Enzymatic assay for the quantitative determination of phospholipase A1 or A2 activity in a sample

A technology of phospholipase and activity, applied in the field of enzyme assay for quantitative determination of phospholipase activity in samples, can solve the problem of low coverage of fluorescent phospholipid molecules, radioactive labeling is not suitable for diagnostic applications, and molecular/nm2 packing density is not suitable for fluorescence assays And other issues

Inactive Publication Date: 2012-03-21
ATEROVAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This assay suffers from the following disadvantages: (i) radiolabeling is not suitable for diagnostic applications, (ii) as shown in the present examples, 1.3 molecules / nm 2 The packing density of is not suitable for use in the fluorimetric assay, and (iii) the detection limit of the method is 1 ng
[0030] A few examples of phospholipid-coated solid phases are disclosed in the art: however, as shown below, the molecular coverage of fluorescent phospholipids is generally lower compared to the present invention, and said coated solid phases phase is not disclosed for use in assays for phospholipase A1 or A2 activity

Method used

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  • Enzymatic assay for the quantitative determination of phospholipase A1 or A2 activity in a sample
  • Enzymatic assay for the quantitative determination of phospholipase A1 or A2 activity in a sample
  • Enzymatic assay for the quantitative determination of phospholipase A1 or A2 activity in a sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0213] 1-Preparation of the phospholipid-coated solid phase according to the inventive method

[0214] Materials and Reagents

[0215] β-py-C 10 -glycerophosphate (β-py-C 10 -PG) was from Molecular Probes / Invitrogen. [mu]m polystyrene microspheres (beads) were purchased from Merck / Estapor. 96-well plates were purchased from Nunc. All other reagents were from Sigma-Aldricht.

[0216] Experimental program

[0217] All steps are performed in glassware not directly exposed to light.

[0218] [beta]-py-C10-Glycerol phosphate was dissolved in 10% methanol / 90% chloroform (v / v) in a glass vessel using ultra-sonication. The exact molar concentration of β-py-C10-phosphoglycerol was determined by means of a standard curve prepared by means of a five-point standard curve (1 / 100 and 1 / 1600 dilution in methanol , prepared from the absorbance at 342 nm of two-fold serial dilutions), and the blank was defined as a 1 / 100 dilution of 10% methanol / 90% chloroform (v / v) solution in me...

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Abstract

The invention relates to a new method for measuring phospholipase A1 or A2 activity in a sample, using a solid phase coated with a fluorochrome-labelled phospholipase A1 or A2 substrate, wherein the molecular coverage is in the range from 8 to 30 fluorochrome-labelled phospholipase A1 or A2 substrate molecules / nm2, and kit for carrying out said method.

Description

technical field [0001] The present invention relates to an enzyme assay for the quantitative determination of phospholipase activity in a sample. Background technique [0002] Phospholipids are the main structural components of biological membranes. In addition to this structural role of phospholipids, the importance of phospholipids as mediators in cellular signaling processes is becoming increasingly apparent. As a result, research into metabolic processes such as phospholipase action and lipid sorting and transport has rapidly expanded. [0003] Phospholipases belong to the most familiar family of interfacial enzymes that express their catalytic activity once present in an interfacial system. Phospholipases also belong to the class of hydrolytic enzymes, however they advantageously and specifically open the ester bonds of phospholipids present in aqueous systems, giving free fatty acids, lysophospholipids, glycerophospholipids, phosphatidic acid and free alcohols, depen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/58
CPCC12Q1/44G01N2333/918
Inventor E·瓦伦廷C·西比拉
Owner ATEROVAX
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