Development of a nitrocellulose-enzyme immunoassay for the detection of yellow-head virus from penaeid shrimp

A nitrocellulose-enzyme immunoassay (NC-EIA) employing horseradish peroxidase (HRP) was developed for the detection of yellow-head virus in the tissues of infected penaeid shrimp. Two substrates, DAB (3,3¢-diaminobenzidine retrahydrochloride) and TMB (3,3¢5,5¢-tetramethylbenzidine) for HRP were evaluated. The TMB was found to produce a more distinct and easily recognizable colour product and provided enhanced sensitivity (0.4 ng for TMB versus 0.8 ng viral protein for DAB). Under the present format, the NC-EIA was capable of detecting as little as 100 TCID₅₀ of virus in infected tissues. Uninfected tissue homogenates yielded no significant background colouration.