Free forms of light chains (FLCs) in serum are clinically useful markers, but their practical use in laboratories has been limited due to the cross-reaction with light chains associated with heavy chains (intact immunoglobulins) on FLC assays. In this study, we attempted to use anti-FLC antiserum and anti-FLC monoclonal antibody (mAb) in a sandwich enzyme-linked immunosorbent assay (ELISA) for serum FLCs. This combination resulted in a synergistic effect, showing 10(5)-fold specificity for FLC kappa and 10(4)-fold for FLC lambda compared to IgG. The specific ELISAs had good sensitivity, detecting 0.78 microg/l of FLC kappa and 31.3 microg/l of FLC lambda. We then measured serum samples before and after the absorption of IgG, IgA, and IgM and found that there was no appreciable difference in the FLC values between them. These results indicate that the ELISAs quantify serum FLCs more precisely even in the presence of about a thousand-fold amount of intact immunoglobulin. Meanwhile, the use of two anti-FLC mAbs in an ELISA showed no response for FLCs probably because of the conflict of the two mAbs at the specific epitope in FLC. Thus, ELISAs with anti-FLC antiserum and anti-FLC mAb can provide a reliable method for the clinical test of serum FLCs.