Use of immunoaffinity chromatography for purification of 125I-labeled human prolactin.
We assessed a simple method for purifying 125I-labeled human prolactin, taking advantage of the abundant supplies of monoclonal antibodies available. 125I-Labeled human prolactin purified by immunoaffinity chromatography is compared with that purified by gel filtration on Sephadex G-100. We used monoclonal antibodies to prolactin to prepare the affinity chromatography columns. Prolactin was radiolabeled by the Chloramine T method, purified by each of the above procedures, and the binding and displacement characteristics were studied in radioimmunoassays in which either monoclonal antibodies or a rabbit anti-prolactin serum was the first antibody. A nonimmune fraction of 125I-labeled prolactin that co-eluted with the immunoreactive hormone from Sephadex G-100 was removed by affinity chromatography, which increased the antibody binding of 125I-labeled prolactin in the radioimmunoassay in the absence of unlabeled antigen (B/T0, in percent) twofold or more, increased the assay sensitivity, and increased the slope of antigen displacement measured by the 50% intercept. Several advantages make this the purification method of choice.
D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006801:Humans; D007457:Iodine Radioisotopes; D011388:Prolactin / Q000276:immunology / Q000302:isolation & purification*; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D011388:Prolactin