Production of monoclonal antibodies to Tomato leaf curl Bangalore virus

Abstract

Purified Tomato leaf curl Bangalore virus (ToLCBV) was injected into mice and the splenocytes were used for establishing hybridoma lines. Initial screening of culture supernatants showed that 13 lines produced antibody, and after further screening four produced functional monoclonal antibodies. Upon characterisation, these were found to be of low affinity, probably due to host protein contamination and poor yield of native virus in the original preparations. In order to circumvent these problems, the coat protein of ToLCBV was over-expressed in Escherichia coli. Fusion experiments using recombinant coat protein as antigen yielded two primary hybridoma clones G₁₁ and E4 that exhibited good affinity of binding to the antigen. Sub-cloning yielded four monoclonal antibodies G₁₁E₇E₇, G₁₁E₇G₁₂, E₄E₂ and E₄G₆. G₁₁E₇E₇ and G₁₁E₇G₁₂ successfully detected ToLCBV in infected leaf extracts of tomato and Nicotiana benthamiana, viruliferous whiteflies and weed samples. These monoclonal antibodies could also detect other type III geminiviruses such as Pumpkin yellow vein mosaic virus and Bhendi yellow vein mosaic virus. Thus these monoclonal antibodies can be used for testing field-collected samples.