Validation of HTRF insulin assay conditions.

<p><b>(A)</b> pH significantly affected the ratiometric HTRF signal across a range of human insulin concentrations [F(2, 40) = 21.35, p<0.001]. Antibody incubation at pH 7 yielded the greatest HTRF signal compared to other pHs (p<0.001). <b>(B)</b> 2 h antibody incubation was sufficient to produce a robust HTRF signal, with longer antibody incubation times (12 and 48 h) further increasing HTRF signal. <b>(C)</b> There was a temperature-dependent difference in HTRF values when antibodies were incubated for 2 h at room temperature (RT, 25°C) versus 37°C [F(1,29) = 16.57, p<0.001] with higher signal observed at RT. <b>(D)</b> There was no significant difference in HTRF signal between human, rodent, porcine and bovine insulin across a range of concentrations (0.01–10 nM; p>0.05). <b>Panels A-C:</b> Data are represented as the mean emitted HTRF ratio ± SEM. <b>Panel D:</b> Data are represented as %ΔF of the HTRF signal for the respective species. For all panels, the data are from experiments performed in triplicate in 384-well plates.