Sensitive interferon assay based on immunoenzymatic quantification of viral antigen synthesis

Abstract

A sensitive enzyme immunoassay (EIA) for determining the biological activity of human interferon was developed. Green monkey kidney (Vero) cells and human embryonic lung (HEL) cells were grown in microtitre plates, treated with leukocyte interferon (IFN α) and infected with vesicular stomatitis virus (VSV). Cells were fixed with paraformaldehyde and permeabilized with Triton X-100. Viral antigen synthesis was measured by labelling the cells with VSV antiserum followed sequentially by protein A horseradish peroxidase conjugate and o-phenylenediamine. Interferon activity was detected as a lowering of the absorbance value from that of the virus control wells, reflecting the inhibition of virus protein synthesis by interferon. The minimum amount of interferon producing statistically significant ( P < 0.01) decrease of absorbance in Vero cells was 1–5 international units (I.U.)/ml as in the standard plaque reduction test the detection limit was 7.5 I.U./ml or more. In HEL cells the detection limit was 1 I.U./ml measured by EIA. The EIA for interferon activity is at least as sensitive as the traditional plaque reduction test. It is reproducible, easy to automatise and requires 7–10 times less cell culture materials than the plaque reduction test. We find it preferential especially when large numbers of specimens with limited volumes are to be analysed for interferon activity.