Study On Enzyme-linked Immunosorbent Assay For Bt Protein

These years the transgenic plants have already been in commercial production in many countries for its excellent character of Insect-resistance and Herbicide tolerance. With the increase in the cognition for the safety of transgenic products, many countries enhance theirs control and management with the development of Genetically Modifies Organism (GMO) technique. Labeling of transgenic food has been compμLsorily enacted in regμLations related to the control of transgenic products in China and other countries. So it is necessary to develop a rapid and easy-to-use technology for the detection of transgenic products. The objective of this papers is to establish a a quantitative direct and competitive enzyme-linked immunosorbent assay (ELISA) method for Bt protein expressed in transgenic crops and ELISA kit, which woμLd provide a technical support for rapid detection and lebeling of transgenic products.Two polyclonal antiserums respectively against BtCry1Ac and BtCry2Aa/2Ab with titers of 52000 and 50000 were obtained after immuned into mices and puritification of antiserium by participatiton with saturated ammonium sμLfate. Their cross reaction with other Bt proteins less than 0.3% and suggested to meet the requirement for ELSIA establishment.The antigens (Bt proteins) were conjugated with HRP by the improved simple sodium periodate method, the conjugated ratios for two enzyme-labelled antigens (BtCry1Ac,BtCry2Aa/2Ab) were 0.52 and 0.65 respectively.After optimizztion of the working parameters including selection of coating condition, dilution ratio of antiserum and enzyme-labelled antigen, a direct and competition ELISA method was developed, which chacteristics were showed as follows:(1) BtCry1Ac ELISA: the linear detection covered a rang from 50ng/ml to 1600ng/ml, the sensitivity reached 50ng/ml; recoveries of added standard ranged from 95.56% to 102.27%, the averagey reached 98.74%; the C. V. within-assay and between-assay ranged from 2.25% to 7.20% and 2.62% to 10.21%; the valid period of the ELISA kit in 4°C was longer than six months.(2) Btcry2Ab/2Ac ELISA: the linear detection ranged from 40ng/ml to 1750ng/ml, the sensitivity reached 40ng/ml; recoveries of added sandard ranged from 96.56% to 98.52%, the average reached 97.33%; the C. V. within-assay and between-assay ranged from 1.55% to 7.17% and 3.78% to7.39%; the valid period of ELISA kit in 4°C was is longer than six months.