From 2003 to 2006, we surveyed the seroprevalence of amoebic infection in female outpatients at a gynecologist's office, which was designated as a sexually transmitted disease sentinel clinic by the Tokyo Metropolitan Government, using an enzyme-linked immunosorbent assay (ELISA). The annual rate of anti-Entamoeba histolytica (HM-1:IMSScl6 strain; HM-1) antibody-positive cases as detected by ELISA increased during that period, and anti-Chlamydia trachomatis antibodies were detected in 60%, i.e., 24 of 40 anti-HM-1 antibody-positive individuals, suggesting sexual transmission of E. histolytica. We designed an ELISA with better sensitivity using the antigen extracted from the virulence-augmented E. histolytica strains (LHM-1 and LLA526 strains) by liver-passaging in hamsters. The average ratios of the S/N value (optical density [OD] of sample/OD of negative control) of ELISA with either the LHM-1 or LLA526 antigen and that of ELISA with the HM-1 antigen were significantly higher in intestinal amoebiasis cases with low S/N values than in amoebic liver abscess cases. In the present study of the seroprevalence of E. histolytica infection, the sera testing positive with low S/N values (<10) by ELISA with HM-1 antigen exhibited higher S/N values by ELISA using LHM-1 and LLA526 antigens. This modification of the antigen preparation for ELISA is expected to be effective in detecting anti-E. histolytica antibodies from such asymptomatic patients who have low antibody titers.