Use of hybridoma technology to assess the antibody repertoire to a negatively charged hapten

Antihapten antibodies can be characterized on the basis of their fine specificity, which is established by the ability of a panel of cross-reactive haptens to inhibit the binding of the antibody to the homologous hapten (i.e., the one used for immunization). Fine specificity has been utilized previously as a phenotypic marker to study the genetics of immunoglobulin expression and to compare directly the hapten-specific receptors on a cell to its secreted product. We have combined the hybridoma technology and fine specificity analysis in an initial attempt to asses the antibody-forming cell repertoire specific for the hapten phthalate. By utilizing a family of haptens that are cross-reactive with phthalate and a replica-plating technique, we are able to screen and to characterize large numbers of antibody-forming cell hybrids very rapidly. We report here five unique patterns of hapten inhibition. The patterns exhibited by antibody secreted by cloned hybrids remain stable for periods of at least 6 months. Identical fine specificities appear repeatedly in our cell fusion studies from one BALB/c mouse to another, and the same patterns observed in the hybridomas have also been demonstrated in spleen cells from BALB/c mice immunized with KLH-4-azophthalate according to the same protocol used to produce the spleen cells for the cell fusion experiments (Bankert, unpublished). Moreover, the frequency of expression of each fine specificity pattern is essentially the same for both the hybridomas and the antibody-forming cells from the spleen. These observations suggest that the hybridomas do reflect random and accurate samples of the hapten-specific antibody-forming cells. We recognize that multiple clonotypes may occur in some or all of the different fine specificity sets. Accordingly, we have begun to select prototypes from each set and to characterize the antibodies further by immunochemical and idiotypic analysis.