Use of Horseradish-Peroxidase Labelled Antibodies in ELISA for Plant Virus Detection

Detection of plant viruses by ELISA using horseradish peroxidase for antibody labelling (ELISA-peroxidase) has been standardized by evaluating variants of the procedure, regarding composition and concentration of buffers and additives. Immunoglobulins (IgG) are isolated from antisera by precipitation with ammonium-sulphate and by purification with DEAE-52 (Whatman) cellulose. IgG are conjugated with horseradish peroxidase by a modified oxidation-periodate method. In ELISA-peroxidase 0.05 M carbonate-bicarbonate coating buffer pH 9.6 has been substituted by 0.01 M carbonate buffer pH 9.2. Extraction buffer is used with 0.5% bovine serum albumin (BSA), without polyvinylpyrrolidone (PVP). Samples are diluted in, phosphate buffered saline (PBS) pH 7.2 with 0.05% Tween 20 and 0.5% BSA. IgG are conjugated with horseradish peroxidase, diluted in 0.1 M Tris-HCl, pH 7.4 with 0.05% Tween 20 and 1% BSA. The substrate is incubated in the darkness for 20 min at room temperature. ELISA-peroxidase proved to be equivalent in sensitivity and specificity with ELISA using alkaline phosphatase for antibody labelling. Its advantage is a lower cost of chemicals used in the test.