Modification of ELISA by Replacing Incubation of Microtiter Plates in an Incubator with Their Shaking in PVY,PVM and PLRV Detection

ELISA (enzyme-linked immunosorbent assay) is a sensitive and reliable method of plant virus detection. It is commonly used in daily research carried out by scientific institutions and laboratories working on the certification of potato tubers. The key stage in this method is a 3–4-h-long incubation of microtiter plates with IgG and with a conjugate in an incubator at a temperature of 37 °C. The aim of the research was to replace this type of incubation process with a technique of mechanically shaking the plates using a shaker to induce a vibrating movement. Three durations of shaking, performed at room temperature, were adopted: 30, 60 and 90 min with two incubation periods at a temperature of 37 °C: 60 and 180 min which were applied at the stage of coating the IgG plates, following addition of the conjugate. The assessment was made for three dilutions of lyophilized sap from leaf of potatoes (1:10, 1:100, 1:1,000). Replacing the stages of plates incubation with IgG and conjugate at 37 °C with mechanical shaking allowed the whole process of DAS-ELISA to be reduced below 3–4 h without any significant impact on its quality. The process turned out to be equally efficient as the 3-h-long incubation in an incubator for PVY, PVM and PLRV detection by means of DAS ELISA. Applying the 90-min-long incubation on a shaker in comparison to a 3-h-long incubation in an incubator gave comparable or even slightly improved results. The reaction background, i.e. the value of absorbance for sap from healthy plants (negative control) was very low in all the combinations irrespective of the time of reading after the substrate was placed. No significant differences for this parameter were found between the combinations and times of reading. Only in the case of PLRV was a clearly visible decrease in test sensitivity found (no positive reactions) at diluted sap over 1:10. Moreover, it was observed that an increase in dilutions impacted the length of reaction. The dilution 1:10 seemed to be the most favorable (maximum 1:100 for PVY and PVM), wherein the sensitivity and pace of staining the substrate for each of the methods did not provoke any doubts regarding the reliability of the test.