Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum

A low-density lipoprotein and chemiluminescence technology, used in biological testing, material testing products, etc., can solve the problems of single component detection, difficult clinical promotion, expensive kits, etc. The effect of low sample consumption

Inactive Publication Date: 2014-03-12
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above detection methods can only detect a single component in a single analysis process, the analysis time is long, the sensitivity is low, and the linear range is narrow, and the foreign ox-LDL kit is expensive, which is not easy to promote clinically

Method used

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  • Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum
  • Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum
  • Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of Immunosensing Array

[0037] First, piranha acid solution (H 2 SO 4 / 30%H 2 o 2 , the volume ratio is 7:3) to soak the glass slide, and let it stand at room temperature for 12 hours to make the glass slide hydroxylated; then rinse it with ultrapure water, dry it with nitrogen, and immerse the activated slide in 1% (v / v) GPTMS / toluene solution (that is, the volume percentage of the GPTMS solution in the toluene solution is 1%), at room temperature (25°C) overnight to make it silanized. Then, rinse with toluene and absolute ethanol in order to remove silane physically adsorbed on the surface of the slide, and blow dry with nitrogen. Finally, use screen printing to process the glass slide to form 48 microwells (diameter 2mm, hole spacing 4mm, 4 rows x 12 columns) on the surface (see figure 1). The rabbit anti-human ox-LDL antibody was diluted to 0.33μg / mL with pH9.6, 0.05M carbonate coating solution, and added to the microwells of 48-well s...

Embodiment 2

[0038] Embodiment 2: the preparation of standard series solution

[0039] Lipoprotein separation: Density gradient ultracentrifugation separated 100 cases of normal people mixed fresh plasma LDL (d=1.019-1.063g / mL). Lp(a) was separated by ultracentrifugation to separate 1.055-1.110g / mL human plasma lipoprotein components, and then subjected to SePharose6B column chromatography.

[0040] Oxidation of lipoproteins: lipoproteins were fully dialyzed by pH 7.4, 0.01mol / L PBS, adjusted protein concentration to 0.5mg / mL, added CuSO 4 To a final concentration of 40 μmol / L, after incubation at 37°C for 20 h, use 1 mmol / L EDTA-Na 2 , pH7.4, fully dialyzed with 0.01mol / L PBS. The Lowry method was used to determine the protein content of purified ox-Lp(a) and ox-LDL.

[0041] Fresh sera from 50 cases of healthy people were collected and mixed, and the mixed sera were calibrated by ELISA method with known concentrations of purified ox-Lp(a) and ox-LDL. It is stipulated that 1U=1mg oxid...

Embodiment 3

[0043] Example 3: Optimization of immune reaction conditions

[0044] (1) Effect of coating antibody concentration Dilute the coating antibody concentrated solution at a ratio of 1:375, 1:750, 1:1500, 1:3000, 1:6000 with pH 9.6, 0.05M carbonate buffer 1.5 μL of antibody solutions of different concentrations were added to the microwells of the array, and after overnight at 4° C., they were blocked at room temperature for 1 hour. Preferably, the concentration of the coating antibody is 0.33 μg / mL (see figure 2 ).

[0045] (2) The influence of the dilution concentration of the enzyme conjugate Dilute the concentrated solution of the enzyme conjugate at the ratio of 1:80, 1:100, 1:200, 1:400, 1:800, 1:1000 (the dilution is containing 5% fetal bovine serum in PBS). As a preference, the dilution ratio of horseradish peroxidase-labeled apolipoprotein (a) [HRP-apo (a)] monoclonal antibody and HRP-apoB polyclonal antibody is 1:100 (see image 3 ).

[0046] (3) Influence of immune...

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Abstract

The invention discloses a method for chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoproteins (a) and low-density oxidized lipoproteins of human serum. According to an analysis system, a multi-component immunosensor modified by silanization is used as a solid-phase carrier; a solid-phase antibody is prepared by covering the carrier with a capturing antibody; a sample to be measured or a standard series solution is added into the solid-phase antibody; after the solid-phase antibody and the sample to be measured or the standard series solution fully react, an antibody labeled with horse radish peroxidase is added to form a sandwich immune compound; finally a chemiluminescence substrate, namely luminal-hydrogen peroxide, is added for reaction; the luminous intensity is detected through a high-resolution charge coupler, so that the oxidized lipoproteins (a) and the low-density oxidized lipoproteins of the human serum can be measured simultaneously, and the immune reaction condition, the enzyme conjugate dilution degree, the detection time and the like are optimized; furthermore, the indexes such as linear range, precision, accuracy and detection limit of the method are checked. The analysis method has the advantages of simplicity in operation, high sensitivity, narrow linear range, high stability and the like and has a certain clinical application value.

Description

technical field [0001] The invention relates to the technical field of immune analysis, in particular to a chemiluminescent imaging immunoassay method for simultaneously measuring human serum oxidized lipoprotein (a) and oxidized low-density lipoprotein. Background technique [0002] Oxidized lipoprotein is currently considered to be an important factor in the development of atherosclerosis (As) and cardiovascular diseases. Both oxidized lipoprotein (a) [ox-Lp(a)] and oxidized low-density lipoprotein (ox-LDL) can cause the accumulation of cholesteryl ester in macrophages through the scavenger receptor pathway, leading to the formation of foam cells; It can also be directly phagocytized by macrophages. At the same time, ox-Lp(a) and ox-LDL can stimulate monocytes to synthesize and secrete adhesion molecules, promote monocyte aggregation, adhere to vascular endothelium, and then transform into macrophages. In addition, ox-Lp(a) and ox-LDL can also stimulate the production of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/92
Inventor 汪俊军于瑞杰宋佳希吴嘉牛冬梅马丽娟
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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