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 κ and λ light chain variable-region (VL) subgroups. Mice were injected with heat-precipitated human Bence Jones proteins or VL-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-VL MoAbs. These methods have led to the generation of MoAbs that by ELISA react specifically with each of the four Vκ subgroups—VκI, VκII, VκIII, and VκIV or five Vλ subgroups—VλI, VλII/V, VλIII, VλIV, and VλVI. These reagents have been used successfully to establish, on the basis of VL subgroup, the monoclonal na...