Insecticidal crystal protein CrylAc enzyme linked immunosorbent detection kit

A technology of enzyme-linked immunosorbent adsorption and insecticidal crystal protein, which is applied in the direction of measuring devices, material analysis and instruments through observation of the impact on chemical indicators, to achieve the effect of reducing detection costs

Inactive Publication Date: 2011-06-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR method can accurately detect genetically modified ingredients from crop seeds to crop maturity, and is not affected by sample processing methods, but it is prone to false positives and false negatives. For highly processed genetically modified products and their Derivatives, such as vegetable oil, sugar, or starch, do not contain any DNA components, so it is difficult for PCR methods to detect such genetically modified foods; enzyme-linked immunosorbent assays are based on the specific combination of antigens and antibodies and enzyme substrates It is a rapid detection technology for high-efficiency catalytic properties. It is fast, sensitive, accurate, and low-cost. It can detect both qualitatively and quantitatively the target transgenic protein.

Method used

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  • Insecticidal crystal protein CrylAc enzyme linked immunosorbent detection kit
  • Insecticidal crystal protein CrylAc enzyme linked immunosorbent detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Production of insecticidal crystal protein Cry1Ac rabbit monoclonal antibody

[0027] 1. Cry1Ac protein immunized rabbits

[0028] Mix 500 μg of Cry1Ac protein with the same amount of complete Freund’s adjuvant, emulsify it as the immunogen, and inject it subcutaneously at five points on the back of rabbits (three New Zealand white rabbits). On day 56, 200 μg of Cry1Ac protein was mixed with incomplete Freund's adjuvant and immunized in the same way.

[0029] 2. cell fusion

[0030] Blood was collected from the rabbit after the fifth immunization and the serum was separated to measure the immune titer, and a rabbit with a higher titer was selected for cell fusion. Ten days before the rabbit spleen was taken for cell fusion, a booster immunization was carried out to the rabbit, and then a sterile spleen was taken. Spleen cells and myeloma cells in the logarithmic growth phase were selected for fusion, the fusion agent was 50% PEG, and HAT was used as ...

Embodiment 2

[0033]Example 2: Production of insecticidal crystal protein Cry1Ac rabbit polyclonal antibody

[0034] Mix 500 μg of Cry1Ac protein with the same amount of complete Freund's adjuvant, emulsify it completely as the immunogen, and inject subcutaneously at five points on the back of rabbits (two New Zealand white rabbits). On day 56, 200 μg of Cry1Ac protein was mixed with incomplete Freund's adjuvant and immunized in the same way. Blood was collected from the rabbit after the fifth immunization and the serum was separated to determine the immune titer. A rabbit with a higher titer was selected for two booster immunizations, and then the whole blood of the rabbit was obtained. Rabbit whole blood was first separated from serum, and then purified by protein A gel column to obtain anti-Cry1Ac rabbit polyclonal antibody.

Embodiment 3

[0035] Example 3: Horseradish peroxidase labeling of anti-Cry1Ac rabbit polyclonal antibody

[0036] a. Antibody treatment: Dissolve 2mg of antibody in CBS (50mM, pH 9.6), dialyze overnight in CBS solution at 4°C, and change medium twice in between.

[0037] b. Oxidation of horseradish peroxidase (the following steps are all carried out under dark conditions):

[0038] 2mg HRP + 0.4mL ddH 2 O to mix;

[0039] 10mg NaIO 4 + 5mL ddH 2 O to mix;

[0040] Take NaIO 4 (aq) 45μL was slowly added to HRP(aq), vortexed and mixed, and reacted at room temperature in the dark for 20 minutes;

[0041] Add 40 μL of ethylene glycol to the above solution, mix well, and continue to react in the dark for 30 minutes at room temperature;

[0042] c. Cross-linking: Add the oxidized HRP to the antibody dialysis bag, mix well, and dialyze (50mM CBS) at 4°C for 2 hours in the dark;

[0043] d. Reduction: Take out the cross-linked product and put it in a light-...

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Abstract

The invention discloses an insecticidal crystal protein CrylAc enzyme linked immunosorbent detection kit. In the CrylAc protein detection of the kit, the lowest detection limit is 7.81ng / mL, the linear detection range is 62.5-500ng / mL, the linear interval fitted equation y is equal to 1.1136Ln(x)-4.164, and R2 is equal to 0.9991. No cross reaction occurs among the kit and CrylAb and Crylc proteins. The kit is suitable for qualitative detection of the CrylAc protein in transgenic plant leaves, fruits and derivatives. The kit can detect large batch of samples simultaneously, is convenient and fast, has very important realistic significance in analyzing related transgenic articles, simultaneously ensures the detection cost to be greatly reduced and has potential economic value.

Description

technical field [0001] The invention relates to a kit, in particular to an enzyme-linked immunosorbent detection kit for insecticidal crystal protein Cry1Ac. technical background: [0002] Among transgenic crops, Bt-Cry transgenic protein is the most researched. Bt gene is currently the most widely used insect resistance gene, and its role in pest control is becoming more and more important. Eight kinds of rice Lepidoptera pests had high resistance. According to reports, there are 70 known Bt insect-resistant genes, and the research and application of Bt insect-resistant crops are developing rapidly. While carrying out research, development and commercialization of genetically modified foods, in order to give a comprehensive assessment of the safety of genetically modified foods and enable consumers to quickly distinguish genetically modified foods from natural foods, it is of great practical significance to develop corresponding rapid detection kits for genetically modif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N21/78
Inventor 郑晓冬倪庚沈金儿冯劲松陆蕾刘娜
Owner ZHEJIANG UNIV
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