Separation of free and bound radio labeled antigen using protein a sepharose cl 4b in human chorionic gonadotropin radio immunoassay

Kato, K.; Shimokawa, M.; Sunahori, K.; Fujiwara, A.; Tanaka, K.; Kawamura, H.; Miyachi, Y.

Folia Endocrinologica Japonica 57(8): 1167-1174


Accession: 006391707

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Protein A isolated from Staphylococcus aureus can bind the Fc portion of IgG of several species. Protein A coupled to Sepharose CL-4B (Protein A-S) was used for the separation of antibody-bound and free radiolabeled hCG containing 7 mg of Protein A was dissolved in 17.5 ml of 1/15 M phosphate buffer saline pH 7.4. A total of 250 .mu.l of this suspension were added to the assay tubes 24 h after mixing 125I-hCG and hCG standards or serum samples. After further incubation for 10 min 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 100-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 2nd antibody.