: Hybridoma Core (Scripps): The responsibilities of the Hybridoma Core are to generate, characterize and maintain monoclonal antibodies for use by all investigators. Specific services include: 1) immunization and bleeding of mice; 2) cell fusion; 3) primary and secondary screening assays (in collaboration with UCSD as needed); 4) cell cloning; 5)ascities fluid production; 6) cell maintenance, freezing and storage; 7) cataloging antibody storage; 8) antibody purification (in collaboration with the UCSD facility); and 9) Immunoglobulin heavy chain identification. Detailed methodologies for each of these techniques have been published previously. More than 250 hybridomas have been generated, cloned, characterized and maintained over the lifetime of this SCOR collaboration, and more than 24 in the last 4 years. Immunology Core (UCSD): The Immunology component at UCSD is responsible for preparation of immunogens used to generate antisera, as well as those used to immunize mice at the Hybridoma Core at Scripps. We use guinea pigs almost exclusively for antisera generation as we have found that they reliably yield high-titered antisera. In fact, we routinely use only 2 guinea pigs per immunization. The Core lab immunizes guinea pigs and determines titers, characterizes the pre- and post-immune antisera, purifies specific immunoglobulins when necessary and stores and catalogues them. Antigens range from isolated apoproteins to fusion proteins to putative receptors, etc. Often such highly purified antisera prove superior even to monoclonal antibodies, for example for cell-injection studies conducted by Drs. Glass, Rosenfeld and colleagues. The UCSD component of the Core also prepares a wide variety of immunological reagents to be used by the other Units, such as primary or, secondary antibodies to be used in immunoassays or Western blots or FACS analysis, and also participates in the purification of antibodies as needed. In addition, the Core will facilitate the selection of polypeptides to be synthesized to make reagent-specific antibodies, which are generated by conjugation to appropriate carriers, such as KLH. For example, we generated peptide-specific antibodies to macrosialin as noted above. During the past four and a half years, we have used 226 guinea pigs to generate antisera to 113 antigens for use by SCOR investigators. We also label antibodies with enzymes, such as alkaline phosphatase or with biotin which can then be used by the different Units for various chemiluminescent assays, Western blots, or for FACS analysis. Finally, an important component of the Immunology Core is to assist investigators and to train new postdoctoral fellows in immunological techniques, both by providing reagents as well as assistance in setting up assays and instructions in use of the chemiluminescent methodologies that we have adapted for all assays. For example, we have utilized commercial reagents for analysis of cytokines, and by use of chemiluminescent techniques greatly increased the sensitivity and linearity of the assays.
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