Immunofluorescence for detection of antibodies against human cytomegalovirus-induced membrane antigens

Abstract

This paper describes an improved method for the in vitro detection of antibodies specifically directed against human cytomegalovirus (CMV)-induced membrane antigens present on the surface of CMV-infected fibroblasts (CMV-MA). Viable cells were found to be essential for specific visualization of CMV-MA staining. The addition of divalent cations (2.6 mM Ca2+ and 2.2 mM Mg2+) and glucose (180 mM) to the incubation and washing buffers improved the viability and morphology of the cells and increased the cell yield at the end of the assay. Clustering of antigen-antibody complexes on the surface of viable CMV-infected cells was prevented by low-temperature incubation (0 to 4 degrees C) rather than by the addition of agents which act on the metabolism of the cell. No interaction with the CMV-induced Fc receptor was observed at 0 degrees C with either human sera or murine monoclonal antibodies. The specificity of the CMV-MA reaction was confirmed by using monoclonal antibodies to CMV nuclear, cytoplasmic, and membrane-associated antigens. Furthermore, a microplate modification of the membrane fluorescence test is described which is suitable for multisample screening purposes. This method can be applied to the determination of anti-CMV-MA antibody titers in human sera and to the screening of hybridoma supernatants for the presence of antibodies with specificity for CMV-MA.