S-65. Recombinant DNA technology-based antibodies for detection and diagnosis of plant viruses

Plant pathogenic viruses are one of the major constraints of crops production world wide. Virus detection methods of high sensitivity, specificity and reliability are essential to devise effective plant virus disease management practices. Bioassays, electron microscopy, serological tests, dot-blot hybridization, polymerase chain reaction-based tests and DNA/RNA microarray -based assays are in use for detection, discrimination and diagnosis of plant viruses. An array of serological tests like different variants of ELISA, dot-blot ELISA, tissue print immunoassay, immuno specific electron microscopy and western blot analysis are widely used in plant virus detection and diagnosis. ELISA-based tests are preferred as they have several advantages. Serological assays require either conventionally produced polyclonal antibodies (PAbs) or monoclonal antibodies (MAbs). Purified viruses or their isolated proteins free from host contaminants are used as immunogens to raise these antibodies in laboratory animals or by hybridoma technology.

The purification of certain viruses like gemini-, tospo-, plant rhabdo-, nano-, luteo-, tenui-, badna-, capilloviruses and certain potyviruses to the required quality and quantity is an uphill task due to certain inherent properties of these viruses and therefore difficult to produce quality antibodies to such viruses by conventional approach. The hybridoma methods for the generation of MAbs against plant viruses also have certain limitations. Three alternative approaches based on recombinant DNA technology and molecular immunology have been used to overcome the limitations of conventional antibody raising in experimental animals. The first approach involves the cloning and expression of specific either structural or non structural gene of the plant viruses in bacterial expression system, purification of expressed viral recombinant protein, usually by affinity chromatography and/or gel electrophoresis, and raising of PAbs against such recombinant virus protein in a rabbit. The yeast (Pichia pastoris) is an alternative to bacterial expression system to produce biologically active recombinant proteins of plant viruses. The PAbs produced to several plant viruses (e.g., Sugarcane streak mosaic virus, SCSMV; Dasheen mosaic virus, DsMV; Banana bract mosaic virus, BBrMV; Groundnut bud necrosis virus, GBNV; Potato leaf roll virus, PLRV) based upon this approach have been found to be superior as compared to conventionally generated PAbs. This approach can generate large quantity of recombinant antigen and recombinant clone can also be universally distributed. Since it is non-infectious and thus poses no quarantine problems. The second approach is DNA-based immunization for generating antibodies in laboratory animals for diagnostic purposes. It involves cloning of DNA sequence of the protein of interest into a mammalian cell expression vector and intramuscular injection of recombinant vector DNA into a rabbit. Specific immune response was detected in such immunized animals. This approach has several advantages over the conventional protein-based immunization. It was used to produce antibodies to certain plant viruses like Tobacco mosaic virus (TMV) and Potato virus Y (PVY). The third approach involves the cloning of variable (V) antibody genes and linking them to a flexible peptide in a single chain Fv (scFv). Such constructs are fused with the capsid proteins of filamentous bacteriophages (e.g., M13) and expressed in bacteria as soluble proteins. Further, antibody genes can be expressed fused with other proteins like alkaline phosphatase or amphipathic helices. The expression of antigen-binding proteins in bacterial cultures provides diagnostic reagents that are able to replace conventional PAbs or MAbs and conjugates. This approach has advantages and limitations and has been used to generate antibodies for detection of certain plant viruses like Cucumber mosaic viruses (CMV), Citrus tristeza virus (CTV) and Beet necrotic yellow vein virus (BNYVV). The details of above three approaches and their relative advantages and limitations will be discussed.