In situ immunoassay for detection of Citrus tristeza virus

An in situ immunoassay (ISIA) is described for detection of Citrus tristeza virus (CTV). Sections from stems, petioles, or leaf veins of citrus plants that were healthy or infected with CTV were fixed with 70% ethanol and incubated with specific polyclonal antiserum (PCA) 1212 or with monoclonal antibodies (MAbs) MCA13 or 17G11. Bound antibodies were labeled with enzyme-conjugated species-specific secondary antibodies and exposed to a substrate mixture (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate). Presence of CTV antigens was indicated by the development of a purple color, which could be visualized by light microscopy, in the phloem tissues of infected citrus plants. No purple color was observed in the phloem tissues of healthy plants. All isolates used in this study, both severe and mild, were detected by ISIA with the PCA 1212 and the broad spectrum MAb 17G11, but only severe isolates were detected by the strain selective MAb MCA13. Location of CTV antigens could be determined directly and accurately by ISIA in both fresh tissues and samples stored in plastic bags at 4 degrees C or frozen for 4 weeks. Sensitivity of ISIA for detecting CTV in infected plants compared favorably with that of direct tissue blot immunoassay (DTBIA). ISIA is a simple, rapid, specific, and practical procedure for CTV identification applicable to both research and diagnostic needs.