Affinity chromatography-enzyme immunoassay method for PAT protein, and dedicated kit

An enzyme-linked immunosorbent assay and reagent technology, applied in the field of immunological detection, can solve the problems of false positives and negatives, decreased reaction efficiency, etc., and achieve the effects of low detection limit, simple pretreatment, and convenient and fast sample extraction.

Inactive Publication Date: 2005-11-16
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used technology in the international detection of bar gene and / or pat gene food is PCR technology, but in the PCR reaction, many factors will affect the reaction, resulting in the decrease of reaction efficiency, the amplification of non-specific fragments, false positives and Negative appearance etc.

Method used

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  • Affinity chromatography-enzyme immunoassay method for PAT protein, and dedicated kit
  • Affinity chromatography-enzyme immunoassay method for PAT protein, and dedicated kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] 1. Sample pretreatment:

[0077] Weigh 2 g of leaf tissue of transgenic corn Bt11, Bt176 and genetic rapeseed MS1 / RF1, MS8 / RF3 respectively, add 5 ml of PBS / 0.01M EDTA (pH7.5), and crush by ultrasonic. Then centrifuge at 12000rpm / min for 30min, repeat twice to remove insoluble tissue fragments, extract the supernatant and filter it through a Whatman membrane with a pore size of 3MM. The filtrate should be frozen at -20°C until testing; overheating should be avoided during the whole operation to cause protein denaturation.

[0078] 2. Preparation of enzyme-labeled antigen: the activated horseradish peroxidase (HRP) was mixed with the purified PAT protein, and the labeling reaction was carried out in a carbonic acid buffer system, and then sodium borohydride was added to reduce the unbound site.

[0079] 3. Preparation of polyclonal PAT antibody

[0080] a. Antigen for immunization: 1 mg / ml PAT protein per immunization, emulsified with 1 ml of complete Freund's adjuvant...

Embodiment 2

[0101] The composition of the PAT protein detection kit:

[0102] A reagent: standard PAT reagent; B reagent: diluent; C reagent: enzyme-labeled antigen reagent; D reagent: enzyme-labeled antigen diluent; E reagent: phosphate PBS solid containing Tween-20; F reagent: phthalate Amine reagent; reagent: sodium acetate-citric acid buffer; H reagent: 30% H 2 o 2 ; Reagent I: 1M sulfuric acid solution; Reagent J: glycine solution; Reagent K: conjugated antibody sepharose-4B gel; ELISA plate coated with PAT antibody.

Embodiment 3

[0104] 1. Sample pretreatment:

[0105] Weigh 2g of transgenic corn Bt11, Bt176, transgenic rapeseed MS1 / RF1, MS8 / RF3 leaf tissues respectively, add 5ml PBS / 0.01M EDTA (pH7.5), and crush by ultrasonic. Then centrifuge at 12000rpm / min for 30min, repeat twice to remove insoluble tissue fragments, extract the supernatant and filter it through a Whatman membrane with a pore size of 3MM. The filtrate should be frozen at -20°C until testing; overheating should be avoided during the whole operation to cause protein denaturation.

[0106] 2. Preparation of reagents

[0107] Standard PAT reagent: PAT standard substance and B reagent are mixed to form a gradient solution.

[0108] Enzyme-labeled antigen reagent: Dissolve reagent C in 10ml reagent D and mix well.

[0109]Preparation of washing solution E: add 200ml of distilled water to reagent E to prepare washing solution E, which is used for washing the microtiter plate.

[0110] Preparation of the substrate mixture: dilute 40mg o...

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Abstract

A detection method includes using PAT protein as antigen to immunize animal for obtaining specific poly antibody, cross - linking the antibody on agarose gel to prepare immuiaffinity chromatographic column, using affinity column to enrich PAT protein after detected sample is processed, collecting affinity column eluent and carrying out enzyme - linked immunosorbant assay for confirming PAT protein content in it. The kit of the method is also disclosed.

Description

technical field [0001] The invention belongs to the field of immunological detection, and relates to an affinity chromatography-enzyme-linked immunological detection method for detecting PAT protein and a special kit thereof. Background technique [0002] At present, the food raw materials involved in genetically modified crops include soybeans, corn, rapeseed, cotton, tomato, potato, zucchini, papaya, sweet pepper, etc. In 2000, genetically modified soybeans, corn, rape, and cotton accounted for more than 99.99% of the planting area of ​​genetically modified crops, accounting for 16% of the global planting area. Among the agronomic traits of transgenic crops, herbicide-resistant transgenic crops accounted for the largest proportion, followed by insect resistance, and then double resistance (herbicide resistance and insect resistance). [0003] Both the Bar gene and the pat gene are herbicide-resistant genes, and the two have only a slight difference in the nucleic acid seq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
Inventor 许文涛黄昆仑邓爱科罗云波
Owner CHINA AGRI UNIV
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