Ultrasensitive mouse insulin elisa

Various concentrations of IDE inhibitors were incubated with 1.5 µg/ml rhIDE for 1/2 h at RT. Next, recombinant human insulin (41-975-100, Biological Industries, Kibbutz Beit Haemek, Israel) diluted in Mercodia ultrasensitive mouse insulin ELISA (10-1249-01) calibrator 0 was added to the tubes and incubated for 1 h at 37°C. From each tube, 25 µl were taken and evaluated for residual insulin concentration using the Mercodia ultrasensitive mouse insulin ELISA kit. Absorbance at 450 nm was recorded by a Spectrafluor plus microplate reader (Tecan, Männedorf, Switzerland).

For plasma GLP-1 quantification, blood samples were collected in test tubes containing a dipeptidyl peptidase IV inhibitor (Millipore). Colonic GLP-1 was extracted according to the method described by Cani et al.33 (link). The homogenate was centrifuged at 2000 × g for 20 min at 4 °C, and the supernatant fraction was decanted and diluted 1000-fold in assay buffer. The active GLP-1 concentration was determined using an enzyme-linked immunosorbent assay (ELISA) method (Active GLP-1 ELISA Kit, Shibayagi). Plasma and caecal acetate levels were enzymatically measured in duplicate using a commercial kit (Acetate Colorimetric Assay Kit, Sigma-Aldrich). The analysis of other caecal SCFAs was performed as follows: Caecal samples were suspended in 5% (w/v) metaphosphate-PBS (49 mL/g sample). Suspensions were subsequently filtered using a 3-kDa MWCO spin column (Pall Corporation) and analysed on a gas chromatography-flame ionisation detector system (Shimadzu GC2014, Shimadzu, Kyoto, Japan) equipped with a glass column packed with 60/80 SHINCARBON A coated with Themon-3000 (Shinwa-kakou). Plasma triglyceride (Wako) and insulin (Ultrasensitive Mouse Insulin ELISA, Mercodia) levels were determined using the corresponding commercial kits.

Blood glucose levels were quantified after 6 h of fasting using a OneTouch Vita^® meter (Lifescan). Plasma Human alpha-synuclein levels were measured using the Human alpha-Synuclein Kit (Meso Scale Discovery) according to the manufacturer’s instructions. Intraassay coefficient of variation was < 14%. Plasma insulin was assayed using the Mercodia Ultrasensitive Mouse Insulin ELISA (Mercodia). Intra- and interassay coefficients of variation were <3.4 and <3.0%, respectively. Plasma leptin was measured using the Quantikine^® ELISA kit (R&D Systems). Intra- and interassay coefficients of variation were <4.5 and <4.7%, respectively. All the samples were analyzed in duplicate.