NOVEL IMMUNIZATION PROTOCOL AND ELISA SCREENING METHODS USED TO OBTAIN AND CHARACTERIZE MONOCLONAL-ANTIBODIES SPECIFIC FOR HUMAN LIGHT-CHAIN VARIABLE-REGION SUBGROUPS

We have developed a novel immunization protocol for the production of a panel of high-affinity murine monoclonal antibodies (MoAbs) that are specific for each of the major human kappa and lambda light chain var iable-region (V(L)) subgroups. Mice were injected with heat-precipitat ed human Bence Jones proteins or V(L)-related fragments emulsified in monophosphoryl lipid A (MPL) and trehalose dimycolate (TDM) at two- to four-week intervals over a seven-month period. A unique direct captur ing enzyme-linked immunosorbent assay (ELISA) employing biotinylated m onoclonal light chains was designed to select optimally immunized anim als for hybridoma preparation and to screen culture supernatants for h igh-affinity anti-V(L) MoAbs. These methods have led to the generation of MoAbs that by ELISA react specifically with each of the four V(kap pa) subgroups-V(kappaI), V(kappaII), V(kappaIII), and V(kappaIV) or fi ve V(lambda) subgroups-V(lambdaI), V(lambdaII/V), V(lambdaIII), V(lamb daIV), and V(lambdaVI). These reagents have been used successfully to establish, on the basis of V(L) subgroup, the monoclonal nature of ser um or urinary immunoglobulins as well as those found in the cytoplasm or on the cell surface of monoclonal plasma cell or B-lymphocyte popul ations, respectively. The availability of anti-V(L) subgroup-specific MoAbs will facilitate the immunodiagnosis and study of patients with m ultiple myeloma, AL amyloidosis, and related B-cell proliferative diso rders.