FIG. 1.

Competitive binding of thyrotropin (TSH) receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R) antibodies versus Alexa-M22-647. Binding assays on parental HEK-EM cells that do not express TSHRs but endogenous IGF-1Rs and on HEK-TSHR cells were performed by FACS competitive binding of Alexa-647-M22 by incubating cells with 1:25 ratio of Alexa-647-M22 to unlabeled TSHR antibodies M22 and KSAb1, TSH, and IGF-1R antibodies AF305 and 1H7 and IGF-1. Competing ligands were added at 4°C 15 min prior to the addition of Alexa-647-M22 and incubation for 1 h at 4°C. Gray histograms represent auto-fluorescence. Solid-black histograms show Alexa-647-M22 fluorescence intensity. (A) and (B) Alexa-647-M22 non-specifically bound to HEK-EM cells. (C)–(F) The >10-fold peak shift in Alexa-647 fluorescence in HEK-TSHR cells (C) was markedly reduced by unlabeled TSHR antibodies M22 (D) and KSAb1 (E), and partially reduced by TSH (F). (G) and (H) Anti-IGF-1R antibodies AF305 (G), 1H7 (H), and IGF-1 (not shown) did not reduce peak fluorescence.