We have described a group of cytokines that modulate mediator release from basophils and mast cells. Histamine releasing factors (HRF)-mediated activation of mast cells and basophils may represent a amplification mechanism of allergic process. The clinical importance of these cytokines can not be established until their chemical structure is better defined. We have purified one species of HRF (44 kD) by sequential HPLC. The objective of this grant is to purify this and other species of HRF to homogeneity and sequence them. In addition, we plan to raise monoclonal antibodies against HRF, develop immunoassays and affinity columns to aid purification. Crude supernatants containing HRF will be generated by culturing two cell lines, U937 and RPMI 8866, both of which produce HRF similar to that synthesized by mononuclear cells (MNC). Furthermore, MNC-derived HRF will be generated by culturing cells isolated from leukocytes concentrates obtained from Blood Bank donors. HRF will be purified by using sequential HPLC. We have recently demonstrated that a major fraction of HRF binds to heparin-agarose. We have also optimized the running conditions of a C4 reverse phase column, and recovered HRF from the column with satisfactory yield. The strategy for the purification of HRF is to apply sequential HPLC involving affinity, ion exchange and reverse phase columns. The homogeneity will be verified by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Purified HRF will be electroblotted onto Immobilon PVDF filters and used directly for sequencing. The latter will be accomplished using an updated Applied Bio-system Sequenator. Monoclonal antibodies will be raised in mice using purified HRF. Hybridoma cells will be grown in the peritoneum of pristane-treated mice. Purified antibody will be used to develop ELISA and/or RIA as well as affinity columns. Our final goal is to conduct a biochemical and immunological comparison of various species of HRF produced by different subpopulations of MNC. This will be accomplished by comparing the elution profile from HPLC columns and by testing the antigenic cross-reactivity among various species of HRF obtained from different sources.
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