Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas.

Abstract

During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic rabbits carrying two transgenes, c-myc and v-abl. These rabbits developed plasmacytomas, and we obtained several plasmacytoma cell lines from which we isolated hypoxanthine/aminopterin/thymidine-sensitive clones. One of these clones, when fused with spleen cells of immunized rabbits, produced stable hybridomas that secreted antibodies specific for the immunogen. The hybridomas can be cloned and propagated in nude mice, and they can be frozen without change in their ability to secrete specific monoclonal antibodies. These rabbit-rabbit hybridomas will be useful not only for production of monoclonal antibodies but also for studies of immunoglobulin gene rearrangements and isotype switching.Monoclonal antibodies (mAbs) from rabbits have not been available because no rabbit plasmacytomas, from which a hybridoma fusion partner could be generated, have been identified. The availability of rabbit mAbs is, however, highly desirable for several reasons. First, rabbits are known to produce antibodies to many antigens that are not especially immunogenic in mice (1-5). For example, Bystryn et al. (2) directly compared rabbit and mouse antibodies directed against human melanoma cells and showed that they recognize different epitopes. Second, rabbit antibodies are generally of high affinity. Third, because most mAbs are generated in mice and rats, relatively few mAbs are available that react with mouse or rat immunogens. Because of this desire for rabbit mAbs several laboratories developed mouse-rabbit heterohybridomas, but this technology has had limited success. The earliest mouse-rabbit heterohybridomas were unstable and/or secreted only light (L) chain (6-9). Raybould and Takahashi (5) reportedly overcame this problem by using normal rabbit serum (NRS) instead of fetal calf serum (FCS) as a supplement to the culture medium. However, Verbanac et al. (10) described major problems with this method. For example, they found that the heterohybridomas were highly unstable and had to be subcloned every 4-6 weeks to avoid loss of antibody secretion. In our laboratory, we obtained no more than two to five hybridomas per fusion when using the method described by Raybould and Takahashi (5). Further, these heterohybridomas were difficult to clone, and the clones were generally unstable and did not secrete antibody over a prolonged period of time. Thus it became clear that heterohybridomas were not a satisfying solution and that rabbit-rabbit hybridomas were needed to stably produce monoclonal rabbit antibodies. We have now developed a fus...

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Preparation of high-affinity rabbit monoclonal antibodies for ciprofloxacin and development of an indirect competitive ELISA for residues in milk

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