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Lipoprotein phospholipase A2 assaying reagent and preparation method thereof

A technology of phospholipase and lipoprotein, which is applied in the field of reagents for detecting lipoprotein phospholipase A2 by latex-enhanced immunoturbidimetric method, which can solve the problems of inconvenient development in conventional laboratories, low luminous efficiency, and patient costs, and achieve improved detection The effects of work efficiency, easy access to raw materials, and easy operation

Active Publication Date: 2013-04-10
YESEN BIOTECH SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two kinds of kits in the market, tube type and plate type, but this method has low luminescence efficiency of acridinium ester, luminol, and isoluminol directly labeled antibodies, and the markers are unstable. This direct labeling method belongs to instantaneous luminescence. type, it is difficult to guarantee the stability and repeatability of test results, and requires special testing instruments
Not convenient for routine laboratory development
However, the electrochemiluminescence immunoassay kit using the biotin-avidin system to detect LP-PLA2 needs to be equipped with an expensive automatic electrochemiluminescence detector, which cannot be carried out in conventional laboratories, and it brings unnecessary costs to patients and increases patient burden
Enzyme-linked immunoassay (ELISA) is not highly automated, and is greatly affected by human factors, has poor repeatability, and the entire reaction measurement time is also very long (at least 40 minutes)

Method used

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  • Lipoprotein phospholipase A2 assaying reagent and preparation method thereof
  • Lipoprotein phospholipase A2 assaying reagent and preparation method thereof
  • Lipoprotein phospholipase A2 assaying reagent and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] a) Lipoprotein Phospholipase A2 Reagent 1

[0061]

[0062] After dissolving the above raw materials with 800ml of purified water, add purified water to make the volume to 1000ml, and then adjust the pH to 7.2 with hydrochloric acid or sodium hydroxide.

[0063] b) Lipoprotein Phospholipase A2 Reagent 2

[0064] 1. Dilute 80nm carboxyl latex microspheres with 50mmol / L, PH6.0 MES buffer to a suspension of 0.01mg / ml;

[0065] 2. Add 20 mg EDAC and 40 mg NHS (N-hydroxysuccinimide sulfonate sodium salt) to 1 ml carboxyl latex microsphere suspension, add EDAC and NHS and mix immediately and react the mixture at room temperature for 15- 30 minutes, stirring constantly;

[0066] 3. Wash the carboxyl latex microspheres with 50mmol / L, PH6.0 MES buffer or purified water to remove unreacted NHS and EDAC, and suspend the latex microspheres in purified water to obtain carboxyl latex microspheres with a concentration of 0.01mg / ml Latex microsphere suspension;

[0067] 4. The g...

Embodiment 2

[0081] a) Lipoprotein Phospholipase A2 Reagent 1

[0082]

[0083] After dissolving the above raw materials with 800ml of purified water, add purified water to make the volume to 1000ml, and then adjust the pH to 8.0 with sodium hydroxide.

[0084] b) Lipoprotein Phospholipase A2 Reagent 2

[0085] 1. Dilute 150nm carboxyl latex microspheres with 50mmol / L, PH6.0 MES buffer to a 0.01mg / ml suspension;

[0086]2. Add EDAC and NHS according to the ratio of 1ml carboxyl latex microsphere suspension plus 20mg EDAC and 40mg NHS, mix immediately and react the mixture at room temperature for 15-30 minutes, stirring constantly;

[0087] 3. Wash the carboxyl latex microspheres with 50mmol / L, PH6.0 MES buffer or purified water to remove unreacted NHS and EDAC, and suspend the latex microspheres in purified water to obtain carboxyl latex microspheres with a concentration of 0.01mg / ml Latex microsphere suspension;

[0088] 4. Dissolving the rabbit anti-human lipoprotein phospholipase ...

Embodiment 3

[0103] a) Lipoprotein Phospholipase A2 Reagent 1

[0104]

[0105]

[0106] After dissolving the above raw materials with 800ml of purified water, add purified water to make the volume to 1000ml, and then adjust the pH to 8.0 with sodium hydroxide.

[0107] b) Lipoprotein Phospholipase A2 Reagent 2

[0108] 1. Take 300nm carboxyl latex microspheres and dilute them with 50mmol / L, PH6.0 MES buffer to form a suspension of 0.01mg / ml;

[0109] 2. Add EDAC and NHS according to the ratio of 1ml carboxyl latex microsphere suspension plus 20mg EDAC and 40mg NHS, mix immediately and react the mixture at room temperature for 15-30 minutes, stirring constantly;

[0110] 3. Wash the carboxylated latex microspheres with 50mmol / L, PH6.0 MES buffer or purified water to remove unreacted NHS and EDAC, and suspend the latex microspheres in purified water to obtain carboxylated latex with a concentration of 0.01mg / ml microsphere suspension;

[0111] 4. Dissolve the mouse anti-human lipop...

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Abstract

The invention relates to a lipoprotein phospholipase A2 assaying reagent and a preparation method thereof, and aims to ensure the characteristics of high reagent accuracy and convenience in preparation method. The invention adopts the technical scheme that the lipoprotein phospholipase A2 assaying reagent comprises the following components: a, a lipoprotein phospholipase A2 reagent 1; b, a lipoprotein phospholipase A2 reagent 2; and c, a liquid lipoprotein phospholipase A2 reference product; the preparation method comprises the following steps: (1) for the lipoprotein phospholipase A2 reagent 1: uniformly mixing; (2) for the lipoprotein phospholipase A2 reagent 2: (1) taking a suspension, (2) reacting with a mixture, (3) obtaining the suspension, (4) regulating the concentration, (5) adding the suspension in step (3) into the solution obtained in the substep (4), (6) reacting, (7) adding ethanol amine, and (8) performing centrifugal treatment; and (3) for the liquid LP-PLA2 (lipoprotein phospholipase A2) reference product: mixing according to a formulation, arranging according to contents, or adding a purified product in the mixed solution.

Description

technical field [0001] The invention relates to a reagent for detecting lipoprotein phospholipase A2 and a preparation method of the reagent, in particular to a reagent for detecting lipoprotein phospholipase A2 by using latex-enhanced immune turbidimetry, which can be widely used in the technical fields of medicine and biochemistry. Background technique [0002] Lipoprotein-associated phospholipase A2 (lipoprotein-associated phospholipase A2, Lp-PLA2), also known as platelet activating factor acetylhydrolase (platelet activating factor-AH, PAF-AH), belongs to a kind of PLA2 in the phospholipase family. dependent phospholipase. Human plasma Lp-PLA2 is mainly secreted by mature macrophages, monocytes, T lymphocytes, mast cells, liver cells, etc., and is regulated by inflammatory mediators; the main function of Lp-PLA2 is to produce eicosanic acid Inflammatory mediators, participate in phospholipid reconstruction and stable balance of biomembrane, lipoprotein metabolism, cell...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
Inventor 陈开华其他发明人请求不公开姓名
Owner YESEN BIOTECH SHANGHAI
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