Elisa kit

Blood was collected in tubes with or without anticoagulant (EDTA). Plasma or serum was separated by centrifugation and stored at −20 °C.
The plasma PP level was determined by an ELISA kit P (Millipore, Saint Charles, MO, USA). The sensitivity was 12.3 pg/mL and the intra and inter-assay coefficients of variation (CVs) were 3.3% and 9.8%, respectively.
The total plasma PYY level, including both PYY_1-36 and PYY_3-36, was measured by an ELISA kit (Millipore, Saint Charles, MO, USA). The sensitivity was 6.5 pg/mL and the intra- and inter-assay CVs were 2.66% and 6.93%, respectively.
The total plasma GLP-1 level, including GLP-1_{7-36 amide}, GLP-1_7-37, GLP-1_{9-36 amide}, GLP-1_9-37, GLP-1_{1-36 amide}, and GLP-1_1-37, was determined by an ELISA kit (Millipore, Saint Charles, MO, USA). A DPP-IV (dipeptidyl protease IV) inhibitor (protease inhibitor cocktail, Sigma Aldrich-Merck, Darmstadt, Germany) was added to tubes (50 µl) in order to prevent GLP-1 breakdown. The sensitivity was 1.5 pmol/l and the intra and inter-assay CVs were 1% and <12%, respectively.
Serum insulin concentration was measured by a chemiluminescent immunometric assay (Immulite 2000, DPC, Los Angeles, CA, USA). The sensitivity was 2 µIU/mL and the intra and inter-assay CVs were 22–38% and 14–23%, respectively.
The serum glucose level was determined by the glucose oxidase enzymatic method (Roche Diagnostics, Monza, Italy).

The subject’s blood samples were collected after 10–12 h of fasting then centrifuged, and the serum was immediately stored in small aliquots in a freezer at −20 °C. The results of insulin and plasma glucose were used in the following formula to calculate the Homeostasis Model Assessment for Insulin Resistance (HOMA-IR): HOMA-IR = Glucose (mg)/18 × Insulin (mUI/L)/22.5 [28 (link)]. Values <2.7 were considered normal [29 (link)].
Duplicate insulin measurements were performed in serum with an immunoenzymatic assay (ELISA method) using commercial kits of high sensitivity and specificity. Insulin: immunoenzymatic method, ELISA kit (MILLIPORE- Billerica, USA). Sensitivity: 1 µU/mL. Glucose: Enzymatic method, automated using YSI 2300 glucose bioanalyzer equipment. Total cholesterol, Low-Density Lipoprotein cholesterol (LDL-c), High-Density Lipoprotein cholesterol (HDL-c), Very Low-Density Lipoprotein cholesterol (VLDL-c), and Triglycerides (TG): automated colorimetric enzyme method, Roche Diagnostics, on Hitachi 917 equipment (Roche Diagnostics, Mannheim, Germany). Leptin: enzimatic method, ELISA kit (MILLIPORE- Billerica, MI, USA). Sensitivity: 0.195 ng/mL. hs-CRP: immunonephelometry using the nephelometer method, BN II Systems (Siemens DadeBehring Inc., Newark, DE, EUA). Sensitivity: 0.02 mg/dL.

Blood glucose levels in tail blood samples were measured before and after STZ injection using a glucometer (OneTouch Ultra 2, LifeScan, Inc., Milpitas, CA, USA). The cumulative incidence of diabetes was calculated as the percentage of hyperglycemic mice (non-fasting blood glucose level ≥ 250 mg/dL) per treatment group at each time point. For the oral glucose tolerance test (OGTT), mice were given glucose (2 g/kg body weight) by oral gavage after a 16 h fast. Blood glucose levels were monitored at indicated time points before and after glucose administration. The area under the curve (AUC) for glucose during OGTT was calculated for each experimental group. Plasma insulin levels were determined using an ELISA kit (Millipore Co., Billerica, MA, USA) following the manufacturer’s instructions.
The pancreas was homogenized in acidified ethanol and incubated for 16 h at 4 °C. Pancreas extracts were centrifuged at 3000× g for 10 min at 4 °C. The insulin content of the supernatant was determined using an ELISA kit (Millipore).

Random-fed blood glucose was measured from blood obtained from the tail vein by using a OneTouch Ultra 2 glucose meter (LifeScan, Inc., Milpitas, CA, USA). Hyperglycemia was defined as a non-fasting blood glucose level ≥ 300 mg/dL. The cumulative incidence of diabetes was calculated as the percentage of hyperglycemic mice at each time point. An oral glucose tolerance test (OGTT) was performed at 2 weeks after STZ treatment. Mice were fasted overnight (16 h), and a glucose load (2 g/kg) was administered orally. Blood glucose and plasma insulin levels were measured from the tail vein at the indicated time points after administration of glucose. The area-under the curve (AUC) for glucose and insulin was calculated for each group of animals during the OGTT. Following overnight fasting, plasma was separated and used to determine the insulin levels by using an ELISA kit (Millipore Co., Billerica, MA, USA).
The pancreas was isolated, homogenized in acidified ethanol, extracted overnight at 4°C, and centrifuged. The insulin content of the supernatant was determined using an ELISA kit (Millipore) and expressed as ng/mg pancreas.

Circulating plasma levels of insulin, corticosterone, adiponectin, and leptin were determined by ELISA using specific commercial kits:-

Corticosterone was evaluated with the ELISA kit from the Cayman Chemical Com-pany, MI, USA, No. 501320.

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Insulin with ELISA kit EMD Millipore Corporation, MI, USA, Cat. # EZRMI-13K.

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Adiponectin with ELISA kit EMD Millipore Corporation, MI, USA, Cat. # EZMADP-60K.

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Leptin with EMD ELISA kit Millipore Corporation, MI, USA, Cat. # EZML-82K.

All samples were worked with in duplicate, according to the manufacturer’s instructions.

The plasma adiponectin level was assessed using adiponectin enzyme-linked immunosorbent assay (ELISA) kits (Millipore Corporation, Billerica, MA, USA). The serum levels of TNF-α and IL-6 were measured with ELISA kits according to the manufacturer’s instructions (Sigma-Aldrich, Saint Louis, MO, USA).