A comparison of four serological tests for the detection of banana bunchy top virus in banana

Four different serological tests for detection of banana bunchy top virus (BBTV) in banana sap are compared: (i) a triple-antibody sandwich ELISA for BBTV, utilising anti-BBTV polyclonal antibodies for virus capture, and anti-BBTV monoclonal antibodies, alkaline phosphatase-labelled sheep anti-mouse antibodies, and p-nitrophenyl phosphate for detection (ELISA-NPP); (ii) an alternative enzyme-substrate system for ELISA 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 nitroblue 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 membrane with anti-BBTV polyclonal antibodies, by using 0.05 M sodium carbonate (pH 9.6) as the coating buffer, and by clarifying the sap by centrifugation and extraction with chloroform or dichloromethane. Treatment 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 on sensitivity; heating at 70 degrees C for 10 min eliminated antigenicity. 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.