Rapid magnetic microsphere enzyme immunoassay for potato virus X and potato leafroll virus

A magnetic microsphere enzyme-linked immunoassay was developed for detection of potato virus X (PVX) and potato leafroll virus (PLRV) in potatoes. Analyte, microspheres with covalently coupled antibody and antibody-enzyme conjugate, were mixed, incubated together for 10 min, magnetically separated from sap, and washed with buffer three times; finally, substrates containing a 5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium or p-nitrophenyl phosphate were added. Color development (A562) or (A405) occurred in positive samples within 15-20 min. Detection sensitivity for PVX was 1-3 ng of purified virus diluted into buffer or healthy leaf sap or PVX-infected potato sap diluted X1,000 in healthy potato sap. Detection sensitivity for PLRV was approximately equal to 10 ng of purified virus diluted into healthy sap or PLRV-infected sap diluted X1,000 in healthy potato sap. This assay can be completed within 30-45 min and provides assay sensitivities comparable to double antibody sandwich enzyme-linked immunosorbent assays on polystyrene or nitrocellulose solid phase carriers for these viruses.