Novel immunization protocol and ELISA screening methods used to obtain and characterize monoclonal antibodies...

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 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 V kappa subgroups--V kappa I, V kappa II, V kappa III, and V kappa IV or five V lambda subgroups--V lambda I, V lambda II/V, V lambda III, V lambda IV, and V lambda VI. These reagents have been used successfully to establish, on the basis of VL 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-VL subgroup-specific MoAbs will facilitate the immunodiagnosis and study of patients with multiple myeloma, AL amyloidosis, and related B-cell proliferative disorders.