An ELISA for RNA helicase activity: Application as an assay of the NS3 helicase of hepatitis C virus

A convenient enzyme-linked immunosorbent assay (ELISA) for RNA helicase act ivity was developed with principles similar to the standard assay. The heli case ELISA utilizes a non-radioactive double-stranded substrate with a biot in-labeled template (long) strand hybridized to a digoxigenin (DIG)-labeled release (short) strand. The template strand binds to the wells of streptav idin-coated microtiter plates (SA-MTP) where the helicase catalyzes the unw inding reaction. Substrate not unwound retains the DIG-labeled release stra nd and is detected using anti-DIG coupled to horseradish peroxidase. Chromo genic detection follows. Absorbance measurement allows determination of unw inding efficiency of reactions. To demonstrate effectiveness, the ELISA-bas ed assay was used to study the unwinding activity of the hepatitis C virus (HCV) NS3 helicase. Using a known inhibitor of NS3 helicase activity and tw o mutant HCV helicases, the ability of the assay to screen potential anti-h elicase drugs and putative helicases is illustrated. The helicase ELISA is more convenient than the standard helicase assay and is especially suited f or the testing of large numbers of samples. (C) 1998 Academic Press.