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Development of RapiDot immunoassays for the detection of banana bunchy top virus
2nd International Conference on Plant Science & Physiology
June 26-27, 2017 Bangkok, Thailand
Moger Narayan, Namrutha S D, Prashanthi S K, Jagadeesh K S and Kiran Mirajkar
University of Agricultural Sciences, India
Posters & Accepted Abstracts: J Plant Pathol Microbiol
Abstract:
Banana (Musa spp.) belongs to the family Musaceae and is one of the globally important fruit crops grown in more than 125 countries with production of 99.99 MT (faostat.fao.org) providing major source of carbohydrates for over 400 million people in tropical countries. Banana Bunchy Top Viral (BBTV) coat protein 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 expressed in pQUANTabody 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 795bp long and they found to share similar homology sequence. BLASTn and BLASTx analysis results indicated 85per cent 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. These specific antibodies conjugate were used to develop a RapiDot Immunodiagnostic kit to detect BBTV in infected banana at field level as low as 0.9 g/ml of antigen concentration.