Establishment And Application Of ELISA To The Detection Of The Protoxin Of Bacillus Thuringiensis HBF-1 In Soil

The purpose of this research was to study on the preparation of antigen of theinsecticidal crystal protein of Bt (Bacillus thuringiensis)strain HBF-1, the polyclonalantibody and an enzyme-linked immunosorbent assay (ELISA) was developed andperformed to determine the Bt protein in soil. The specific HBF-1 strain from Bt whichshowed high toxicity to Anomala corpulenta and A. exoleta.In this study, the crystal protein was obtained by isoelectric point deposition, and theextracted protein was purified by SDS-PAGE. The highly purified protein with molecularweight of 130 kDa was used as antigen to immune New Zealand Rabbit for four times, andthen specific antibody was acquired. Mainly immunoglobuling (IgG) of rabbit anti-Bt waspurified by caprylic acid-ammonium sulphate precipitation. The result of SDS-PAGEsuggested that it's one simple, efficient, rapid and low cost method for the purification ofIgG from antiserum, and the concentration of the purified IgG was 5.6344mg/mL. With thesodium periodate method antibody was labeled by HRP to make enzyme-labelled antibody.After tested, In the combination of enzyme-labelled antibody, HRP/Ab was 0.798, thecombination rate of enzyme was 0.28.In this research, Direct—ELISA, ID—ELISA, DAS—ELISA were experimented toassay the content of Bt protein in soil, Through comparison of CV, EC₅₀ and the rate ofdetection, DAS—ELISA is the best method, and the DAS—ELISA for detection of the Btprotein was established. Through the specificity test blocking test, cross test, andduplication test, we can see clearly that the method of double sandwich ELISA is veryspecific, sensitive and stable. It offers that the assay has a promising application future.The method of DAS—ELISA was applied to detect Bt protein in the peanut field. Theresult showed that the content of the Bt protein which was detected by this method canincarnate the dynamics of the Bt protoxin in soil. when manuring the fermentation liquid ofHBF-1 in sowing time, concerned the sample of original liquid and double diluted liquid in the depth of 20cm, the detection of Bt protein reached the peak on the 3rd day, and then gotinto slowly degradation stage. Concerned the sample of 2cm, the dynamics of Bt protoxinis not stable before the 21st day, and reached the peak on the 21st day, and then slowlydegraded till 134th day, this experimentation was over. The result showed that in the periodof 3rd～14th day, the detection of Bt protoxin in the depth of 20cm is significantly higherthan that in the depth of 2cm, and then except the sample of double diluted in the period of21st～31st, the Bt protoxin in the depth of 20cm is more than 2cm. When manuring thefermentation liquid of HBF-1in flowering early time, the detection of protoxin reached thepeak on the 3rd day, and then the content ofprotoxin in the depth of 20cm degraded rapidly,though in the depth of 2cm the protoxin degraded slowly. When manuring the fermentationliquid of HBF-1 in flowering later time, the detection of Bt protoxin reached the peak onthe 3rd～7th, and then degraded all along with lower speed.Finally, it will be a best control to the larvae of scarbaeoid beetles when manuring thefermentation liquid of HBF-1 in flowering early time or later time, because in this periodthe expressing levels of Bt protoxin reached the peak and in time at peak egg stage, theduration of the 1st instar and 2nd instar of Anomala corpulenta Motschulsky.