ELISA on Virus-Infected Cells

The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells by endocytosis from the cell surface and fusion of the viral envelope with the membrane of late endosomes. The viral envelope glycoproteins undergo antigenic changes both upon virion endocytosis and upon fusion of the viral envelope with the endosomal membrane. These changes in virion antigenicity during virus entry were first described by immunofluorescence of infected cells. Although immunofluorescence provides valuable information on the subcellular distribution of the viral glycoproteins, the quantification of immunofluorescence signals in a large number of cells is not only dependent on relatively expensive microscopy equipment, but is also relatively time-consuming. In order to quantify the antigenicity of MuHV-4 virions entering NMuMG epithelial cells in a reliable, as well as time- and cost-effective way, we have developed an ELISA with infected cells as the solid phase. In this assay, cells are grown on 96-well tissue culture plates, exposed to virions at 4 °C, followed by incubation at 37 °C allowing virion endocytosis. Cells are fixed either directly after virion binding at 4 °C or after incubation at 37 °C. After subsequent permeabilization, the cells are incubated with monoclonal antibodies specific for the viral envelope glycoproteins, followed by detection with an alkaline phosphatase-coupled secondary antibody. Upon incubation of cells with p-nitrophenyl phosphate substrate, the absorbance is measured on a conventional ELISA microplate reader. The different ways of data interpretation are discussed.