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A liquid chip for detecting bt cry1 Ac protein and its application

A liquid phase chip and protein technology, applied in the field of protein detection, can solve problems such as difficult detection

Inactive Publication Date: 2011-12-21
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been more than ten years of research on the detection methods of genetically modified crops at home and abroad, due to the large number and variety of genetically modified crops, some genetically modified products undergo deep processing, treatment and storage under various conditions, and the genetically modified components are partially or completely degraded, so the detection is difficult. Big

Method used

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  • A liquid chip for detecting bt cry1 Ac protein and its application
  • A liquid chip for detecting bt cry1 Ac protein and its application
  • A liquid chip for detecting bt cry1 Ac protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1Bt

[0030] Example 1 Preparation of Bt cry1 Ac monoclonal antibody

[0031] According to the method disclosed in the literature "Qiao Yanhong, Zhang Wei, Lin Min, Zhang Jie, Pan Jiarong; Establishment of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against Bt Cry1 Ac Protein[J]; High-Tech News; 2005, 02, 91-93", BALB / c mice were immunized with Bt cry1 Ac as the antigen, and SP2 / 0 myeloma cells were fused with the spleen cells of the immunized mice. Three pairs of monoclonal antibodies that recognize different epitopes were screened out, and one pair was selected for protein The production of liquid-phase chip, named this pair of monoclonal antibodies AB3 and AE3.

Embodiment 2B

[0032] Example 2 Coating of polystyrene fluorescent magnetic microspheres of Bt cry1 Ac monoclonal antibody AB3

[0033] Adopt Bio-Plex TM Protein amino coupling kit and No. 37 blank carboxyl fluorescent magnetic microspheres coat Bt cry1 Ac monoclonal antibody on polystyrene fluorescent magnetic microspheres. The specific steps are as follows:

[0034] Select No. 37 carboxylated magnetic fluorescent microspheres (Bio-Rad) (1.25×10 7 / ML), vortex for 30 seconds, ultrasonically shake for 30 seconds, take 100μL microspheres into the reaction tube (using imported 1.5mL eppendorf tube), centrifuge at 14000×g for 4 minutes, carefully remove the supernatant, add 100μL microspheres The ball cleaning solution, vortex for 10 seconds, ultrasonic vibration for 10 seconds, 14000×g centrifugation for 4 minutes, carefully remove the supernatant, add 80 μL microsphere activation buffer to resuspend the microspheres, vortex for 30 seconds, ultrasonic vibration for 30 seconds, Prepare EDC (50mg / mL...

Embodiment 3A

[0035] Example 3 Biotinylation of AE3 (1.5 mg / mL) antibody

[0036] This step was performed according to the instructions of the Pierce kit. Finally, the biotinylated antibody was dialyzed overnight (PBS, pH 7.4) to remove unbound biotin, and stored in aliquots at -20°C for later use. The specific steps are: take the frozen biotin out of the refrigerator to room temperature, prepare the antibody solution, and prepare a 10 mM biotin solution (prepared for use now), then add a certain volume of the biotin solution to the antibody solution, and incubate on ice for 2 hours. The dialysis process is as follows: put the dialysis bag in the pretreatment solution, boil it for 10 minutes, wash it with distilled water three times, boil it in distilled water for 10 minutes, and store the processed dialysis bag in distilled water at 4°C. Put the antibody solution into the dialysis bag and change the PBS buffer continuously, usually at least one day for dialysis.

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Abstract

The invention discloses a liquid phase chip for detecting Bt cry1 Ac protein and its application. The liquid phase chip provided by the present invention includes fluorescent microspheres coated with anti-Bt cry1 Ac monoclonal antibodies, biotinylated monoclonal antibodies against different antigenic epitopes of Bt cry1 Ac, and streptavidin phycoerythrin. The present invention also provides using the liquid phase chip to detect whether the plant is a transgenic Bt cry1 Ac plant. The present invention utilizes a monoclonal antibody against Bt cry1 Ac and adopts a double antibody sandwich method to establish a liquid phase chip detection method for Btcry1 Ac protein in transgenic crops (GMO). Good repeatability.

Description

Technical field [0001] The invention belongs to the field of protein detection, and particularly relates to a liquid phase chip for detecting Bt cry1 Ac protein and its application. Background technique [0002] With the gradual maturity of transgenic technology, the industrialization of genetically modified crops has shown a good momentum. While genetically modified crops bring huge economic and social benefits to human beings, they also have potential environmental and food safety risks. In consideration of the safety of genetically modified crops, China, the European Union, Japan, South Korea and other countries and organizations have formulated corresponding laws and regulations to require the labeling of genetically modified crops and processed foods in order to improve the health of humans and animals and the diversity of the ecological environment. protection. Since genetically modified crops and their products may have potential adverse effects on human health and the e...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 韩雪清王慧煜梅琳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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