Elisa for dog plasma

Blood samples were collected in microtubes pre-coated with lithium-heparin (Becton Dickinson, Franklin Lakes, NJ) containing 50μL EDTA (Sigma Chemicals, St Louis, MO). Blood samples were centrifuged immediately, and the supernatant was assayed for glucose with a YSI 2700 autoanalyzer (Yellow Springs Instrument Co., Yellow Springs, OH), then frozen at -20°C. Plasma insulin was measured with an ELISA for dog plasma (Alpco, Salem, NH). Samples for measuring [3-³H]glucose were deproteinized with barium hydroxide and zinc sulfate, evaporated to remove radiolabeled water, and counted in Ready Safe scintillation fluid (Beckman Instruments, Fullerton, CA). Tracer-determined whole body glucose disposal (Rd) and endogenous glucose production (EGP) were calculated using Steele’s equation modified for use with labeled glucose infusion [9 (link)] after smoothing plasma glucose and tracer data by optimal segments. Steady state was defined as the final thirty minutes of the hyperinsulinemic euglycemic clamp. Peripheral insulin sensitivity is calculated from the peripheral insulin action (ΔRd), the change in plasma insulin (ΔIns), and the glucose concentration at steady state (Gluc_SS) by the equation ΔRd/(ΔInsxGluc_SS). Similarly, hepatic insulin sensitivity is calculated using the hepatic insulin effect (ΔEGP), with the equation ΔEGP/(ΔInsxGluc_SS) (9).