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Method and special enzyme-linked immunoassay kit for detecting cowpea trypsin inhibitor

A technology of trypsin inhibition and enzyme-linked immunosorbent reagents, applied in the direction of protease inhibitor immunoglobulin with anti-peptide structure, methods based on microorganisms, biochemical equipment and methods, etc., to achieve the effect of broad application prospects

Inactive Publication Date: 2013-08-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high-throughput, fast and high-sensitivity enzyme-linked immunoassay technology has been widely used in the identification of transgenic plants, etc., but the double-antibody sandwich enzyme immunoassay method of CpTI has not been reported, so an accurate The CpTI immunoassay method is of great significance

Method used

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  • Method and special enzyme-linked immunoassay kit for detecting cowpea trypsin inhibitor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, preparation detects the enzyme-linked immunosorbent assay kit of cowpea trypsin inhibitor

[0040] 1. Preparation of kits

[0041] (1) Preparation of CpTI immunogen and CpTI standard

[0042] CpTI immunogen and CpTI standard were obtained through the steps of cowpea protein extraction, salting out, gel filtration and affinity chromatography, the specific methods are as follows:

[0043] (1) CpTI protein extraction and crude purification

[0044] Carry out cowpea total protein extraction with high-salt solution, concrete method is: get 80 grams of Heimei No. 1 cowpea seeds (purchase Bai Yinong Vegetable Seed Firm) and grind into powder and soak with 400ml 0.3% sodium chloride aqueous solution, soaking condition is: temperature is 4°C for 16 hours, shake and centrifuge during the period, and take the supernatant to obtain the extract.

[0045]The obtained extract was subjected to rough purification by salting out with saturated ammonium sulfate solution. ...

Embodiment 2

[0100] Embodiment 2, the application of kit

[0101] 1. Make a standard curve

[0102] (1) Coating of polyclonal antibody: Dilute 1 mg / mL anti-CpTI rabbit polyclonal antibody with coating buffer, according to the volume ratio of anti-CpTI rabbit polyclonal antibody to coating buffer: 1:1000 After proportional dilution, add it to the microplate, 100 μl per well, and incubate at 37°C for 3 hours; pour off the solution in the microplate, wash the plate 4 times with PBS buffer, and shake dry.

[0103] (2) Add different concentrations of CpTI protein standard solution (experimental well) respectively in the microplate of step (1), 100 μ l in each well, and add 100 μ l PBS buffer solution without adding standard solution in the control well; Incubate for 30min; pour off the solution in the ELISA plate, wash the plate 4 times with PBS buffer, and shake dry;

[0104] The CpTI protein concentrations in the CpTI protein standard solution of different concentrations are respectively 10...

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Abstract

The invention discloses a method and special enzyme-linked immunoassay kit for detecting a cowpea trypsin inhibitor. The enzyme-linked immunoassay kit disclosed by the invention contains a cowpea trypsin inhibitor monoclonal antibody, wherein the cowpea trypsin inhibitor monoclonal antibody is generated by the secretion of a cowpea trypsin inhibitor monoclonal antibody hybridoma cell line CpTI (cowpea trypsin inhibitor) 3D6 of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.4617. The invention adopts an anti-CpTI multiclonal antibody and an enzyme-labeled anti-CpTI monoclonal antibody and uses double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) for the preparation of the kit; and the prepared kit is used to identify the truth of transgenic crops, and has the advantages of high identification speed and highsensitivity.

Description

technical field [0001] The invention relates to a method for detecting cowpea trypsin inhibitor and a special ELISA kit. Background technique [0002] Cowpea trypsin inhibitor (CpTI) belongs to the Bowman-Birk type of double-headed protease inhibitors in the serine protease inhibitor family, that is, one inhibitor molecule can inhibit the catalytic activity of two trypsin molecules. Since the 1980s, the CpTI gene, as an insect-resistant gene, has become a candidate gene for transgenic plants, and after expression, it has shown excellent insect-resistant characteristics compared with non-transgenic plants. genes carried. To accurately evaluate the ecology and food safety of CpTI transgenic crops, carry out mandatory labeling of genetically modified products, test and monitor the purity of genetically modified crop seeds, etc., it is necessary to establish a set of rapid and accurate detection of exogenous gene expression proteins with independent intellectual property rights...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/38C12N5/20G01N33/577G01N33/543G01N21/31C12R1/91
Inventor 王保民谭桂玉南铁贵赵洪伟高巍王敏孙硕曹振李召虎
Owner CHINA AGRI UNIV