Kit for detecting lipoprotein phospholipase A2 protein concentration

A phospholipase and lipoprotein technology, which is applied in the field of kits for detection of lipoprotein phospholipase A2 (Lp-PLA2) protein concentration, can solve the problem of not truly reflecting the true concentration level of Lp-PLA2, the detection limitations of limited sample types, etc. To achieve the effect of facilitating large-scale promotion and application, satisfying emergency and outpatient rapid diagnosis, and expanding the scope of application

Inactive Publication Date: 2019-04-12
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for accurately detecting lipoproteins for the defects that the Lp-PLA2 mass method detection kit in the prior art cannot truly reflect the true concentration level of Lp-PLA2 and is limited by the detection limitations of sample types. Phospholipase A2 Protein Concentration Kit

Method used

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  • Kit for detecting lipoprotein phospholipase A2 protein concentration
  • Kit for detecting lipoprotein phospholipase A2 protein concentration
  • Kit for detecting lipoprotein phospholipase A2 protein concentration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of a kit for detecting the protein concentration of lipoprotein phospholipase A2

[0033] 1. Preparation of calibrators and quality controls

[0034] The concentration of Lp-PLA2 antigen was determined by traceability, and diluted with calibrator diluent to prepare Lp-PLA2 calibrator and Lp-PLA2 quality control substance. For example, the concentration of the calibrator is 0, 50, 100, 500, 800, 1000ng / ml, and the concentration of the quality control substance is 400ng / ml.

[0035] The specific components of the calibrator diluent used are: 20mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 300mM NaCl, 1% bovine serum, 0.5mM Proclin-300, and its pH is 6.0.

[0036] 2. Preparation of pretreatment solution

[0037] The pretreatment solution uses 25mM Tris as the matrix solution, which contains 1.9mM pyridine, 0.5-5.5% X-100; also includes 150mM NaCl, 1% sucrose, 5% glycerol, 0.1% BSA, 0.05% Tween-20 and 0.2% Proclin-300, its pH is 7.0.

[0...

Embodiment 2

[0077] Example 2 Determination of Lp-PLA2 antigen in whole blood using the kit prepared in Example 1

[0078] The sample to be tested is whole blood from fingertips, and the specific steps are:

[0079] 1. Add 10 μl of Lp-PLA2 calibrator, Lp-PLA2 quality control substance and test sample to three reaction tubes respectively;

[0080] 2. Add 40 μl of pretreatment solution to each reaction tube and mix well;

[0081] 3. Add 50 μl enzyme conjugate working solution and 50 μl magnetic bead working solution to each test tube and mix well; react at 42°C for 3 minutes;

[0082] 4. Magnetically separate the solution after the reaction in step 3 to collect the magnetic beads; add 300 μl of cleaning solution to each reaction tube to wash the magnetic beads, repeat 3-5 times, and remove the cleaning solution;

[0083] 5. Add 100 μl of substrate solution to each reaction tube, mix well and detect the luminescence value.

[0084] 6. Use the concentration and luminescence value of the sta...

Embodiment 3

[0087] Embodiment 3 The performance measurement of kit of the present invention

[0088] 1. Linear verification

[0089] Take a traceable standard or quality control product with a concentration as high as possible, dilute the sample with normal saline, measure each point 3 times, take the average value, draw a regression line between the result and the expected concentration, and calculate the regression coefficient r > 0.99, It shows that the dilution linearity of the kit provided by the invention is good.

[0090] 2. Precision verification

[0091] Take a traceable serum high-value quality control product and a low-value quality control product, perform 10 tests on each quality control product, and calculate the average value and standard value of a total of 10 test results.

[0092]

[0093] According to the coefficient of variation CV=(standard deviation / mean value)×100%, it is calculated: CV1 (350 ng / mL)=4%, CV2 (800 ng / mL)=3%. It can be known that the kit provided...

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Abstract

The invention discloses a kit for detecting lipoprotein phospholipase A2 protein concentration. The kit for detecting the lipoprotein phospholipase A2 protein concentration comprises a calibrator, a cleaning solution, a substrate solution, a pretreatment solution, an enzyme conjugate working solution and a magnetic bead conjugate working solution. The pretreatment solution contains a surfactant, the enzyme conjugate working solution contains an enzyme labeled Lp-PLA2 antibody, and the magnetic bead conjugate working solution contains magnetic beads coated with the Lp-PLA2 antibody. According to the kit for detecting the lipoprotein phospholipase A2 protein concentration, chemiluminescent immunoassay is combined with magnetic particle separation technology, high detection sensitivity, highspecificity and accurate result are achieved, and wide range detection of 50-1000 ng / ml is achieved. Finger tip whole blood or anticoagulated venous whole blood is directly used as a sample to be detected, direct detecting can be carried out without pretreatment of a sample in advance, so that the detection speed is greatly improved, the operation steps are simplified, and the application range ofthe kit is expanded; the kit can be operated in a fully automatic and one-touch mode, and a test result can be obtained in about 7 minutes, the requirements of hospital emergency and outpatient rapiddiagnosis can be well met, and wide-scale popularization and application are facilitated.

Description

technical field [0001] The invention relates to immunochemical detection technology, in particular to a kit for detecting the protein concentration of lipoprotein phospholipase A2 (Lp-PLA2). Background technique [0002] Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a member of the phospholipase A2 (phospholipase A2, PLA2) superfamily, consisting of 441 amino acid residues and a relative molecular weight of 45.4kD. PLA2 is a family of enzymes that can hydrolyze short-chain acyl groups on the Sn-2 position of the phospholipid glycerol scaffold. According to the location, substrate specificity, cofactor requirements and physiological functions, PLA2 is mainly divided into three types: secreted (sPLA2), cytoplasmic (Cpla2) and calcium-independent (iPLA2). Lp-PLA2 belongs to iPLA2 and does not require calcium ions to maintain its catalytic activity. It is a serine-dependent phospholipase that mainly acts on oxidized phospholipids rather than unoxidized phospholipids with...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 毛宁宇李双法于林李奎刘功成付光宇吴学炜苗拥军
Owner AUTOBIO DIAGNOSTICS CO LTD
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