Identification of Naturally Occurring Follistatin Complexes: MATERIALS AND METHODS(4)


The inhibin B standard was prepared by immunoaf-finity purification from human follicular fluid and was provided by Dr. Nigel Groome. No cross-reactivity between heterologous ligands was found for any of the assays using purified recombinant proteins. Recombinant human ligands were added at two dilutions into human urine from a pool of men. Inhibin A recovery was 100% and 110% for high and low standards. Inhibin B recovery was 90% and 92% for high and low standards. The assays were sensitive (11.7 pg/ml inhibin A; 31.1 pg/ml inhibin B) and precise. The intraassay variation was 4.8% and 4.2% for inhibin A and inhibin B assays, respectively. The interassay variation was 8.2% and 9.8% for inhibin A and inhibin B assays, respectively. buy ventolin inhalers

The activin A concentration was measured using a one-step (simultaneous) monoclonal antibody-based ELISA (2F8:6H5) as described elsewhere. Briefly, Nunc (Nalge Nunc International, Rochester, NY) Immunlon Max-isorp microtiter plates were coated overnight (2F8, 4 ^g/ ml). Standards, controls, or diluted samples (1:5 or 1:10) and freshly diluted HRP-conjugated 6H5 were added and incubated for 2 h at room temperature. The bound conjugated antibody was measured at 490-nm absorption after addition of orthophenylene diamine and hydrogen peroxide. The assay limit of detection was 100 pg/ml. The ELISA had inter- and intraassay coefficients of variation of less than 11% and 10%, respectively. Activin A was recovered (>95%) in urine samples.

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