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Kit for rapidly diagnosing lipoprotein-associated phospholipase A2 and use method of kit

A rapid diagnosis, lipoprotein technology, applied in the field of medical biological immunological detection reagents, can solve the problems of unstable material properties, low sensitivity, unsuitable for large batches, automatic detection, etc.

Active Publication Date: 2017-05-31
ANHUI IPROCOM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, 1) spectrophotometry mainly uses PAF thioesters or their analogs as substrates. After the substrate is hydrolyzed by Lp-PLA2, it releases a substance with a 4-nitrophenol group. It is decomposed into 4-nitrophenol, and the change of absorbance caused by this change can be measured at 405nm, so that the activity of the enzyme can be determined, but the detection sensitivity of this method is not high
2) Latex-enhanced turbidimetric method, Lp-PLA2 reacts with the sensitized latex particles with anti-Lp-PLA2 antibody in the reagent in the phosphate buffer system, and agglutination occurs under the action of the aggregation accelerator polyethylene glycol Increase the turbidity and detect the change in the absorbance of the reaction solution at a wavelength of 546nm. The degree of change is proportional to the content of Lp-PLA2 in the sample. However, this method is prone to false positives due to the influence of blood lipid concentration, and is not suitable for large-scale and automated detection.
Currently, there is no relevant research on kits based on ECLI technology for the detection of Lp-PLA2

Method used

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  • Kit for rapidly diagnosing lipoprotein-associated phospholipase A2 and use method of kit
  • Kit for rapidly diagnosing lipoprotein-associated phospholipase A2 and use method of kit
  • Kit for rapidly diagnosing lipoprotein-associated phospholipase A2 and use method of kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 Composition and preparation of detection kit of the present invention

[0048] The test kit of the present invention comprises the following components:

[0049] Reagent a: standard and quality control

[0050] Reagent b: liposome complex

[0051] Reagent c: Magnetic separation reagent

[0052] Reagent d: Assay buffer

[0053] Reagent e: cleaning solution

[0054] The preparation of each reagent in the kit of the present invention comprises the following steps:

[0055] (1) Preparation of lipoprotein-associated phospholipase A2 standard substance and quality control substance

[0056] raw material factory Potassium dihydrogen phosphate Shenzhen Excellence Biotechnology Co., Ltd. Sodium chloride Shenzhen Excellence Biotechnology Co., Ltd. BSA Sigma-Aldrich Sigma-Aldrich (Shanghai) Trading Co., Ltd. Tween-20 Shanghai McLean Biochemical Technology Co., Ltd. lipoprotein-associated phospholipase A2 antigen Abnov...

Embodiment 2

[0086] Example 2 Using the detection kit of the present invention to detect Lp-PLA2

[0087] (1) Preparation of the sample to be tested: the collection of the sample refers to the routine operation, specifically adding the collected venous blood into a test tube or a heparin anticoagulant tube, centrifuging, and taking the supernatant for the experiment.

[0088] (2) Dilute the liposome complex at a ratio of 1:5 with PBS buffer containing 2% (w / v) BSA.

[0089] (3) Take 25 μL diluted liposome complex, 25 μL magnetic separation reagent, 50 μL sample to be tested or lipoprotein-related phospholipase A2 standard, mix, vortex at room temperature for 1 hour, fully react, and form magnetic particles- For the antigen-liposome complex, the reaction solution is sucked into the electrochemical reaction cell, the magnetic particle-antigen-liposome complex is magnetically adsorbed on the electrode surface, and 100 μL of cleaning solution is added to gently rinse the electrode surface for ...

Embodiment 3

[0093] Embodiment 3 The methodological examination of detection kit of the present invention

[0094] The detection kit prepared in the embodiment is tested according to the routine manufacturing and testing procedures in the art, and the results are as follows:

[0095] ① Analytical sensitivity

[0096] Repeat the determination of the zero calibration product (or sample diluent) for no less than 10 times, calculate the mean (x) and standard deviation (s) of the response, and substitute the response of (x+2s) into the dose-response curve to calculate The corresponding concentration value is the minimum detection limit, and the minimum detection limit of the self-made kit obtained is 0.01ng / ml.

[0097] ②Linear relationship

[0098] Taking the logarithm of the concentration of the reference standard in the self-made kit as the abscissa, and the logarithm of the signal value count as the ordinate, the log-log function of the double-logarithmic mathematical model is used to mea...

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Abstract

The invention relates to a kit for rapidly diagnosing lipoprotein-associated phospholipase A2 and a use method of the kit. The kit comprises a standard substance, a quality control substance, a liposome complex, a magnetic separation reagent, an assay buffer solution and a cleaning solution, wherein the inner part of the liposome complex is coated with N-hydroxysuccinimide ester of tris (bipyridine) ruthenium; an antibody for resisting the lipoprotein-associated phospholipase A2 is connected to the surface of the liposome complex by interaction of streptavidin and biotin; the magnetic separation reagent takes magnetic particulates containing amino terminals as a carrier and is connected with the antibody for resisting the lipoprotein-associated phospholipase A2 through the interaction of the streptavidin and the biotin. Compared with an enzyme linked immunosorbent assay kit produced in a foreign Diadexus company, the kit prepared by the invention has the advantages that sensitivity and precision for analyzing the lipoprotein-associated phospholipase A2 are greatly improved; in addition, the kit disclosed by the invention is simple and convenient to operate, quick and easier to popularize.

Description

technical field [0001] The invention belongs to the field of medical biological immunological detection reagents, and in particular relates to a kit for rapid diagnosis of lipoprotein-related phospholipase A2 and a use method thereof. Background technique [0002] Lipoprotein-associated phospholipase A2 (Lp-PLA2) belongs to the phospholipase A2 superfamily, is mainly synthesized and secreted by mature macrophages and T lymphocytes, and is regulated by inflammatory mediators. Lp-PLA2 mainly exists in the form of binding to lipoproteins. It can not only achieve the effect of anti-arteriosclerosis by hydrolyzing platelet activating factor, oxidized phospholipids and other inflammatory mediators, but also hydrolyze oxidized phospholipids in oxidized low-density lipoproteins. pro-atherogenic effect. Its dual characteristics make it a hot spot of attention. [0003] In recent years, more and more studies have proved that Lp-PLA2 is an independent risk factor for vascular endothe...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/573
CPCG01N33/54326G01N33/573
Inventor 陈立国邹伟权张亚丽李庆祥母润红王涛苏焱
Owner ANHUI IPROCOM BIOTECH CO LTD
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