Development, comparison and utilization of enhanced immunoassays for the detection of lettuce mosaic virus

Abstract

<p>Enzyme-linked immunosorbent assay (ELISA) employing alkaline phosphatase, (beta)-galactosidase, and biotin to conjugate the anti-LMV Immunoglobulin (L-IgG) was used to detect lettuce mosaic virus (LMV). Substrates, 4-methylumbelliferyll phosphate (MUP), and 4-methylumbelliferyll-(beta)-D-galactopyranoside (MUG) and p-nitrophenylphosphate (NPP) were used to react with the appropriate labeled antibody. Polystyrene microtiter plates and polystyrene beads were used as the solid-phase in the immunoassays. Optimal conditions for coating the solid-phase, specificity of the immune reaction, and other sensitivities of the immune reaction were also defined for standardization. A sensitivity level of 25 and 50 ng/ml of purified LMV was obtained when beads and plates were used as the solid-phase in s-ELISA, respectively. The b-ELISA was sensitive at 1 and 10 ng/ml of purified LMV when beads and plates were used, respectively. Fluorescence assays with beads as the solid-phase were sensitive at 1 ng/ml of purified LMV when either the substrate MUP for alkaline phosphatase or MUG for (beta)-galactosidase were used. The use of a fluorogenic substrate and beads as the solid-phase can enhance sensitivity for detection of LMV. The addition of seed extracts did not interfere with the ability of the assay to detect purified virus. This indicates a potential for seed assay. In the seed assay using ELFA-MUP, LMV was detected in at least one infected seed from a contrived sample containing 17 infected (virus source was predetermined to have 2% LMV infection) and 483 healthy seeds with a probability of 0.30. The chances of detecting the virus using the bead ELFA-MUP increases with increasing number of infected seeds to be tested;The simplicity and reproducibility of an ELFA-MUP system has potential utility for routine screening of seed samples.</p>