Diagnostic Reagents and ELISA Kits for Plant Viruses Affecting Horticultural Crops
In the present innovation, recombinant fusion proteins were used as antigens to raise polyclonal antibodies for plant viruses. Polyclonal antibody (PAb) generated using the classical approach of antigen preparation, is difficult to be reproduced if the original virus culture is lost. The recombinant coat protein constructs can be stored and maintained for longer periods and can be used for antibody production whenever required. It is indigenous technology and the market price of the product will be about 50% of the price of the currently available diagnostic reagents and kits, which at present are largely imported. The expression construct of different viruses (PVX, PVS, PVM, PVY, PVA, BBTV, BSMyV, ToLCNDV, CMV, BBrMV, ORSV, CyMV, GLRaV-3 and GLRaV-4) were developed at the Division of Plant Pathology and were provided to Imgenex, Bhubaneswar to raise the antiserum against the constructs. Purified recombinant coat proteins of viruses (200 µg) were used to prepare polyclonal antiserum in New Zealand white rabbits and monoclonal antibodies in Swiss albino mice. The polyclonal antibody developed was validated using PCR positive samples as well as purified proteins by direct antigen coating (DAC)-ELISA. Monoclonal antibody developed from different clones and subclones of PVX, PVS, PVM, PVY, PVA, BBTV, BSMyV, ToLCNDV, CMV, BBrMV, ORSV, CyMV were used to detect the respective viruses in PCR positive plant samples as well as purified proteins by DAS-ELISA. Antibodies with good titre against PVX, PVS, PVM, PVY, ToLCNDV, CMV, BBrMV, ORSV, CyMV, GLRaV-3, and GLRaV-4) were used for the preparation of ELISA kits.