Cytomegalovirus Antigen Detection: A Comparison of EIA Assays Utilizing Avidin-Biotin-Peroxidase Complex (ABC) and Unlabeled Antibody (PAP)

Abstract

Five Enzyme Immunosorbent Assays (EIA) were developed for in vitro Cytomegalovirus antigen detection. The noncovalent binding properties of avidin and biotin were utilized in introducing biotinylated peroxidase as the enzyme moiety in four assays. Peroxidase anti-peroxidase was the enzyme carrying molecule in the fifth assay. In assays employing biotinylated peroxidase, avidin was used to link enzyme to antigen site by binding with biotinylated secondary antibody, or anti-avidin antibody with specificity to a secondary antibody. Avidin and biotinylated peroxidase were employed in sequential steps, or as a preformed complex. Relative assay sensitivities were determined by detection of viral antigen in cells infected with various doses of virus, at progressive time points post-inoculation. Assays in which biotinylated secondary antibody was bound by preformed or nonpreformed avidin biotinylated peroxidase complexes were shown to be more sensitive than an assay employing peroxidase anti-peroxidase (PAP). The PAP assay was more sensitive than assays in which anti-avidin antibody linked the antigen site to preformed or non-preformed avidin-biotinylated peroxidase complexes. Assays employing preformed avidin-biotinylated peroxidase complexes were more sensitive than their counterparts using avidin and biotinylated peroxidase in sequential steps. All five assays were able to detect Cytomegalovirus antigen prior to the appearance of virus cytopathic effect. The more sensitive assay detected viral antigen in cells infected with 12.5 and 100 TCID₅₀ at 12 hours, and 3 hours post inoculation, respectively.