Abstract
Early pregnancy factor (EPF) is a pregnancy-associated protein detected in the maternal serum by using the rosette inhibition assay and by evaluating the suppression of adoptive transfer of contact sensitivity. Because of its inhibitory effect on the functional reactivity of immunocompetent cells, EPF is thought to be involved in immunoregulation of the maternal immune system during early pregnancy. EPF was purified six million-fold from the serum of pregnant women between 5 and 12 weeks of gestation. The specific activity of purified EPF was approximately 8 x 10(8) units/mg. The purification scheme involved sequential DEAE-cellulose chromatography, S-Sepharose chromatography, concanavalin A-Sepharose chromatography, heparin-Sepharose chromatography, Mono S fast protein liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein has an apparent molecular weight of 21,500 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 28,000 by gel permeation high pressure liquid chromatography. The isoelectric point of purified EPF moiety is 6.5. The biological activity was susceptible to the proteolytic enzyme trypsin, acidic pH conditions, organic solvents, and sodium dodecyl sulfate, but stable to heat treatment at 56 degrees C for 30 min and the reducing agent dithiothreitol. The biological and physicochemical properties of EPF appear to be distinct from other pregnancy-associated and immunoregulatory proteins.
Highlights
DEAE-cellulosechromatography,S-Sepharosechro- incubation of lymphocytes with ALS, in a dose-dependent matography,concanavalinA-Sepharosechromatog- manner
Early pregnancy factor (EPF) has been reported to inhibit the adoptive and sodium dodecyl sulfate, but stable to heat treat- transfer of contact sensitivity to thesoluble antigen, trinitroment at56 “Cfor 30 min and the reducing agent dithi-fluorobenzene [16]
Evidence for the presence of immunomodulating substances in the maternal blood during pregnancy emerges from the observation that serum from pregnant animals is capable of inhibiting lymphocyte responses in several assay systems
Summary
PrFegancatonrcy to obtain the protein purified to homogeneity. Earlier at- flow column equilibrated with Buffer A. The column was washed with tempts by several workers to purify and characterize the EPF molecule(s) from the serum of pregnant sheep [20,21,22], pigs [23], humans [24], as well as conditioned medium from mouse embryos cultured in vitro [25], have met with limited success. This could be partly due to heterogeneity in the molecular the same buffer and eluted with a linear gradient from 0.1 to 1.0 M sodium chloride in Buffer A. From heparin-Sepharose column was concentrated and dialyzed against Buffer Ain an Amicon ultrafiltration cell using PM-10
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