PURIFICATION AND PARTIAL CHARACTERIZATION OF AN EARLY-PREGNANCY FACTOR-INDUCED SUPPRESSOR FACTOR (EPF-S-1)

PROBLEM: The immunomodulatory properties of early pregnancy factor (EP F) are mediated through induction of at least two lymphokines, designa ted EPF-S-1 and EPF-S-2 (previously estimated M(r) 15,000 and 55,000 r espectively). The activity of the former is MHC-restricted while the l atter is restricted to a locus (or loci) outside the MHC. The present study established further criteria by which EPF-S-1 and EPF-S-2 might be distinguished from each other and compared with other suppressor fa ctors. In addition, techniques have been developed to purify EPF-S-1 t o homogeneity. METHOD: Congenic mouse strains were used to map the gen etic restriction of EPF-S-2 in the rosette inhibition test and high pe rformance gel permeation chromatography was used to demonstrate that E PF-S-1 induces EPF-S-2 but not vice versa. Further studies then focuse d on isolation of this first component of the cascade, EPF-S-1, from i mmune ascites (from growth in athymic mice of the anti-EPF-S-1-produci ng rat-mouse hybridoma R2T gamma, in which EPF-S-1 is complexed to ant ibody). Techniques used were acidification followed by application to Sep-pak C-18 cartridges, high performance cation-exchange chromatograp hy and two reversed-phase HPLC steps on a C-3 column. Purified materia l was analyzed by SDS-PAGE and Edman degradation. RESULTS: Approximate ly 10 mu g EPF-S-1 were isolated from 60 ml ascitic fluid. Homogeneity of the purified material was demonstrated by SDS-PAGE, where it ran a s a single band of approximate M(r) 12,000 coincident with biological activity. Attempts at Edman degradation indicate that the molecule is N-blocked. CONCLUSION: Definitive primary characterization of EPF-S-1 must await the preparation and isolation of proteolytic fragments of t he molecule, but the present studies establish conditions which make s uch structural analysis possible.