Purification of human urine colony-stimulating factor by affinity chromatography.
Antisera from rabbits immunized with murine macrophage colony-stimulating factor (CSF-1) were evaluated for cross-reactivity with human urine CSF-1. One cross-reactive antiserum was used to purify CSF from human urine. The IgG fractions from normal rabbit serum and the anti-CSF serum were bound to cyanogen bromide-activated sepharose. Ten-liter pools of human urine were concentrated by ultrafiltration and applied sequentially to the normal IgG and antiserum columns. Cross-reactive proteins were removed by the IgG column, whereas CSF was bound by the anti-CSF column. After extensive rinsing of the antibody column, the CSF was eluted with 4 M sodium thiocyanate. This fraction contained four to five contaminating proteins as judged by migration in sodium dodecyl sulfate-acrylamide gel. In a further purification step, the CSF was retained selectively by concanavalin A sepharose and eluted with alpha-methylglucoside. This purified CSF had a specific activity of 0.8-2.3 x 10(7) U/mg protein. A single major contaminant was removed by reversed phase high performance liquid chromatography. Final specific activity of the purified CSF ranged from 2.5 to 4.4 x 10(7) U/mg protein. Each 10-liter pool of urine yielded 18-20 micrograms of pure material with a 15%-25% recovery. This technique is more rapid and provides a higher yield of pure human CSF-1 than the more tedious multi-step procedures that have been described previously.
Montefiore Hospital, University of Pittsburgh School of Medicine, Pennsylvania.
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Waheed
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RK
Shadduck
D000741:Anemia, Aplastic / Q000652:urine; D002846:Chromatography, Affinity; D003115:Colony-Stimulating Factors / Q000276:immunology / Q000302:isolation & purification*; D003429:Cross Reactions; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007938:Leukemia / Q000652:urine; D008264:Macrophages; D008970:Molecular Weight; D014556:Urine / Q000032:analysis*
D003115:Colony-Stimulating Factors; D007074:Immunoglobulin G