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Protocols in ecological and environmental plant physiology

 

Article << Previous     |     Next >>   Contents Vol 47(3)

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

ADW Geering and JE Thomas

Australian Journal of Agricultural Research 47(3) 403 - 412
Published: 1996

Abstract

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 (AmpakTM 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 dichloroniethane. Treatment of the sap before centrifugation by snap-freezing at -70¦C, or heating at either 30 or 50¦C for 10 min, had no effect on sensitivity; heating at 70¦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. Keywords: Musa; ELISA; dot immunobinding assay; chemiluminescence; enzyme amplifiction



Full text doi:10.1071/AR9960403

© CSIRO 1996

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