Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof

A chemiluminescence method and chemiluminescence technology, applied in the field of immunoassay, can solve the problems such as the inability to be widely used in clinical diagnosis and scientific research work, the low sensitivity of the ELISA method, and the difficulty in realizing full automation, and achieve a simple and reliable pretreatment process without radioactivity. contamination, the effect of ensuring sensitivity

Inactive Publication Date: 2015-08-05
南京格耀生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latex-enhanced immunoturbidimetric method has the disadvantages of narrow detection linear range and low positive rate; the ELISA method has defects such as low sensitivity, narrow linear range, and difficulty in realizing full automation; thus limiting its popularization and use, it cannot be widely used in clinical diagnosis and scientific research Work

Method used

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  • Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof
  • Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof
  • Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Preparation of a kit for detecting Lp-PLA2 and CRP.

[0026] (1) Preparation of Lp-PLA2 and CRP calibrator:

[0027] Dilute the pure Lp-PLA2 and CRP with pH=7.5 and 0.1M Tris-HCl buffer solution into six levels of lyophilized calibrator, and the target concentrations after reconstitution with pure water are 0, 0.25, 2, and 10 respectively. , 25 and 50 ng / ml. Wherein, the raw materials of the Lp-PLA2 and CRP calibrator are standard grade, and the purity is not lower than 99%.

[0028] (2) Preparation of magnetic particle solution coated with anti-fluorescein isothiocyanate (FITC) polyclonal antibody:

[0029]Apply a magnetic field to magnetic particles with a particle size of 1 μm, let them stand for 15 minutes, pour out the supernatant, wash 3 times with 25mM MES buffer solution with pH=4.7, and suspend with the buffer solution, the concentration is 50mg / mL; Add 2 mg of anti-FITC polyclonal antibody to the suspension, and mix uniformly at room temperature; ...

Embodiment 2

[0041] Example 2 Preparation of a kit for detecting Lp-PLA2 and CRP.

[0042] Basically the same as Example 1, the difference is that the magnetic particles coated with anti-fluorescein isothiocyanate polyclonal antibody are suspended in the diluent to prepare a 0.5 μg / mL magnetic particle solution; the fluorescein isothiocyanate-labeled Dissolve Lp-PLA2 monoclonal antibody in diluent to prepare 0.25 μg / mL FITC-labeled Lp-PLA2 monoclonal antibody solution; FITC-labeled CRP monoclonal antibody is dissolved in diluent Prepare 0.25 μg / mL fluorescein isothiocyanate-labeled CRP monoclonal antibody solution; alkaline phosphatase-labeled Lp-PLA2 monoclonal antibody is dissolved in diluent to prepare 0.25 μg / mL alkaline phosphatase-labeled Lp -PLA2 monoclonal antibody solution; alkaline phosphatase-labeled CRP monoclonal antibody was dissolved in diluent to prepare 0.25 μg / mL alkaline phosphatase-labeled CRP monoclonal antibody solution; chemiluminescence substrate solution was 0.1M, ...

Embodiment 3

[0043] Example 3 Detection of the kit prepared in the present invention (the kit prepared in Example 2).

[0044] 1. Sampling: Take 1ml of whole blood or serum;

[0045] 2. Sample testing:

[0046] The reagents in the kit should be equilibrated to room temperature after being taken out from storage conditions before being used for detection; before use, the magnetic particle solution coated with anti-FITC polyclonal antibody should be thoroughly mixed to ensure that the magnetic particles are evenly suspended, but a magnetic stirrer cannot be used Stir; cleaning solution: dilute the cleaning solution 15 times with deionized water and mix well; set up a 37°C water bath; put the chemiluminescence detector in the waiting state; prepare test tubes and mark them as needed.

[0047] Mix 50 μl of calibrator or sample to be tested with 50 μl of FITC-labeled Lp-PLA2 monoclonal antibody solution and ALP-labeled Lp-PLA2 monoclonal antibody solution, and incubate 50 μl of calibrator or s...

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Abstract

The invention discloses a kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, wherein the kit comprises: magnetic particles coated by an anti-fluorescein isothiocyanate polyclonal antibody, an Lp-PLA2 monoclonal antibody marked by fluorescein isothiocyanate, a CRP monoclonal antibody marked by fluorescein isothiocyanate, an Lp-PLA2 monoclonal antibody marked by alkaline phosphatase, a CRP monoclonal antibody marked by alkaline phosphatase, a chemiluminescent substrate liquid with alkaline phosphatase catalytic luminescence, a diluent liquid, a cleaning agent, an Lp-PLA2-series standard substance, and a CRP-series standard substance. The kit of detecting the content of Lp-PLA2 and CRP is used in a reaction mode of a double antibody sandwich method, so that the technical principle of chemiluminescent detection with a magnetic particle immune-separation technology is effectively utilized, by that the content of the Lp-PLA2 and the CRP in human serum or blood plasma samples are quantitatively measured and detection sensitivity is ensured. The kit is low in pre-treatment requirement on samples, is simple and reliable in the pre-treatment of the samples, can quickly detection large batches of samples in high throughput and is convenient to operate and produce.

Description

technical field [0001] The invention relates to the field of immune analysis, in particular to a kit, method and application for detecting the contents of Lp-PLA2 and CRP based on a chemiluminescent method. Background technique [0002] Ischemic stroke is a common and frequently-occurring disease in clinical practice. The common cause is atherosclerosis leading to stenosis of the lumen and cerebral thrombosis, resulting in interruption of blood flow in the local cerebral blood supply area, resulting in cerebral ischemia, Hypoxia, softening and necrosis. A large number of studies have shown that atherosclerosis itself is an inflammatory process, and continuous low-level inflammation indicates the possibility of cardiovascular and cerebrovascular diseases. Lp-PLA2 is a newly discovered inflammatory marker, which belongs to the non-calcium ion-dependent phosphatase in the phospholipase A2 superfamily. It is mainly secreted by inflammatory cells (such as macrophages, lymphocyte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
CPCG01N33/6893G01N33/573
Inventor 杨子学王书奎
Owner 南京格耀生物科技有限公司
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