A COMPARISON OF 4 SEROLOGICAL TESTS FOR THE DETECTION OF BANANA BUNCHY TOP VIRUS IN BANANA

Four different serological tests for detection of banana bunchy top vi rus (BBTV) in banana sap are compared: (i) a triple-antibody sandwich ELISA for BBTV, utilising anti-BBTV polyclonal antibodies for virus ca pture, and anti-BBTV monoclonal antibodies, alkaline phosphatase-label led sheep anti-mouse antibodies, and p-nitrophenyl phosphate for detec tion (ELISA-NPP); (ii) an alternative enzyme-substrate system for ELIS A involving an amplification step (Ampak(TM) enzyme amplification kit) (ELISA-A); (iii) a colorimetric dot immunobinding assay (DIBA-C), in which the enzyme-substrate system was alkaline phosphatase and nitrobl ue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate; (iv) an enhanced chemiluminescent form (DIBA-ECL), in which the enzyme-substrate system was horseradish peroxidase and luminol. For both DIBA-C and DIBA-ECL, maximum sensitivity was obtained by pre-coating the nitrocellulose me mbrane with anti-BBTV polyclonal antibodies, by using 0.05 M sodium ca rbonate (pH 9.6) as the coating buffer, and by clarifying the sap by c entrifugation and extraction with chloroform or dichloromethane. Treat ment of the sap before centrifugation by snap-freezing at -70 degrees C, or heating at either 30 or 50 degrees C for 10 min, had no effect o n sensitivity; heating at 70 degrees C for 10 min eliminated antigenic ity. ELISA-NPP, ELISA-A, and DIBA-ECL had equivalent sensitivity, but DIBA-C was up to 8-fold less sensitive than the former 3 assays. ELISA -NPP was adjudged to be the best compromise between sensitivity, cost and completion time.