CORE--Monoclonal Antibody Facility

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The hybridoma core facility provides a cell culture facility to assist the investigators in the completions of the projects. We are available to produce monoclonal and in some cases polyclonal antibodies for their use. This includes the immunization of mice, performing test bleeding to determine the degree of immunization, removal of spleens and lymph nodes for fusion and the actually fusion. Hybridomas are selected in HAT medium and tested in a number of assays for specificity. We screen using radioimmunoassay, ELISA, and western blotting. Hybridomas producing antibodies of the appropriate specificity are cloned two times by limiting dilution. To produce large quantities of antibodies we grow the hybridomas in tissue culture, or injected them into pristane primed mice. The antibodies are purified from these solution by Protein A or Protein G chromatography, DEAE ion exchange or on separose columns with covalently attached anti- immunoglobulins. All of these procedures are available to the members of the program project. In addition to production of monoclonal we prepare widely used reagents such as antibodies to HA tag, C-Myc tag, antibodies to mouse isotypes, antibodies to the proteosomes, and any other control antibodies matched for isotype and allotype. We are also trying to develop antibodies to receptor known to be involved in the discriminating Th1 and Th2 immunity. Common scientific thinking at this time suggests that autoimmune disease stems from overactivation of Th1 immune response. We are trying to prepare mouse monoclonals against the IL-12B2 receptor, believed to play an important role in expansion of Th1 cells and the interferon Gamma Beta receptor believed to be involved in generation of Th2 cells. We have produced and tested monoclonal anti-IL-12B2 produced against a peptide derived from the known sequence. This failed to bind to Th1 cells as determined by a number of assays. Therefore, we prepared a GST fusion protein containing the extracellular domain of the IL-12B2 chain. This was done by RT-PCR from RNA collected from mice following immunization with LPS, IL-12 and gamma interferon. We successful prepared PCRed this fragment and have now expressed it in bacteria. We will use Hamster for the hybridoma in order to avoid the tolerance which might be present in the mouse. The hybridomas will be tested by ELISA screening on the fusion protein with another GST fusion as a negative control. Positive hybridoma supernatnats will be tested on spleen cell populations prepared to express primarily Th1 cells.