Determination of Herpes Simplex Virus-Induced Alpha Interferon-Secreting Human Blood Leucocytes by a Filter Immuno-Plaque Assay

Abstract

A filter immuno-plaque assay was developed which detects alpha interferon (IFN-.alpha.)-secreting human peripheral blood leucocytes (PBL). Polyclonal anti-IFN-.alpha. antibodies were fixed to the nitrocellulose membrane bottoms of 96-well Millititer plates, which also contained monolayers of glutaraldehyde-fixed human WISH amnion cells infected by Herpes simplex virus type 1 (HSV). Such cells are potent IFN inducers and during a 16 h cocultivation with PBL, IFN-.alpha. was absorbed by the membrane-bound polyclonal antibodies around IFN-.alpha.-secreting cells. This IFN-.alpha. was detected with murine monoclonal antibodies against IFN-.alpha. and peroxidase-labelled antibodies against murine immunoglobulin, using diaminobenzidine as substrate. Distinctly stained plaques were seen, the frequency of which gave a minimal estimate of approximately 10 IFN-.alpha.-producing cells in 104 PBL (range in 12 blood donors 2.95-25.1). Fewer plaques than expected were seen at low PBL numbers per culture, one explanation being that cell interactions then limit the IFN-.alpha. response. The immuno-plaque assay should be useful in futher studies of the cellular basis of the IFN-.alpha. response.