Enzyme linked immune detection method of GOX protein in transgenosis plant and its special agent kit

A technology of transgenic plants and enzyme-linked immunosorbent adsorption, which is applied in the field of immunological detection, can solve the problems of non-specific fragment amplification, reaction efficiency reduction, false positive and negative, etc., and achieve the effect of strong specificity and low detection limit

Inactive Publication Date: 2007-02-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used technology for GOX genetically modified food detection in the world is PCR technology, but in the PCR reaction, many factors will affect the progress of the reaction, resulting in the decrease of reaction efficiency, the amplification of non-specific fragments, the appearance of false positives and negatives, etc.

Method used

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  • Enzyme linked immune detection method of GOX protein in transgenosis plant and its special agent kit
  • Enzyme linked immune detection method of GOX protein in transgenosis plant and its special agent kit

Examples

Experimental program
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Effect test

Embodiment 1

[0067] 1. Sample pretreatment:

[0068] Weigh 2 g of leaf tissue of transgenic corn GT73, add 5 ml of PBS / 0.01 MEDTA (pH 7.5), 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.

[0069] 2) Preparation of enzyme-labeled antibody: the activated horseradish peroxidase was mixed with the purified GOX protein antibody, and the enzyme-labeled antibody was prepared by sodium periodate labeling method;

[0070] (3) Preparation of polyclonal GOX antibody.

[0071] a. Antigen for immunization: 1 mg / ml GOX protein per immunization, emulsified with 1 ml of complete Freund's adjuvant for the first immunization, and 1 ml of incomplete Freund's adjuvant for booster immunization. b. Animal immu...

Embodiment 2

[0086] The composition of GOX protein detection kit:

[0087] A reagent: standard GOX protein reagent; B reagent: enzyme-labeled antibody reagent; C reagent: antigen diluent; D reagent: antibody diluent; E reagent: phosphate PBS solid containing Tween-20; F reagent: ophthalmic diluent Amine reagent; G reagent: sodium acetate-citric acid buffer; H reagent: 30% H 2 o 2 ; I reagent: sulfuric acid solution; J reagent: GOX antibody IgG pre-coated plate, 48T / 96T

Embodiment 3

[0089] 1. Sample pretreatment:

[0090] Weigh 2 g of transgenic GT73 leaf tissue respectively, add 5 ml of PBS / 0.01M EDTA (pH 7.5), 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.

[0091] 2. Preparation of reagents

[0092] Standard GOX reagent: GOX standard substance and C reagent are mixed to form a gradient solution.

[0093] Enzyme-labeled antibody reagent: Dissolve reagent B in 10ml reagent D and mix well.

[0094] 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.

[0095] Preparation of the substrate mixture: dilute 40mg of F reagent with 20ml of G reagent, then ad...

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Abstract

An enzyme linked immunization method for detecting GOX protein in transgenosis plant includes using GOX protein as antigen to carry out animal immunization to obtain specific polyclone antibody and monoclone antibody, carrying out enzyme linked immunoadsorption detection to confirm GOX protein content after detected sample is processed by preliminary means. The special kit for realizing said method is also disclosed.

Description

technical field [0001] The invention belongs to the field of immunological detection and relates to biotechnology products, in particular to an enzyme-linked immunological detection method and a special kit for GOX protein in transgenic plants. 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] GOX gene is a herbicide resistance gene, which has been widely used at present. At present, transgenic crops th...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): G01N33/543G01N33/535
Inventor黄昆仑许文涛罗云波芦云杨加佳
OwnerCHINA AGRI UNIV