Purification of uteroglobin using monospecific antibodies coupled to divinylsulphone-activated agarose.
As a model for the isolation of a labile or trace protein, the purification of uteroglobin (UGL) by immunoaffinity chromatography is described. Antibody was isolated from sheep antiserum by immunoprecipitation, and coupled to divinylsulphone-activated agarose (Mini Leak). For the immunoabsorption stage rabbit uterine mucosal scrapings were defatted and incubated directly with the immunosorbent. After washing and desorption, the UGL preparation contained relatively few high molecular weight impurities and these were removed by gel chromatography. Purification was monitored at each step by two-dimensional SDS polyacrylamide gel electrophoresis and immunoelectrophoresis. Furthermore, affinity-purified UGL was tritiated with N-succinimidyl[2,3-3H]propionate and assayed by fluorography. In order to determine absolute UGL concentrations a competitive ELISA was developed.
Dannhorn
D R
DR
Department of Biology, Philipps University, Marburg, F.R.G.
Wirth
B
B
Kirchner
C
C
D000818:Animals; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000302:isolation & purification*; D007106:Immune Sera / Q000032:analysis; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G; D009092:Mucous Membrane; D011233:Precipitin Tests; D011817:Rabbits; D012685:Sepharose; D012756:Sheep; D013450:Sulfones; D014598:Uteroglobin / Q000276:immunology / Q000302:isolation & purification*
D003432:Cross-Linking Reagents; D006023:Glycoproteins; D007106:Immune Sera; D007074:Immunoglobulin G; D013450:Sulfones; C009873:divinyl sulfone; D012685:Sepharose; D014598:Uteroglobin