[Separation of free and bound radiolabeled antigen using protein A-sepharose CL-4B in hCG radioimmunoassay (author's transl)].
Protein A isolated from Staphylococcus aureus can bind the Fc portion of IgG of several species. We used Protein A coupled to Sepharose CL-4B (Protein A-S) for the separation of antibody-bound and free radiolabeled hCG in the radioimmunoassay, and the results were compared to the double antibody method. One gram of Protein A-S containing 7 mg of Protein A was dissolved in 17.5 ml of 1/15M phosphate buffer saline pH 7.4. Two hundred fifty microliters of this suspension were added to the assay tubes 24 hours after mixing 125I-hCG, anti-hCG and hCG standards or serum samples. After further incubation for 10 minutes at room temperature, the tubes were centrifuged and the radioactivity in the precipitates was measured by a gamma spectrometer. The standard curve obtained by the Protein A-S method was virtually identical to that obtained using the double antibody method. The intra- and interassay coefficients of variation in the hCG radioimmunoassay using the Protein A-S method ranged from 6.6 to 8.2% and from 7.2 to 11.9% respectively, which were close to those obtained by the double antibody method. IgG in serum inhibited the binding of Protein A-S to anti-hCG, and thus it was necessary to dilute the samples more than one-hundred fold. Under these conditions a good correlation exists in the serum hCG levels determined by either the Protein A-S method or the double antibody method. Thus, Protein A-S permitted the rapid separation of antibody-bound and free radiolabeled hCG in the radioimmunoassay and could be substituted for the second antibody.
D000941:Antigens / Q000032:analysis; D006063:Chorionic Gonadotropin / Q000097:blood*; D005260:Female; D006801:Humans; D007141:Immunoglobulin Fc Fragments; D008297:Male; D011247:Pregnancy; D011863:Radioimmunoassay / Q000379:methods*; D013205:Staphylococcal Protein A / Q000276:immunology*
D000941:Antigens; D006063:Chorionic Gonadotropin; D007141:Immunoglobulin Fc Fragments; D013205:Staphylococcal Protein A