Rapid, quantitative Tube immunoassays for on-site detection of Botrytis, Aspergillus and Penicillium antigens in grape juice Original Research Article

Two Tube immunoassays have been developed for the detection and quantification of fungal antigens in grape juice. One for Botrytis, the main cause of Bunch rot in grapes in California, and the other for Botrytis plus rot caused by species of Aspergillus and Penicillium, fungi that can also be involved in Bunch rot. The assays, which are rapid, user friendly and do not require laboratory facilities were designed and tested for on-site use at wineries during harvest. Both assays employ highly specific monoclonal antibodies, BC12.CA4, for Botrytis and an hitherto unreported antibody, AF-CA2, for all species of Aspergillus and Penicillium. The protocol for both assays is the same and mirrors that used in plate-trapped antigen (PTA) ELISAs. The antibodies detect water-soluble fungal antigens present in juice from infected grapes that bind rapidly to the walls of Nunc immunosorbent tubes. Saline extracts from freeze-dried mycelial cultures of Botrytis cinerea and Aspergillus niger are used as standards. The assays are very sensitive detecting between 1 and 3% levels of rot in grapes corresponding to 10–30 μg/ml extracts from freeze-dried mycelium. Absorbance values from the Tube assays parallel but do not equal those from PTA-ELISA tests. No problems were encountered with tests carried out on the testing stands at wineries at harvest time by personnel with no scientific background.