Purification of antigen-dependent macrophage migration inhibition factor (MIF) from lymph draining a tuberculin reaction.
An antigen-dependent factor showing migration inhibition (MIF) and gold uptake stimulation (GUS) activities which has been previously described (Lowe & Lachmann, 1974) has been further purified from efferent lymph collected from cannulated nodes of BCG-sensitized sheep undergoing a delayed hypersensitivity response to PPD. During purification, fractions containing MIF activity also exhibited GUS activity. Initial purification by salt precipitation showed that antigen-dependent MIF activity was in the 40-90% ammonium sulphate precipitate. Non-specific activity and contaminating immunoglobulin were found in the 0--20% and 20--40% precipitates. Gel filtration on Sephadex G-200 and affinity chromatography on Concanvalin A-Sepharose have shown that antigen-dependent MIF is a glycoprotein of approximately 70,000 molecular weight (Lowe & Lachmann, 1974). Traces of contaminating antibody in the glycoprotein fraction were removed by immuno-adsorption on monospecific anti-sheep IgG-Sepharose. Antigen-dependent MIF was also purified by affinity chromatography on PPD-Sepharose. The eluted fractions with all the antigen-specific activity, contained less than 1% of the applied material. Analysis by polyacrylamide gel electrophoresis showed that the major protein component in the purified MIF preparation has a molecular weight and electrophoretic mobility identical with that of sheep albumin. Although this represents a high degree of purification of antigen-dependent MIF it seems that albumin is still present as a contaminant and that the protein associated with MIF activity is present in trace quantities.
D000645:Ammonium Sulfate; D000818:Animals; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006168:Guinea Pigs; D008196:Lymph / Q000276:immunology*; D008263:Macrophage Migration-Inhibitory Factors / Q000302:isolation & purification*; D008264:Macrophages / Q000276:immunology*; D008297:Male; D012709:Serum Albumin / Q000302:isolation & purification; D014373:Tuberculin / Q000276:immunology*; D014374:Tuberculin Test
D008263:Macrophage Migration-Inhibitory Factors; D012709:Serum Albumin; D014373:Tuberculin; D000645:Ammonium Sulfate