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Partial Purification and Characterization of a Lymphocyte-inhibitory Factor(s) in Ascitic Fluids from Ovarian Cancer Patients¹

Division of Immunology, Department of Microbiology and Immunology [A. D. H., J. R. D.], and Department of Obstetrics and Gynecology [S. A. G.], Duke University Medical Center, Durham, North Carolina 27710

Attempts were undertaken to purify and characterize the lymphocyte-inhibitory activity found in the ascitic effusions from ovarian cancer patients. The active moiety responsible for the inhibition of in vitro lymphocyte function was partially purified from several ascitic fluids by means of concanavalin A-Sepharose 4B (negative) affinity and DE52 ion-exchange chromatography, with final yields of 68 to 74% and 3- to 8-fold increases in specific activity. Analytical polyacrylamide gel electrophoresis showed that the active fraction contained mostly albumins and -globulins. Further purification by semipreparative electrophoresis in polyacrylamide gels revealed that the majority of the lymphocyte-inhibitory activity migrated with the albumins, although minor levels of activity were detected in the material which eluted from the -globulin region. The lymphocyte-inhibitory activity was found to be separable from albumin by means of a rabbit anti-human serum albumin immunoadsorbent column. The active factor which lost activity upon storage at -20° was found to have a molecular size of 40,000 to 80,000 daltons, as estimated by Sephadex G-200 filtration. Attempts to separate an active, small-molecular-size component from fractions containing the inhibitory activity by acid ultrafiltration were largely unsuccessful. Immunodiffusion analysis of the purified fractions following removal of albumin revealed the presence of containating amounts of ₁-acid glycoprotein and ₁-antitrypsin. However, levels of these two proteins did not correlate with the presence of lymphocyte-inhibitory activity.

¹ This investigation was supported in part by American Cancer Society Grant IM-94, the Ettie Stettheimer Memorial Fund, and NIH Grant CA-14049.

² NIH Postdoctoral Fellow on Training Grant T32 CA-09058-03. Present address: Oncology Center 3-130, Johns Hopkins Hospital, Baltimore, Md. 21205. To whom requests for reprints should be addressed.

Received 3/26/79. Accepted 3/ 5/80.