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Abstract Because Bt Cry 1Aa, Cry 1Ab and Cry 1Ac share high sequence identity(82%~90%), it is difficult to prepare Bt Cry1Ab monoclonal antibody that has highly specific by using conventional method. In order to prepare monoclonal antibody against Bt Cry1Ab, we acquired the amino acid sequence of Bt Cry1Ab protein from NCBI. According to the antigenicity, hydrophilicity and accessibility analyzed results by ANTHEPORT and DNAStar computer software, the specific peptide of Bt Cry1Ab was synthesized by a chemical process. The Bt Cry1Ab peptide was linked with carrier keyhole limpet hemocyanin(KHL), and then used for immunized mouse as antigen. Twenty-two strains of hybridoma, which produced monoclonal antibodies to the peptide, had been obtained by cell fusion technique. One hybridoma strain(3A10), which was specific to natural Bt Cry1Ab selected by indirect ELISA. The result of isotype analysis showed that the monoclonal antibody of 3A10 hybridoma strain belong to IgG1 subclass and the light chain was κ chain. The hybridoma chromosome number was 89~108. The monoclonal antibody of hybridoma ascites titers to synthetic peptide was 1∶1×107 and to natural Bt Cry1Ab protein was 1∶1×104. The purified MAB titers to synthetic peptide was 1∶1×108 and to natural Bt Cry1Ab protein was 1∶2×104. The relative affinity of the monoclonal antibody was 0.5 μg/mL. The monoclonal antibody of hybridoma strain could react specifically with the Bt Cry1Ab protein without reacting with Bt Cry1Ac and Bt Cry1Aa proteins by ELISA. The lowest detectable value of the monoclonal antibody to Bt Cry1Ab was 10 ng/mL by indirect ELISA detection. The monoclonal antibody of hybridoma strain can distinguish transgenic Bt CrylAb cotton and general cotton(Gossypium hirsutum L.) by ELISA, and it can recognize specifically Bt Cry1Ab protein in cotton.
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Received: 20 August 2012
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