Preparation of steroid radioligands for ultrafiltration assays by a solid-phase transport globulin method using concanavalin A-Sepharose.
Concanavalin A-Sepharose (Con A-Sepharose) was applied to separate non-protein-bound and albumin-bound radioactive impurities from steroid radioligands. Con A-Sepharose gel, plasma, and steroid radioligand were mixed, incubated, and then washed with buffer. This method was compared with an affinity diafiltration method which separates non-protein-bound radioactive impurities with a filter membrane. 3H-Water and 3H-estrone sulfate, chosen to serve as molecules representative of non-protein-bound and albumin-bound impurities, were removed quite effectively by the Con A-Sepharose method, while 85% of 3H-estrone sulfate could not be removed by the diafiltration method. Plasma unbound cortisol (F) and testosterone (T) values determined by ultrafiltration using 3H-F and 3H-T prepared by the Con A-Sepharose method were significantly lower than those determined using the radioligands unprocessed or prepared by the diafiltration method. The whole procedure of the Con A-Sepharose method takes only 3-4 hours. This method is a simple, rapid, and effective technique for preparation of steroid radioligands for plasma protein binding studies.
D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000097:blood; D008024:Ligands / Q000302:isolation & purification*; D008297:Male; D011485:Protein Binding; D012685:Sepharose / Q000031:analogs & derivatives; D012712:Serum Globulins; D013256:Steroids / Q000302:isolation & purification*; D013739:Testosterone / Q000097:blood; D014316:Tritium; D014462:Ultrafiltration / Q000379:methods*
D008024:Ligands; D012712:Serum Globulins; D013256:Steroids; C028326:concanavalin A-sepharose; D014316:Tritium; D013739:Testosterone; D012685:Sepharose; D006854:Hydrocortisone