Immunochemistry of monoclonal antibodies

We have isolated and evaluated five monoclonal antibodies stimulated by the immunization of BALB/c mice with KLH-4-azophthalate. Although the humoral response in the sera of mice stimulated with the same antigen appeared restricted in isoelectric focusing, each of the hybridomas that have been isolated appears to be unique in isoeletric focusing pattern, binding constant, and fine specificity. The binding constants cover a range of two orders of magnitude, and all appear to be somewhat higher than expected for antihapten antibodies in mice, which often average between 10⁵ and 10⁶. The fine specificity appears to clearly distinguish between each of the five clones. Thus, 42-4A5 is not 50% inhibited by 2.5 x 10² M isophthalate or benzoate, while clone 42-3A2 is not 50% inhibited by 2.5 x 10⁵ M o-bromobenzoate or o-iodobenzoate. Clone 29-4A2 showed greater cross-reactivity than the other three by being better inhibited by several of these haptens. Clone 29-2A4 appears unique, but the data are difficult to interpret, since it is apparently a mixture of two clones as is evidenced by the presence of two isotypes in the preparation and two distinct groups of bands in isoelectric focusing. Although individual monoclonal antibodies have isoelectric points (pIs) in the same region of the gel, the more basic group of bands seen in antibodies isolated from hybridoma 29-2A4, as well as the more basic bands in hybridomas 42-3A2, 29-4A2 and 42-4A5, have no parallel in the normal pattern at a pH above 6.9. The other half of the normal pattern, pH 6.4-6.0, fails to match any of the clones we have isolated. Since all of the monoclonal antibodies are composed of γ₁ heavy chains and κ light chains, the different isoelectric focusing patterns are not the result of isotype differences. Each of these monoclonal antibodies appears to be unique, and we are continuing to examine the B-cell repertoire by isolating other hybridomas and evaluating their monoclonal products. It is hoped that we will begin to see repetitive clones from different mice that have been immunized with KLH-4-azophthalate.