Development of a dot-immunobinding assay for detection of citrus tristeza virus.
The dot-immunobinding assay (DIBA) was adapted for detection of citrus tristeza virus (CTV) and compared with DAS-ELISA and DAS-indirect ELISA. DIBA was easy to perform and as sensitive as either ELISA procedure for CTV diagnosis. The entire test could be performed in 2-3 h using polyclonal antibodies, with minimal laboratory equipment. Three different polyclonal antibodies gave a strong positive reaction with 12 selected CTV isolates; however, each serum had to be cross-absorbed with sap from healthy plants before use. The broad spectrum 3DF1 monoclonal antibody reacted with most of the CTV isolates. The MCA-13 strain-specific monoclonal antibody was specific for most severe CTV isolates. As blocking agents, 3% bovine serum albumin (BSA), 3% gelatin, 0.5% non-fat dry milk or 5% Triton X-100 gave an adequate white background on the nitrocellulose membranes and permitted discrimination between infected and healthy samples. However, 3% gelatin gave the best contrast between green for the healthy samples, and purple color for infected samples.
Rocha-Peña
M A
MA
University of Florida, Citrus Research and Education Center, Lake Alfred.
Lee
R F
RF
Niblett
C L
CL
D002957:Citrus / Q000382:microbiology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005069:Evaluation Studies as Topic; D015151:Immunoblotting / Q000379:methods*; D010942:Plant Viruses / Q000302:isolation & purification*; D012680:Sensitivity and Specificity