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Kit for detecting lipoprotein-associated phospholipase a2 and its preparation and application

A phospholipase and lipoprotein technology, which is applied in the field of biochemical detection, can solve the problems of insufficient Lp-PLA2 binding, affecting the measurement accuracy, and selecting a single method of enzymatic immunoluminescence antibody coating.

Active Publication Date: 2017-04-05
SHENZHEN NEW INDS BIOMEDICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many interfering components in the measurement of Lp-PLA2 in the whole blood sample, which affects the accuracy of the measurement; and the ELISA antibody coating method is single, and the binding to Lp-PLA2 is not thorough enough, so that the detection sensitivity is not high; the reaction on the orifice plate There are many uncertain factors, resulting in poor repeatability of the test; in addition, the enzyme-linked immunosorbent immunoassay is limited by its degree of automation, which makes the reaction time longer

Method used

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  • Kit for detecting lipoprotein-associated phospholipase a2 and its preparation and application
  • Kit for detecting lipoprotein-associated phospholipase a2 and its preparation and application
  • Kit for detecting lipoprotein-associated phospholipase a2 and its preparation and application

Examples

Experimental program
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Effect test

preparation example Construction

[0090] The following is the preparation method of each component of the kit:

[0091] Preparation 1: Preparation of magnetic sphere suspension of monoclonal antibody or polyclonal antibody coated with Lp-PLA2

[0092] The immunomagnetic spheres used in this preparation step are selected from the nano-magnetic microsphere suspension produced by Merck Company with a concentration of 100 mg / ml and a hydroxyl group of 95 mgKOH / g.

[0093] (1) Preparation of buffer:

[0094] Weigh 2.55g of sodium acetate trihydrate, dissolve it in 4500ml of purified water, add 14ml of acetic acid, and mix well to obtain an acetate buffer solution with a pH of 3.6.

[0095] (2) Magnetic microsphere connection (magnetic microsphere connection CMC method):

[0096] Suspend the magnetic microspheres in the above-mentioned pH3.6 acetate buffer solution 5 times of the coating volume, so that the concentration of the magnetic beads is 20mg / ml; then add 1-cyclohexyl-2-morpholinoethylcarbodiimide to Tosy...

Embodiment 1

[0164] Using the first anti-Lp-PLA2 antibody, prepare a suspension of magnetic spheres coated with the first anti-Lp-PLA2 antibody according to Preparation 1 above.

[0165] Using the second anti-Lp-PLA2 antibody, a second anti-Lp-PLA2 antibody solution labeled with ABEI was prepared according to Preparation 4 above.

[0166] Preparation of replacement agent: Measure 1000ml of purified water into a beaker, weigh 15g CHAPS, 0.5g casein, 2gEDTA-2Na, 6g DDT, 1g Tris, 3g MES and 2g NaN 3 Once dissolved in water, after it is completely dissolved, measure and add 150ml of newborn bovine serum, 5ml of acetic acid, 25ml of horse serum, 10ml of glycerol and 25g of BSA, and then filter after fully mixing to obtain the replacement agent solution.

[0167] Preparation of calibrator dilution: Accurately weigh 0.2g KH with an analytical balance 2 PO 4 , 2.9g Na 2 HPO 4 12H 2 O and 8g NaCl, 5g BSA, 2g NaN 3 and 0.125g MgCl 2 , slowly add purified water to adjust the volume to 1000ml, ...

Embodiment 2

[0181] The composition and detection method of the kit in this implementation are roughly the same as those in Example 1, the only difference being that no displacing agent is used in this example. The results are shown in Table 1-4, figure 2 .

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Abstract

The invention provides a kit for detecting lipoprotein-associated phospholipase A2 and a preparation method of the kit. The kit comprises one or more first antibodies for resisting the lipoprotein-associated phospholipase A2, and one or more second antibodies for resisting the lipoprotein-associated phospholipase A2, wherein the first antibodies are used for being combined with enveloped magnetic spheres of to-be-tested lipoprotein-associated phospholipase A2; the second antibodies are combined with the to-be-tested lipoprotein-associated phospholipase A2 on other sites which are different from the combined site of the to-be-tested lipoprotein-associated phospholipase A2 and the first antibodies for resisting the lipoprotein-associated phospholipase A2 and are marked with tracer markers. In a preferred scheme, the kit also comprises a displacing agent; and the detection accuracy of the kit can be further improved. The invention further provides a method for detecting the lipoprotein-associated phospholipase A2 by virtue of the kit. The concentration of the lipoprotein-associated phospholipase A2 in the sample can be determined by adopting serum as a detection sample at high repeatability, high accuracy and high sensitivity.

Description

technical field [0001] The invention relates to the detection field of biochemical substances, in particular to a kit for detecting lipoprotein-associated phospholipase A2 and a preparation method thereof, and a method for detecting lipoprotein-associated phospholipase A2. Background technique [0002] Lp-PLA2 is encoded by the PLA2G7 gene, also known as platelet activating factor acetylhydrolase (PAF-AH), which belongs to PLA2 in the phospholipase family and is a serine-dependent phospholipase whose catalytic activity does not require Ca 2+ . The molecular weight of human plasma Lp-PLA2 is 45KDa, which is mainly secreted by macrophages, monocytes, T lymphocytes, mast cells, liver cells, etc., and is regulated by inflammatory mediators. For example, gamma interferon and lipopolysaccharide inhibit its secretion, and platelet-activating factor promotes its secretion. The main functions of Lp-PLA2 include: producing dodecanoic acid inflammatory substances, participating in ph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/573G01N33/577
CPCG01N33/573G01N2333/914G01N2800/50G01N2800/52
Inventor 饶微袁锦云李钦黄科罗凯林冬霞李婷华
Owner SHENZHEN NEW INDS BIOMEDICAL ENG
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