Partial purification of human leucocyte migration inhibitory factor (LIF) by immunosorption of supernatant protein contaminants detected by crossed immunoelectrophorsis.
Concentrated supernatants from washed human lymphocytes incubated in serum-free medium were investigated by crossed immunoelectrophoresis. Quantitatively, the most important macromolecules were serum proteins, in particular albumin and degraded products of albumin. No gross difference was detectable between supernatants from concanavalin A stimulated and unstimulated lymphocytes. The degraded proteins were considered to arise as a result of proteolytic enzymes present in both stimulated and unstimulated lymphocyte supernatants. These molecules exhibited almost the same electrophoretic mobility and molecular weight as native albumin, and might therefore be expected to be difficult to separate from some lymphokines by conventional biochemical techniques. Rabbit immunoglobulins to whole human serum proteins together with immunoglobulins against crude supernatants of mitogen stimulated lymphocytes were therefore bound covalently to an agarose matrix. This preparation efficiently removed all detectable proteins from concentrated supernatants of activated lymphocytes as determined by crossed immunoelectrophoresis. Leucocyte migration inhibitory factor (LIF) was recovered almost quantitatively, and a 40-fold purification of LIF was achieved. The technique is rapid, economical and well suited as an initial step for purification of large quantities of LIF.
D000818:Animals; D000906:Antibodies; D002474:Cell-Free System; D002850:Chromatography, Gel; D006801:Humans; D007122:Immunoelectrophoresis; D007123:Immunoelectrophoresis, Two-Dimensional; D007163:Immunosorbent Techniques; D008222:Lymphokines / Q000302:isolation & purification*; D011817:Rabbits; D012709:Serum Albumin / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D008222:Lymphokines; D012709:Serum Albumin