Analysis of cell-associated recombinant hepatitis B surface antigen by flow cytometry

Murine L cell lines secreting recombinant hepatitis B surface antigen (rHBsAg) of either the Adw or Ayw subtype were used as a model system to develop procedures for analysis of cell-associated HBV antigens by flow cytometry. Only weak membrane immunofluorescence was observed when viable Ad or Ay cells were reacted with monoclonal antibodies (MAbs) to either subtype specific or the common group specific "a" determinant of rHBsAg. Following fixation and permeabilisation to allow access of MAbs to the intracellular compartment, specific reactivity of cells with both anti-"a" and subtype specific MAbs was readily demonstrated by flow cytometric analysis. Comparison of the fluorescence histograms produced by analysis of Ad and Ay producing cells with the anti-"a" MAb demonstrated an increased proportion of cells with high levels of intracellular rHBsAg in the Ay line. The results of these studies demonstrate that flow cytometric analysis with MAbs is a useful tool for characterizing the expression of viral antigens at the cellular level. The application of this technique to monitoring the production of native viral proteins following in vitro infection should provide valuable insights into the process of viral replication.