Ultra sensitive mouse elisa

Blood and plasma glucose levels were determined by an immobilized glucose oxidase method (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH, USA). Plasma insulin was measured by ELISA (Ultra-Sensitive Mouse ELISA, Crystal Chem, USA). Liver triacylglycerols (TAGs) were extracted using the method of Folch [20 (link)], lipids were dried under N₂ gas, resuspended in absolute ethanol and quantified using an enzymatic colorimetric method (GPO-PAP reagent; Roche Diagnostics).

Blood samples were collected on ice, heparinized and spun at room temperature before storage of plasma at 4°C or allowed to clot and spun at 4°C before flash-freezing of serum in liquid nitrogen. Serum metabolites were measured by small-scale enzymatic assay for glucose (1070; Stanbio Laboratory, Boerne, TX) or using a standard automated glucose monitor. Serum insulin levels were determined by an ultrasensitive mouse-ELISA (Crystal Chem, Downers Grove, IL). Plasma levels for insulin like growth factor -1 (IGF-1) was determined using Quantikine ELISA kit for mouse/rat IGF-1(MG100; R&D Systems, MN, USA). Radioimmunoassay kit for glucagon estimation (GL-32K; Millipore, Billerica, MA) was used. For glucagon, blood samples were collected in tubes containing aprotinin (1 g/ml, Bayer Healthcare, Berkeley, CA), and the serum was stored at -80°C before radio-immunoassay. Serum amylase levels were analyzed using Biovision assay kit (K711-100; Biovision, Milpitas, CA).

glucose and insulin tolerance tests were performed by blood sampling after an intraperitoneal injection of glucose (1 mg/g; Sigma-Aldrich, UK) or human recombinant insulin (0.75 unit/kg: Actrapid; Novo Nordisk, Bagsvaerd, Denmark), as previously described (5 (link), 6 (link), 30 (link), 32 (link), 33 (link)). glucose concentrations were determined in whole blood using a portable meter (Roche Diagnostics, Burgess Hill, UK). Plasma insulin concentrations were determined by enzyme-linked immunoassay (Ultrasensitive mouse ELISA; CrystalChem, Downers Grove, IL). Triglycerides and total cholesterol were quantified as described previously (8 (link)). Liver function was assessed by measuring blood serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) (74707, 74032, Advia Chemistry, Siemens).