Immunodiagnosis of Cucurbit yellow stunting disorder virus using polyclonal antibodies developed against recombinant coat protein

The coat protein (CP) gene of Cucurbit yellow stunting disorder virus (CYSDV) was amplified by reverse transcription - polymerase chain reaction (RT-PCR). The amplicon,vas cloned in pGEM-T, sequenced and subcloned into a bacterial expression vector (pQE-31). The recombinant CYSDV CP was expressed as a fusion protein with an N-terminal hexa-histidine tag, purified by affinity chromatography yielding 8 mg native protein per liter of bacterial culture, and used as an antigen to produce CYSDV CP antiserum in a rabbit. The resulting antiserum was successfully assayed in tissue blot immunoassay TBIA), dot blot immunoassay (DBIA), indirect ELISA and DAS-ELISA, with a titer of about 10(3) for all methods. TBIA was very specific and showed the virus localization in the phloem tissue and is recommended for large-scale surveys.