Jornal de Ciências Agrícolas e Pesquisa de Alimentos
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Abstrato

Development of Rapidot Immunoassays for the Detection of Banana Bunchy Top Virus

Namratha ND, Narayan BM, Chougal A and Chidanand AR

BBTV was used as an antigen to produce single chain fragment variable (scFv) antibody using phage display technology. Two clones showing highest reading in monoclonal ELISA were selected viz., pBSNMAB5 and pBSNMAB40. Single chain antibody fragments of pBSNMAB5 and pBSNMAB40 were subcloned into pQUANTa body expression vector which enabled to transcriptionally fuse with scFv monoclonal antibody fragment and the gene coding for alkaline phosphatase (PhoA) enzyme. The clones were sequenced with LMB3 forward and pHEN reverse primer and characterized. The scFv genes of both clones were 795 bp long and they found to share similar homology sequence. BLASTn and BLASTx analysis results indicated 85 percent homology with Synthetic construct anti-TNF alpha single chain Fv antibody gene, partial cds and further the BLASTx analysis showed that the 86 per cent homology with circulating B cell antibody heavy chain variable region (Homo sapiens). Upon expression of clone, it has produced fusion protein of ALP along with pBSNMAB5 and pBSNMAB40 monoclonal antibody. The scFv-ALP conjugate has been produced against BBTV protein. Using this antibody conjugate, a detection kit was developed. This Rapidot Immunodiagnostic kit detects the BBTV as low as 0.9 g/ml concentration in both NC membrane and membrane cassette and it was also cross checked with other proteins to evaluate the specificity of mono clones raised against BBTV.