A rapid and sensitive radioimmunoassay for the detection of human cytomegalovirus binding and infection of human fibroblasts.
A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.
Shanley
J D
JD
Department of Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Biegel
D
D
Pachter
J S
JS
D002460:Cell Line; D003587:Cytomegalovirus / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D006801:Humans; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D001616:beta-Galactosidase