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Time-resolved fluorescence immunoassay method of Lp-PLA2 and kit

A technology of time-resolved fluorescence and immunoassay methods, which can be applied in biological testing, measuring devices, and analytical materials, etc. It can solve the problems of time-resolved fluorescence analysis, provision, and lack of kits for Lp-PLA2, and eliminate non-specific Fluorescence interference, wide analysis range, easy automation effect

Inactive Publication Date: 2015-02-18
杨子学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No immunoassay specifically for the detection of Lp-PLA2 by time-resolved fluorescence analysis has been reported nor is there a corresponding kit available

Method used

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  • Time-resolved fluorescence immunoassay method of Lp-PLA2 and kit
  • Time-resolved fluorescence immunoassay method of Lp-PLA2 and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of Lp-PLA2 time-resolved fluorescence analysis detection kit.

[0034] (1) Preparation of experimental solution:

[0035] Concentrated washing solution: 0.2M phosphate buffered saline (20×PBST) containing 1wt% TWEEN-20;

[0036] Washing solution: 0.01M phosphate buffer containing 0.05wt% TWEEN-20; the washing solution is obtained by diluting the concentrated washing solution;

[0037] Enhancement solution: 1L pH3.2 potassium hydrogen phthalate buffer containing 15 μmol β-naphthoyltrichloroacetone (β-NTA), 50 μmol tri-n-octylphosphine oxide TOPO and 1 mL Triton X-100 (Triton X100) .

[0038] (2) Preparation and purification of Lp-PLA2 monoclonal antibody A and Lp-PLA2 monoclonal antibody B:

[0039]Lp-PLA2 monoclonal antibody A and Lp-PLA2 monoclonal antibody B recognize different epitopes of Lp-PLA2 respectively and do not affect each other. Among them, Lp-PLA2 monoclonal antibody A and B can be obtained by monoclonal hybridoma cell technology ...

Embodiment 2

[0053] Example 2 The precision, accuracy and stability test of the Lp-PLA2 time-resolved fluorescence analysis detection kit.

[0054] 1. The accuracy and precision analysis experiment of the kit:

[0055] The standard solution of 207ng / ml Lp-PLA2 (pH7.4, 0.05M phosphate buffer) was tested 20 times. 207ng / ml is the critical value of Lp-PLA2 to predict the high risk of cardiovascular and cerebrovascular diseases. The high accuracy of the method is conducive to the accurate judgment of the risk of disease. The average value of 20 test results was 204.64ng / ml, the standard deviation was 8.26, and the variation rate was 4.03%. The results show that the detection result is close to the actual concentration, and the accuracy is high, and the variation rate is less than 5%, which has good accuracy.

[0056] 2. Kit stability test:

[0057] The storage condition of the kit is 2-8°C. After 6 months of storage, various indicators of the kit were measured and found to be within the nor...

Embodiment 3

[0058] Example 3 Comparison of Lp-PLA2 Time-Resolved Fluorescence Analysis Detection Kit and ELISA Detection Kit.

[0059] Select Lp-PLA2 standard solution with three concentrations of high, medium and low, and analyze it with this kit and ELISA detection kit at the same time. Each concentration is tested 5 times, and the results are compared to determine the consistency of the two methods. . Choose 1000ng / ml for high concentration, 300ng / ml for medium concentration, and 80ng / ml for low concentration. The test results obtained are as follows:

[0060]

[0061] The t-test of paired samples was performed on the two groups of data, and the result was P>0.05, and the difference between the two was not statistically significant, indicating that the two methods had good consistency in the test of high, medium or low concentration of Lp-PLA2 sex.

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Abstract

The invention provides a time-resolved fluorescence immunoassay method of Lp-PLA2. The double antibody sandwich method and the time-resolved fluorescence immunoassay method are used together to measure the content of Lp-PLA2. The time-resolved fluorescence immunoassay method comprises the following steps: adding an Lp-PLA2 standard substance or a centrifuged blood serum sample into respective micropores in a microporous plate coated with an Lp-PLA2 resistant monoclonal antibody A; after oscillatory reaction, washing with a concentrated cleaning solution, then adding a biotin labeled Lp-PLA2 resistant monoclonal antibody B; after oscillatory reaction, washing with the concentrated cleaning solution; adding lanthanide labeled streptavidin, and washing with the concentrated cleaning solution after oscillatory reaction; after adding an enhancement solution and oscillating, performing fluorescence emitting under excitation of ultraviolet light, testing the fluorescence strength cps with a time-resolved fluorescence spectrometer, and determining the content of LP-PLA2 in a sample to be tested in comparison with a standard curve. The time-resolved fluorescence immunoassay method can quantitatively test the content of Lp-PLA2 in the sample, and has the characteristics of high sensitivity, wide analysis range, good stability, quick detection and easy automation.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a time-resolved fluorescent immunological detection method for plasma lipoprotein phospholipase A2 (Lp-PLA2). Background technique [0002] Lp-PLA2 belongs to the phospholipase A2 superfamily and is mainly secreted by inflammatory cells (such as macrophages, lymphocytes, etc.). Lp-PLA2 is a protein composed of 441 amino acids with a relative molecular weight of about 45400. Unlike other members of the phospholipase A2 family, Lp-PLA2 does not require calcium ions to maintain its catalytic activity. Lp-PLA2 has the activity of degrading platelet activating factor, so it is also called platelet activating factor acetylhydrolase, which exists in large quantities in atherosclerotic sites, and can be regulated by inflammatory mediators, and has the effect of promoting atherosclerosis. Lp-PLA2 is a new inflammatory response marker, which is a specific marker reflecting va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/533G01N33/577
Inventor 杨子学
Owner 杨子学
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