Comparison of penicillinase-based and alkaline phosphatase-based enzyme-linked immunosorbent assay for the detection of six potato viruses

Abstract

A penicillinase (PNC)-based enzyme-linked immunosorbent assay (ELISA) was compared with one based on alkaline phosphatase (ALP) to detect potato viruses A, S, V, X and Y, and potato leafroll virus. Tests were made with different ratios (w/w) of γ-globulin∶enzyme in the conjugation reaction. The γ-globulin fraction of antisera to the viruses was conjugated to ALP or to PNC by the single step glutaraldehyde bridge method using the ratio of 1∶1 (γ-globulin∶enzyme). The ALP conjugates worked well but PNC conjugates gave substantial non-specific reactions and in general were not suitable for ELISA. However, PNC conjgates made with a ratio of 1∶0.05 (γ-globulin∶enzyme) gave little or no non-specific reaction and were at least as sensitive as ALP conjugates. Both ALP and PNC conjugates were found to be useful in three variants of ELISA (double antibody sandwich; plate-trapped antigen and a simplified plate-trapped antigen form of ELISA) to detect the six potato viruses. Although PNC-based ELISA often required longer incubation with substrate to achieve the same sensitivity of detection as with ALP-based ELISA, this is only a minor disadvantage and PNC should prove to be more economical and safer to use than ALP.