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Study on Immunoassay and It's Application of Paclobutrazol and Heat Denatured Bt Cry1Ac

Author: CaoZhen
Tutor: WangBaoMin
School: China Agricultural University
Course: Crop Physiology
Keywords: pacloburazol heat denatured Bt Cry1Ac monoclonal antibody ELISA
CLC: S482.8
Type: PhD thesis
Year: 2014
Downloads: 7
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Abstract


Paclobutrazol is a widely used plant growth regulator which could reduce the height of plants. The residue of paclobutrazol won’t degradation for more than two years in the applied soil at room temperature. This research was to develop a sensitive and effective method for residue detection and monitor, and study the practical application. The safety was a much concern on transgenic proteins. Bacillus thuringiensis Bt CrylAc is one of the most commercial used insecticidal proteins in China. The common ELISA could not be used to detect heat denatured protein due to lack of antibody. This research has developed monoclonal antibody of heat denatured Bt Cry1Ac, which is a method basis for the development of practical enzyme linked immunosorbent assay (ELISA).The hapten of paclobutrazol was synthesized. Complete antigen was obtained by conjugating hapten to BSA/OVA The indirect competitive enzyme linked immunosorbent assay (icELISA) for paclobutrazol was developed based on the obtained monoclonal antibody (McAb). The immunogen of heat denatured Bt CrylAc was invented. Rabbit antiserum and mouse monoclonal antibody were obtained by the immunization of New Zealand rabbit and BALB/c mouse, respectively. And the sandwich ELISA for heat denatured Bt CrylAc was developedThe main results as follows:(1) An icELISA was developed with monoclonal antibody McAb6H73C9recognizing the plant growth regulator paclobutrazol (PBZ). The icELISA had a half-maximum inhibition concentration (IC50) and working range of approximately8.7and2.0~50.4ng/mL, respectively. Average recoveries of PBZ in the wheat (Triticum aestivum) kernel samples were between84.3and118.9%with relative standard deviations between3.9and14.2%. As determined by the icELISA and further confirmed by liquid chromatography-electrospray ionization quadrupole Orbitrap mass spectrometry (LC-ESI-MS) analysis, the maximum residue concentration was about0.07mg/kg in the kernel samples, which indicated that PBZ could transfer from PBZ-treated seedlings to the kernel samples. The correlation coefficient r2between icELISA and LC-ESI-MS results was0.979, which manifested that the developed icELISA was sensitive enough for monitoring PBZ residues in wheat kernels.(2) The antigen of denatured Bt CrylAc protein was obtained by heat. The1.0mg/mL Bt CrylAc was dissolved in0.01M PBS, pH7.5and heated by100℃boiling for5min, which then was mixed with freund’s complete adjuvant or freund’s incomplete adjuvant, with the same volume. The hybridoma which could secrete antibody against native or heat denatured Bt CrylAc were screened by protein-coating ELISA and rabbit antiserum-protein-mouse antibody sandwich ELISA. Hybridomas1F2and3C9which could recognize coated native Bt Cry1Ac were obtained. And monoclonal hybridomas5E9C6、5E9D8、3E6A3、3E6A5and3E6E2which could recognize heat denature Bt Cry1Ac were developed. The monoclonal antibodies that secreted from5E9C6and3E6E2could apply to Western Blot. This study offers an experience for rapid development of mouse monoclonal antibody for Western Blot. (3) The theory of small molecular compounds or proteins monoclonal antibody development alike each other, however the methods for target hybridoma screening were much different. Hybridoma which secrets antibody of small molecule compounds could be screened by icELISA method, based on the tilter and specificity of antibody that dissolved in nutrient solution. The research found that conformation of protein which adhere to solid phase may different from conformation of its’ dissolved form. This could change the antigenic determinant. So hybridoma secrets antibody of active proteins should be screened by sandwich ELISA. And hybridoma which secrets antibody of denatured or for western blot usage could be developed by heat denatured antigen.

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CLC: > Agricultural Sciences > Plant Protection > Pesticide ( chemical control ) > Various pesticides > Plant growth regulator
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