VIEW ARTICLE

Techniques

Rapid Sample Analysis with a Simplified ELISA. L. W. Stobbs, Research scientist, Agriculture Canada, Vineland Research Station, Vineland Station, Ontario L0R 2E0; D. Barker, graduate assistant, Department of Biology, University of Waterloo, Waterloo, Ontario N1G 2W1. Phytopathology 75:492-495. Accepted for publication 25 October 1984. Copyright 1985. Department of Agriculture, Government of Canada. DOI: 10.1094/Phyto-75-492.

Simultaneous incubation of virus samples with conjugate reduced the assay time and was as sensitive and specific as the standard ELISA procedure. To further simplify this test, procedures were investigated in which virus-specific antibody-coated plates could also be pretreated with conjugate and stored over extended periods. Air- or freeze-drying of conjugate in antibody-coated wells prior to the addition of virus did not provide adequate identification of virus-positive samples, possibly because of enzyme-antibody crosslinking during dehydration. Solidification of the conjugate in the sample wells with 5% gelatin permitted easy recognition of virus and, by reducing the healthy baseline reactions, made visual assessment less subjective. By increasing the concentration of conjugate while proportionately reducing the volume applied to each well, equivalent conjugate concentrations were used as in the standard ELISA procedure, while occupying only a minimal volume of each well. Gelatin-matrixed conjugate remained stable for extended periods at - 20 C and could be stored under refrigeration for several weeks without significant decrease in sensitivity or specificity. This procedure provides a convenient means of preforming the plates with coating antibody and conjugate, such that only sample addition, incubation, and substrate reaction are required to obtain sample diagnosis.