2. 2. The use according to claim 1, wherein the large molecular weight quaternized chitosan, the small molecular weight quaternized chitosan and the soluble salt containing citrate are respectively used in the detection process of oxidized low density lipoprotein, gel particles with different mechanical strength are formed by the reaction of the different molecular weight quaternized chitosan and the citrate, the detection of the content of the oxidized low density lipoprotein is realized by an immunonephelometry, the molecular weight of the large molecular weight quaternized chitosan is 100-200 kDa, and the molecular weight of the small molecular weight quaternized chitosan is 30-50 kDa.
  3. 3. The detection kit for the oxidized low-density lipoprotein is characterized by comprising a reagent R1, a reagent R2 and a reagent R3, wherein the reagents comprise the following components in terms of mass volume concentration: the reagent R1 comprises 0.03-0.6% of large molecular weight quaternized chitosan, 0.6-0.9% of KCl, 0.5-3% of polyethylene glycol 6000 and 50-100 mM of buffer solution; The reagent R2 is citrate buffer solution 2.0% -10.0% and pH 7.0-7.2; The reagent R3 comprises 0.02% -0.3% of small molecular weight quaternized chitosan, 0.01% -0.05% of oxidized low-density lipoprotein antibody marked by latex microspheres, 0.1% -0.5% of ethanolamine, 1% -5% of trehalose and 50-100 mM of buffer solution; The buffer solution in the reagent R1 and the reagent R3 is independently selected from any one of HEPES buffer solution, tris-HCl buffer solution, MES buffer solution, MOPS buffer solution, TAPS buffer solution and glycine buffer solution.
  4. 4. The kit for detecting oxidized low-density lipoprotein according to claim 3, wherein the molecular weight of the large-molecular-weight quaternized chitosan in the reagent R1 is 100-200 kDa, and the molecular weight of the small-molecular-weight quaternized chitosan in the reagent R3 is 30-50 kDa.
  5. 5. The kit for detecting oxidized low-density lipoprotein according to claim 3, wherein the citrate buffer solution in the reagent R2 is selected from one or more of sodium dihydrogen citrate, potassium dihydrogen citrate, disodium hydrogen citrate, dipotassium hydrogen citrate, trisodium citrate and tripotassium citrate buffer solution, and the pH is adjusted to 7.0-7.2 by using hydrochloric acid and sodium hydroxide solution.
  6. 6. The kit for detecting oxidized low-density lipoprotein according to claim 3, wherein the reagent R1 and the reagent R3 further comprise a bacteriostat with a mass volume concentration of 0.05% -0.15%, and the bacteriostat comprises, but is not limited to, nipagin esters, isothiazolinones, quaternary ammonium salts, potassium sorbate and proclin 300.
  7. 7. The kit for detecting oxidized low-density lipoprotein according to claim 3, wherein the pH of the reagent R1 and the reagent R3 are both 7.0.+ -. 0.5.
  8. 8. The method for detecting oxidized low density lipoprotein by using the kit according to any one of claims 3 to 7, wherein a sample to be detected is mixed with a reagent R1, then a reagent R2 is added for incubation for 2 to 5min, then a reagent R3 is added for mixing after the incubation is finished, an absorbance value A1 is immediately read at 570 nm, the reaction is carried out for 5 to 8 min at 35 to 38 ℃, an absorbance value A2 is read again, and the degree of reaction is calculated A=a2-A1, converted to the corresponding content of oxidized low density lipoprotein according to a standard curve.

Description

Detection method and detection kit for oxidized low-density lipoprotein Technical Field The invention belongs to the technical field of biological detection, and particularly relates to a detection method and a detection kit for oxidized low-density lipoprotein. Background Oxidized low density lipoprotein (ox)LDL) is an important marker of atherosclerosis, the level of which is closely related to the occurrence and development of cardiovascular diseases. Accurate and rapid detection of oxLDL is of great importance for early diagnosis and prevention of cardiovascular diseases. Existing oxThe LDL detection method mainly comprises an enzyme-linked immunosorbent assay (ELISA), a chemiluminescent immunoassay (CLIA), a Latex Enhanced Immunoturbidimetry (LEIA) and the like, wherein, latex microspheres are used for marking anti-ox-LDL antibodies, the particle size of a compound formed after the combination of the anti-ox-LDL antibodies is increased, and the ox-LDL quantitative LEIA method is realized through turbidity change, so that the method has the advantages of simple operation and high detection speed, but the problems of weak signal response, insufficient sensitivity, narrow linear range and the like of the traditional single-particle-size latex microsphere marked LEIA are solved, and the accurate detection requirements of low-concentration and high-concentration ox-LDL are difficult to meet, so that the double-particle-size latex microsphere marked detection method is developed. However, existing oxs based on dual particle size latex microsphere markersLDL detection methods still suffer from a number of technical drawbacks, such as: Firstly, the preparation process of the double-particle-size latex microsphere kit is complex, as disclosed in patent CN119667179A, two latex microspheres involved in the kit preparation need to be marked in two steps, the surface modification conditions and antibody marking parameters of the two particle-size latex microspheres need to be optimized independently, the process parameters are difficult to unify, and the production flow of the kit is complex and time-consuming; secondly, the sensitivity and the linear range of detection concentration of the immunoturbidimetry are affected by the concentration ratio of the antigen to the labeled antibody, so that the problems of low detection sensitivity and narrow linear range and incapability of fundamentally changing the imbalance of the concentration ratio between the labeled antibody and the antigen can be solved. Therefore, there is a need for improving the prior art and providing a new idea to further improve the sensitivity of the immunoturbidimetry and expand the linear range of detection. Disclosure of Invention In order to solve the technical problems, the invention provides a detection method and a detection kit for oxidized low-density lipoprotein. The invention controls the reaction proportion of the antigen and the labeled antibody by controlling the release speed of the latex microsphere labeled antibody (namely the oxidized low density lipoprotein antibody labeled by the latex microsphere) and the free antigen in the sample to be detected, so that the labeled antibody and the antigen to be detected can be positioned between the optimized reaction proportion of the antigen and the labeled antibody, namely the equivalent band period, in the reaction process, so as to form a stronger turbidity signal. The technical scheme provided by the invention is as follows: The first aspect of the invention provides a new application of quaternary ammonium salt chitosan, in particular to an application of quaternary ammonium salt chitosan in oxidized low-density lipoprotein detection. Preferably, the application is that the quaternary ammonium chitosan with different molecular weights (comprising large molecular weight quaternary ammonium chitosan and small molecular weight quaternary ammonium chitosan) and soluble salt containing citrate are used in the detection process of oxidized low-density lipoprotein, gel particles with different mechanical strengths can be formed by means of the reaction of the quaternary ammonium chitosan with different molecular weights and the citrate, the mechanical strengths of the gel particles are different, the diffusion resistance of the gel particles to antigen and labeled antibody is different, so that the diffusion speed of the antigen and the labeled antibody is regulated, the antigen and the labeled antibody are contacted in a proper proportion to perform immune reaction to form immune complex, the detection of the content of the oxidized low-density lipoprotein is realized by an immune turbidimetric method, the molecular weight of the quaternary ammonium chitosan with the large molecular weight is 100-200 kDa, and the molecular weight of the quaternary ammonium chitosan with the small molecular weight is 30-50 kDa. In a second aspect of the present invention, there is provided a method for d