Improved ELISA conditions for detection of plant viruses

Abstract

Clover yellow vein virus (CYVV) and homologous antisera were used to test effects of time and temperature on enzyme-linked immunosorbent assay (ELISA) in polystyrene substrate plates. Replicated lattice square and Youden square experimental designs were used to measure and account for variation in absorption values associated with sample position within polystyrene plates. Adsorption of coating antibody to polystyrene was relatively rapid, reaching optimum assay efficiency in 1 h at 5°C when applied at 2.5 μg/ml. Binding of antigen and enzyme-linked antibody (conjugate) in their respective steps during ELISA was also rapid. Incubation of antigen and conjugate for 2 h each was adequate to enable detection of 20 ng CYVV in a 100 μl sample, but longer incubation of either reactant improved results. At this virus concentration, reduction in antigen incubation time by one-half could be compensated by doubling the conjugate incubation time and vice versa. Incubation of conjugates at 5°C rather than 30°C increased final ELISA readings ( A 400nm) more than two-fold. Substrate hydrolysis followed classic first order kinetics at room temperature. Greater efficiency of late antisera in ELISA was demonstrated by comparison of antisera produced relatively early and late during a rabbit's immune response. Alfalfa mosaic virus and peanut stunt virus with their homologous antisera were used to test the effects of antigen and conjugate incubation times for optimum assay efficiency. The results of these time course experiments with both viruses were similar to those obtained with CYVV. These time and temperature effects on ELISA should be applicable to most rabbit serum-virus combinations.