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Liquid-chip kit for quantitatively detecting concentration of lipoprotein phospholipase A2 in sample and preparation method thereof

A quantitative detection and lipoprotein technology, applied in measurement devices, instruments, disease diagnosis, etc., can solve problems such as high false negative rate, and achieve the effects of high accuracy, high speed, and wide linear range.

Inactive Publication Date: 2015-08-05
北京协和洛克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, the immunodiagnostic methods for cardiovascular diseases can only detect a single indicator. Due to the differences in sensitivity, accuracy and specificity of different indicators, high false negative rates often occur in clinical diagnosis.

Method used

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  • Liquid-chip kit for quantitatively detecting concentration of lipoprotein phospholipase A2 in sample and preparation method thereof
  • Liquid-chip kit for quantitatively detecting concentration of lipoprotein phospholipase A2 in sample and preparation method thereof
  • Liquid-chip kit for quantitatively detecting concentration of lipoprotein phospholipase A2 in sample and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The operation method of the lipoprotein phospholipase A2 (LP-PLA2) concentration liquid chip detection kit of the present invention is as follows:

[0036] 1. Reconstitute the standard and quality control with deionized water and mix well, add standard 50 μl / well, quality control and sample 10 μl and 40 μl sample buffer / well to the microplate respectively;

[0037] 2. Dilute the antibody-coated magnetic particles by 25 times, then add 50 μl of magnetic particles to the microwell plate, and shake at room temperature for 120 minutes;

[0038] 3. Wash the plate twice with a plate washer;

[0039] 4. Add 25 μl / well of biotin-labeled monoclonal antibody to the microwell plate, and place it on a shaker for 60 minutes at room temperature;

[0040]5. Add 25 μl / well of streptavidinized phycoerythrin to the microwell plate, and place it on a shaker for 30 minutes at room temperature;

[0041] 6. Wash the plate twice with a plate washer;

[0042] 7. Add 150 μl / well sheath solut...

Embodiment 2

[0046] Prepare the streptavidinized phycoerythrin for this kit by:

[0047] Streptavidinylation of Phycoerythrin:

[0048] 1) Desalination treatment of phycoerythrin

[0049] Purified R-PE is generally stored in 60% saturated ammonium sulfate buffer solution, and the supernatant must be removed by centrifugation before use, and resuspended in 50mmol / L PBS, and ammonium sulfate is removed using a desalting column.

[0050] 2) Derivatization of phycoerythrin

[0051] Dissolve 4.5 mg of phycoerythrin in 1.0 mL of phosphate buffer, add 10 μL of 3-(2-pyridyldimercapto)propionic acid N-hydroxysuccinimide ester (SPDP) anhydrous methanol solution (4 mg / mL), Make the molar ratio of SPDP to phycoerythrin about 10, react at room temperature for 120 minutes, desalt through Sephadex gel chromatography, equilibrate and elute with phosphate buffer, and collect the peak of phycoerythrin solution.

[0052] 3) Thioylation of streptavidin

[0053] Get 0.5 mg of streptavidin, add the above-me...

Embodiment 3

[0059] Prepare the kit by:

[0060] Preparation of microspheres coated with monoclonal antibodies against lipoprotein phospholipase A2: Take 5.0×10 microspheres without coating 6 One, add 60 μL of 100 mM sodium dihydrogen phosphate buffer solution with a pH value of 6.2, 20 μL of 25 mg / mL N-hydroxysulfosuccinimide solution and 20 μL of 25 mg / mL carbodiimide solution for activation, and then add specific 25 μg of specific monoclonal antibody for coating; then add blocking solution containing 1% bovine serum albumin for blocking; finally store in 10mM phosphate buffer saline in the dark at 4°C;

[0061] Preparation of concentration gradient standard and quality control of lipoprotein phospholipase A2: 10 mM sodium phosphate buffer containing 1% mass fraction of newborn bovine serum, 0.01% Proclin300, 1% bovine serum albumin, 0.05% Triton X-100 Solution (PH7.0) The pure product of the marker was prepared into concentration gradient standards with marked concentrations of 0, 50, ...

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Abstract

The invention belongs to the technical field of in-vitro immunodetection and particularly relates to a kit for detecting the concentration of lipoprotein phospholipase A2 in a sample and a preparation method thereof. The kit comprises microspheres coated with lipoprotein phospholipase A2, a micropore reaction plate, a standard product and a quality control product of a concentration gradient of lipoprotein phospholipase A2, a biotin-marked lipoprotein phospholipase A2 antibody, streptavidin-marked phycoerythrin, and a phosphate buffer solution containing a protein protection agent. Detection instruments used for the detection is MAGPIX of Luminex and Luminex 200, 3D; on the platform, measurement for the concentration of lipoprotein phospholipase A2 of human plasma by high flux, high speed, low cost, high accuracy and good repeatability can be realized, and simultaneously, detection markers of different types can be freely combined and added, so that simultaneous detection of multiple markers is realized, the accuracy rate for diagnosis of cardiovascular diseases is increased and the kit has important application prospect.

Description

technical field [0001] The invention belongs to the technical field of in vitro immunoassay, and in particular relates to a kit for detecting the concentration of lipoprotein phospholipase A2 in a sample and a preparation method thereof. Background technique [0002] Lp-PLA2 belongs to the phospholipase A2 superfamily and is a serine lipase composed of 441 amino acid residues with a relative molecular weight of 45kD. Unlike other members of the phospholipase A2 family, Lp-PLA2 does not require calcium ions to maintain its catalytic activity, and it has the activity of degrading platelet-activating factor, so it is also called platelet-activating factor acetylhydrolase (platelet-activating factor acetylhydrolase, PAF-AH). Lp-PLA2 in the blood is mainly synthesized and secreted by mature macrophages and lymphocytes, exists in large quantities in atherosclerotic sites, and can be regulated by inflammatory mediators. Active Lp-PLA2 can preferentially hydrolyze the ester bond i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/573G01N33/531G01N2800/32
Inventor 郑乐民张立峰李晓燕马志军吴建榕
Owner 北京协和洛克生物技术有限责任公司
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