Production Of GNA Monoclonal Antibodies And Application In The GNA Transgenic Soybean Detection

Galanthus Nivalis agglutinin (GNA) is one of the most important members of the plant agglutinin family. The GNA monomer is composed by 109 amino acids, and its molecular weight is 12kDa, Natural GNA is a glycoprotein polymers which formed by four monomers with the non-covalent bond and molecular weight is 50kDa. GNA can specifically combine the mannose, and destruct the epidermal cells of intestines to kill the sap sucking injurious insects of Homopteran and many other insects. It is a compensation to BT regarding the insect spectrum to kill. GNA gene was transferred into more and more plants. The transgenic plants and its safety issue receive more and more attention along with the very fast development of transgenic plants. In this study the anti-GNA monoclonal antibodies were generated through cell fusion and were used to the examination of soybean plants transformed with GNA gene.The pure GNA purchased from SIGMA Corporation was used to immune the BALB/c mouse for four times and 8μg for each time. The cell fusion was conducted by the spleen cells and NS-1 cells after four days of the last immunization, and the fusion rate was 5% and the positive rate was 20.8%. An anti-GNA hybrid cell strain was yielded and named as 2D8 after 4 round of cloning by limit dilutions. The results showed that the antibody 2D8 was specific to GNA protein and its titration was 1: 400 determined by Western Blot and indirect ELISA.The soluble proteins were isolated from the GNA transgenic soybeans and 2D8 antibodies were used to test the proteins by indirect ELISA. The GNA protein was 0.1% of the total soluble proteins.These results will provide an alternative way to detect the transgenic plants more quickly and more accurately in protein level.