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Enzyme linked immunosorbent assay kit used for detecting human ox-LDL (oxidized low-density lipoprotein)

A low-density lipoprotein, enzyme-linked immunosorbent assay technology, applied in the field of medical testing, can solve the problems of poor stability of the kit, affecting large-scale applications, and difficult clinical testing.

Inactive Publication Date: 2015-12-16
GUANGZHHOU HUAHONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the antigen-antibody reaction is very complicated, it is related to many factors. Although the epitope of phosphorylcholine is the same as the epitope presented on the surface of low-density lipoprotein after oxidative modification, anti-phosphorylcholine antibody and oxidized low-density lipoprotein The specificity of antigen-antibody reaction is difficult to predict, and the accuracy of detecting oxidized low-density lipoprotein with anti-phosphorylcholine antibody is not high enough, so it is difficult to apply to clinical detection
[0004] Patent CN201210160797 discloses an ELISA detection kit for human oxidized low-density lipoprotein. The kit has good sensitivity and accuracy, but in practical applications, the kit has poor stability and low yield of monoclonal antibodies. Affect its large-scale application

Method used

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  • Enzyme linked immunosorbent assay kit used for detecting human ox-LDL (oxidized low-density lipoprotein)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Preparation of anti-human oxidized low-density lipoprotein antibody: culture hybridoma cells (preservation number: CCTCCNO: C200304) expressing anti-OxLDL monoclonal antibody in vitro; the number of inoculated cells is 2×10 5 / ml; the composition of the culture medium was 97%DMEM / F12:RPMI1640=1:1; 3%FBS; the rolling speed was 0.5rpm; all the medium was changed once every 96 hours, and the medium was changed twice and harvested three times. And when the cells are cultured, add 10IU / 10ml LIF (leukocyte inhibitory factor), 2IU / 10ml HCG, 2IU / 10ml bovine insulin to the culture medium. The culture supernatant is filtered and clarified, purified, and the antibody is collected;

[0036] (2) Preparation of an ELISA plate coated with anti-human oxidized low-density lipoprotein antibody:

[0037] a. Antibody dilution: Dilute the anti-human oxidized low-density lipoprotein monoclonal antibody to 2.5-20 μg / ml with a 50 mM Tris-HCl buffer solution with a pH of 8.0 to obtain a co...

Embodiment 2

[0061] Embodiment 2 kit sensitivity measurement

[0062] Prepare PBS buffer solution of different concentrations of Ox-LDL standard substance respectively, concentration is respectively 0.1U / L, 0.5U / L, 1U / L, 5U / L, 10U / L, 20U / L, adopts the reagent prepared in embodiment 1 Cartridge is detected, with control buffer as blank control, and use contrast ratio kit to carry out the same detection at the same time, contrast ratio is the ELISA kit that patent CN201210160797 embodiment 1 prepares, and specific detection method is as follows:

[0063] a) Antigen-antibody reaction: Add 50 μl of standard solution and diluent to the microwells of the coated microtiter plate, and incubate in a water bath at 37° C. for 50 minutes. Wash the plate with washing buffer 5 times.

[0064] b) Add the HRP-labeled anti-apoB antibody solution to each well, 100 μl per well, and incubate in a 37° C. water bath for 50 minutes. Repeat the plate washing operation 5 times.

[0065] c) Chromogenic reactio...

Embodiment 3

[0071] The anti-human oxidized low-density lipoprotein antibody was prepared according to the method in Example 1 of patent CN201210160797, and the antibody content in the supernatant before purification was measured by HPLC method. At the same time, the same measurement was performed on Example 1 of the present invention, and the results showed that: Example 1 of the present invention 1 The antibody content in the harvested supernatant was 9.7 mg / L, while the antibody content in the supernatant of Patent CN201210160797 Example 1 was 3.8 mg / L.

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Abstract

The invention belongs to the field of medical detection, particularly relates to an enzyme linked immunosorbent assay kit used for detecting human ox-LDL (oxidized low-density lipoprotein). The linked immunosorbent assay kit comprises components as follows: (1), an elisa plate coated with human ox-LDL antibodies; (2), standard products in human ox-LDL series; (3), an enzyme-labeled antibody solution; (4), a diluent; (5), a washing liquid; (6), a substrate solution; (7), a developing solution; (8), a stop solution.

Description

Technical field [0001] The present invention is a medical testing field, which is specifically involved in an enzyme -linked immune kit used to detect human oxidative low -density lipoprotein. Background technique [0002] Atherosclerosisis refers to a lesions that are characterized by thickening, hardening and decreased elasticity of the arterial wall, and characteristics characterized by the endometrial porridge.At present, the cause of the disease is not completely determined, which may be related to age, gender, blood lipids, hypertension, smoking, diabetes, obesity, infection and other factors.High cholesterol concentration, especially low -density lipoprotein (LDL) is a major risk factor for AS.Research in recent years has found that low -density lipoprotein has greatly strengthened its AS effect after oxidation and modification.At present, it is believed that oxidized low-density lipoprotein (OX-LDL) is mainly involved in the formation of atherosclerosis from endothelial c...

Claims

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Application Information

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IPC IPC(8): G01N33/92
CPCG01N33/92G01N2800/324
Inventor 陈立国
Owner GUANGZHHOU HUAHONG BIOLOGICAL TECH
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