Experimental layout, data analysis, and thresholds in ELISA testing of maize for aphid-borne viruses

Several aspects of enzyme-linked immunosorbent assay (ELISA) procedures and data analysis have been examined in an attempt to find a rapid and reliable method for discriminating between ‘positive’ and ‘negative’ results when testing a large number of samples. A layout of ELISA plates was designed to reduce uncontrolled variation and to optimize the number of negative and positive controls. A transformation using the fourth root ( A 1/4) of the optical density readings corrected for the blank ( A) stabilized the variance of most ELISA data examined. Transformed A values were used to calculate the true limits, at a set protection level, for false positive ( C) and false negative ( D). Methods are discussed to reduce the number of undifferentiated samples, i.e. the samples with response falling between C and D. The whole procedure was set up for use with an electronic spreadsheet. With the addition of few instructions of the type ‘if…then…else’ in the spreadsheet, the ELISA results were obtained in the simple trichotomous form ‘negative/undefined/positive’. This allowed rapid analysis of more than 1100 maize samples testing for the presence of seven aphid-borne viruses—in fact almost 8000 ELISA samples.