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Method for detecting insecticidal crystal proteins Bt Cry1Ab/Ac and special enzyme linked immunosorbent assay kit thereof

A kit and protein technology, applied in the field of detection of insecticidal crystal protein Bt Cry1Ab/Ac, can solve the problems of low recognition rate and affinity

Inactive Publication Date: 2012-07-18
FUJIAN ANXIN RUIJIE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the different post-translational modifications of Bt Cry1Ab / Ac proteins in plants and animals, the protein expressed by strains often has a low recognition rate and affinity for Bt Cry1Ab / Ac in plants

Method used

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  • Method for detecting insecticidal crystal proteins Bt Cry1Ab/Ac and special enzyme linked immunosorbent assay kit thereof
  • Method for detecting insecticidal crystal proteins Bt Cry1Ab/Ac and special enzyme linked immunosorbent assay kit thereof

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1, the acquisition of hybridoma cell line and the preparation of anti-Bt Cry1Ab / Ac protein monoclonal antibody

[0039] Balb / C mice: purchased from the Experimental Animal Center of Academy of Military Medical Sciences.

[0040] SP2 / 0 myeloma cells: purchased from China Veterinary Drug Administration.

[0041] 1. Animal immunity

[0042] 1. Bal b / C mice aged 8-10 weeks were used as experimental animals.

[0043] 2. Basic immunization: Filter the 1mg / mL Bt Cry1Ac protein solution through a sterile filter, add an equal volume of Freund's complete adjuvant, fully stir and emulsify with a magnetic stirrer until it does not spread when dropped into water. The emulsified immunogen was used to inject Bal b / C mice subcutaneously at multiple points in the abdominal cavity and back, and the injection dose was 0.1 mg of BtCry1Ac protein per mouse.

[0044] 3. Booster immunization: 2 weeks after the basic immunization, take 1mL of 1mg / mL Bt Cry1Ac protein solution, ad...

Embodiment 2

[0080] Embodiment 2, the ELISA kit that detects Bt Cry1Ab / Ac protein

[0081] 1. Preparation of kits

[0082] The ELISA kit for the detection of Bt Cry1Ab / Ac protein of the present application consists of a polyclonal antibody against Bt Cry1Ab / Ac protein (abbreviated as coating source), a monoclonal anti-Bt Cry1Ab / Ac protein labeled with horseradish peroxidase Antibody (referred to as enzyme-labeled antibody), coating buffer, washing solution, standard solution, substrate buffer, stop buffer and blocking solution.

[0083] 1. Preparation of coating agent-anti-Bt Cry1Ab / Ac protein polyclonal antibody

[0084] Rabbits were immunized with BtCry1Ac protein as an immunogen, and anti-BtCry1Ab / Ac protein polyclonal antibodies were obtained through serum extraction and antibody purification steps. The specific steps are as follows:

[0085] (1) Three-month-old New Zealand big-eared white rabbits (purchased from the Experimental Animal Center of the Academy of Military Medical Scien...

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Abstract

The invention discloses a method for detecting insecticidal crystal proteins Bt Cry1Ab / Ac and a special enzyme linked immunosorbent assay kit thereof. An anti-Bt Cry1Ab / Ac monoclonal antibody used by the method and the kit is secreted by a hybridoma cell strain Bt2F9, and the application number of the hybridoma cell strain in CGMCC (China General Microbiological Culture Collection Center) is CGMCC No.5162. The monoclonal antibody is obtained by simultaneously using a Bt Cry1Ac protein solution and an extracting solution of Bt Cry1Ac transgenic cotton plant leaves as a coating antigen and screening hybridoma cells through an indirect non-competitive ELISA (Enzyme-Linked Immuno Sorbent Assay) method, the monoclonal antibody has high affinity for Bt Cry1Ab / Ac protein expressed in plants, and the affinity constant is 1.03*10<9>M<-1>. The double-antibody sandwich enzyme linked immunosorbent assay kit prepared by the monoclonal antibody is used for qualitatively or quantitatively detecting proteins Bt Cry1Ab / Ac in transgenic plants such as cotton and corn, the linear detection range is 0.78-50ng / mL, and the kit has the advantages of fastness and sensitiveness.

Description

technical field [0001] The invention relates to a method for detecting insecticidal crystal protein Bt Cry1Ab / Ac and a special ELISA kit. Background technique [0002] The insecticidal crystal protein gene of Bacillus thuringiensis (abbreviated as Bt) has been successfully applied in crops such as cotton and corn. According to the homology of the coding gene sequence and the insecticidal spectrum of the encoded protein, Bt insecticidal crystal proteins are divided into cry family and cryt family, and each class is further divided into different subclasses. Bt genes currently expressed in plants include Bt cry1Aa, cry1Ab, cry1Ac, cry2Aa, cry3Bb, cry9c. The Bt Cry1Ab / Ac protein has a very high similarity, and the BtCry1Ac protein gene in commercial crops currently on the market already contains the Bt Cry1Ab protein gene. [0003] The Bt immunoassay kit has been widely used in the quality detection of transgenic insect-resistant cotton and corn seeds, the authenticity identi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 王保民南铁贵何丽珊谭桂玉孙硕曹振张亮李召虎
Owner FUJIAN ANXIN RUIJIE BIOTECH CO LTD
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