Determination of herpes simplex virus-induced alpha interferon-secreting human blood leucocytes by a filter immuno-plaque assay.
A filter immuno-plaque assay was developed which detects alpha interferon (INF-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 10(4) 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 further studies of the cellular basis of the IFN-alpha response.
Rönnblom
L
L
Department of Medicine, Central Hospital, Boden, Sweden.
Cederblad
B
B
Sandberg
K
K
Alm
G V
GV
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005374:Filtration / Q000295:instrumentation; D006801:Humans; D007370:Interferon Type I / Q000097:blood* / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D018139:Simplexvirus / Q000254:growth & development; D010948:Viral Plaque Assay
D007370:Interferon Type I