Hepatitis B virus surface antigen (HBsAg) found in a commercial vaccine was use as immunogen and antigen for the production and selection of murine monoclonal antibodies against this viral antigen. Antibody relative avidity was determined based on their capture capacity. Competition studies, and the differential recognition pattern of vaccine preparations showed that high avidity IgG1 antibodies were directed against two distinct antigenic regions. Among them, 6F4 was most suitable for the detection of HBsAg in a sandwich ELISA system, using an IgM antibody, 5E8, as capture. However, the combined use of 6F4 with another which does not recognize the same epitope did not improve the sensitivity of the assay. On the other hand, the combination of 6F4 with a lower avidity IgG1 antibody, 6G10, increased the universe of antigenic diversities recognized by this system. An enzyme immunoassay for detection of HBsAg is proposed, using 3 monoclonal antibodies: one IgM as capture and two IgG1 for detection.