Rapid and Simplified ELISA for Routine Field Inspection of Rice Stripe Virus

Rice stripe virus (RSV), which causes the most serious disease of rice in Japan, is transmitted by the small brown planthopper (Laodelphax striatellus) in a persistent manner. The virus is also transovarially transmitted in a high percentage1). The incidence of the disease is known to be correlated with the percentage of the viruliferous insects. Therefore, serological assays, such as the passive hemagglutination test (PHA) and latex flocculation test (LF), have been employed every spring for routine field inspection of RSV in individual insects. Although both methods have sufficient sensitivity, each has its characteristic shortcoming, such as the low stability of the red blood cells in PHA and the difficulty of judging the reaction by naked eye observation in LF. To solve these problems, we considered the alternative of using enzyme-linked immunosorbent assay (ELISA), a reliable and highly sensitive method for detecting RSV in plants and insect vectors2). The major constraint to the more widespread use of ELISA is the time involved; it normally requires 2-3 days to complete. This problem was solved by the simplified ELISA method which should be excellent for mass-indexing RSV detection. The antiserum against RSV used had a titer of 1:2,048 in a preciptin ring interface test. The ƒÁ-globulin and its enzyme conjugate were prepared according to Clark and Adams3). RSV infected rice plants were maintained and proliferated using viruliferous insects that had fed on infected rice plants. We first examined rapid ELISA4), which reduces the incubation time of standard ELISA3). Microplates (Immuron 2, Dynatech Laboratories, Inc.) were coated with 1ƒÊg/ml of ƒÁ-globulin. The conjugate was diluted to 1:2,000. Both healthy and infected leaf extracts were diluted in PBS-Tween. Reciprocals of dilution end points of A410•†0.3 measured by MR580 microplate autoreader (Dynatech Laboratories, Inc.) were definitely judged as positive reactions by naked eye observation and recorded as