Production of a monoclonal antibody specific for Citrus Tristeza Virus

In this work Citrus Tristeza Virus (CTV) was purified from the bark and midrib tissue of infected plants by pulverization of frozen tissue samples in a mortar with extraction buffer, with polyethylene glycol (PEG), and by sucrose gradient centrifugation. In an alternative purification method, CTV was purified by Sephacryl 300 column chromatography after precipitation by PEG. A hybridoma line secreting monoclonal antibody (MAb) highly specific for the CTV was generated by fusion of splenic lymphocytes of immunized BALB/c mice with non-secreting mouse myeloma cells followed by selection in hypoxanthine-aminopterin-thymidine (HAT) medium, and finally, by screening culture fluids for CTV affinity in ELISA. The MAb produced by the CTV-IG8 hybridoma clone was highly specific to CTV and did not react with non-infected citrus plant extracts.