Comparative analysis of procedures used to isolate variant antigen from Trypanosoma brucei rhodesiense.
Comparisons made among various procedures leading to the isolation of variant antigen from Trypanosoma brucei rhodesiense bloodstream trypomastigotes. As a means of parasite disruption, freeze-thawing solubilized 36% more variant antigen than did sonication. Protease inhibitors were important additions to the suspension prior to cellular disruption. If trypanosomal extracts were frozen for at least 1 wk prior to chromatographic isolation of variant antigen, recovery of the antigen was reduced by 70%. Ion exchange chromatography was more efficient in the isolation of variant antigen than either lentil-lectin or antibody-affinity columns. All three methods yielded qualitatively similar variant antigen preparations. Using the most efficient isolation procedure tested, about 4 mg of variant antigen was isolated per 10(10) bloodstream trypomastigotes. The most efficient means of isolating variant antigen from plasma of infected rats began with passage of fresh plasma with protease inhibitors through an ion exchange column followed by antibody-affinity chromatography. This resulted in a preparation that was 52% variant antigen, a 370-fold concentration over plasma levels.
D000818:Animals; D000906:Antibodies; D000941:Antigens / Q000302:isolation & purification*; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005615:Freezing; D037102:Lectins; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000032:analysis; D051381:Rats; D013010:Sonication; D014346:Trypanosoma brucei brucei / Q000276:immunology*
D000906:Antibodies; D000941:Antigens; D037102:Lectins; D011480:Protease Inhibitors; D011506:Proteins