Novel Immunization Protocol and ELISA Screening Methods Used to Obtain and Characterize Monoclonal Antibodies Specific for Human Light Chain Variable-Region Subgroups

Abstract

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 κ and λ light chain variable-region (V_L) subgroups. Mice were injected with heat-precipitated 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 capturing enzyme-linked immunosorbent assay (ELISA) employing biotinylated monoclonal light chains was designed to select optimally immunized animals for hybridoma preparation and to screen culture supernatants for high-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_κ subgroups—V_κI, V_κII, V_κIII, and V_κIV or five V_λ subgroups—V_λI, V_λII/V, V_λIII, V_λIV, and V_λVI. These reagents have been used successfully to establish, on the basis of V_L subgroup, the monoclonal nature of serum or urinary immunoglobulins as well as those found in the cytoplasm or on the cell surface of monoclonal plasma cell or B-lymphocyte populations, respectively. The availability of anti-V_L subgroup-specific MoAbs will facilitate the immunodiagnosis and study of patients with multiple myeloma, AL amyloidosis, and related B-cell proliferative disorders.