Human insulin elisa kit

Cells grown in culture conditions inductive for pancreatic islets specification were preincubated in L15 medium without insulin and supplemented with 2.5 mM glucose in 37 °C for 4 h. Supernatants were collected, filtered through 0.45 µm filter and used for determination of insulin secretion. For ELISA experiments, wells of 96-well plate (Human Insulin ELISA kit, Sigma-Aldrich) coated with antibodies against human insulin were incubated with 100 µL of analysed supernatants along with insulin concentration standards run in parallels, for 2.5 h with gentle shaking. The whole assay was performed at room temperature. After the incubation, solutions were discarded and wells were rinsed three times with 1× Wash buffer. Afterwards, 100 µL of the detection buffer containing biotinylated antibodies was applied to each well and incubated for 1 h. After the washing step (three times with 1× Wash buffer), bound antibodies were detected with streptavidin-conjugated horse-radish peroxidase (100 µL of the solution with 45 min of incubation). The reaction was visualised by incubation with 100 µL of colorimetric TMB substrate (30 min, development was ended with 50 µL of Stop solution. Absorbance was determined spectrophotometrically at 450 nm on NanoPhotometer (Implen).

The anthropometric data (pre-pregnancy weight (pre-weight), height, pre-body mass index (pre-BMI), waist circumference (waist) and hip circumference (hip)), lifestyle factors and history of medication and treatments were obtained from clinical medical records. Pre-BMI was calculated as the subject’s pre-weight (kg) divided by their height squared (m²). Waist and hip were measured in an erect position at the narrowest point between the iliac crest and the lower costal margin and at the level of the pubic symphysis, respectively. Systolic and diastolic blood pressure was measured twice (YuTu Model XJ11D, YuTu Company, Shanghai, China) with the participant in a sitting position, and the mean value was used for further analysis. The subjects were given instructions by a physician to how to be fasting prior to the collection of samples. After 8 h of fasting, venous blood samples were collected between 07:00 and 09:00 for the assessment of metabolic biomarkers (FBG, TC, TG, LDL-C, HDL-C, etc) using a fully automatic biochemical analyser (Hitachi 7000, Tokyo, Japan). HbA1c level was detected by using high-performance liquid chromatography (HLC-723G8, Tosoh Bioscience, Japan) Insulin content was measured using a Human Insulin ELISA Kit (Sigma-Aldrich, St. Louis, MO, USA).

Venous blood was collected from each participant after an overnight fast (at least 8 hours) by a certified phlebotomist using standard laboratory techniques. Two different blood samples were collected: 1) serum glucose determination (Vaccutainer Serum Separator tube), and 2) for glycosylated hemoglobin (A1C, via EDTA collection tube). Glucose levels were measured by conventional hexokinase enzymatic methods with inter coefficient of variation (CV) = 2.2% and intra CV=10.1%. Serum total adiponectin was measured using an enzyme-linked immunosorbent assay (ELISA) with inter and intra CV to be 8.4% and 7.4% respectively (Linco Reearch Inc, MO, US). Percentages for A1c were measured from whole blood samples using Roche Tina Quant method by a certified clinical laboratory with both intra and inter CV=3.57% (Laboratory Corporation of America, LabCorp, FL, US). Serum insulin levels were determined using the Human Insulin ELISA kit from Millipore (St Charles, MZ, U.S.). High-sensitivity C-reactive protein (Hs-CRP) was analyzed in serum using Immulite method with inter CV=5.4% and intra CV=8.2%. A 1:100 manual dilution of the antibody provided a measurable range of 0.1–500 mg/L [37 (link)].

Differentiated cells were pre-incubated for 2h at 37°C in Krebs-Ringer bicarbonate HEPES buffer (KRBH:116 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl_2, 1.2 mM KH₂PO4, 1.2 mM MgSO₄, 24 mM HEPES, 25 mM NaHCO₃, and 0.1% BSA). Then, the cells were incubated for 1h at 37°C in KRBH containing the 2.5 mM D-glucose or 27.5 mM D-glucose or 30 mM potassium chloride (KCl). The human insulin levels in culture supernatants were measured with a Human Insulin ELISA kit (Millipore) according to the manufacturer's instructions. The total protein content was determined with a BCA Protein Assay Kit (Millipore) and served as internal control between groups.

Differentiated cells at terminal day were cultured without insulin for 3 h and thoroughly washed prior to ELISA. Differentiated cells or Groups of 20 similar sized islets were pre-incubated in KRBH buffer for 1 h at 37 °C. The medium was replaced with KRBH buffer containing either 27.7 mM glucose or 5.5 mM glucose for 1 h, and then the supernatant and cells for the determination of insulin secretory ability were collected. Media samples were analyzed using Rat/Mouse insulin ELISA kit (Millipore) or human insulin ELISA kit (Millipore). Released insulin was normalized to total protein content. The insulin secretion level was presented as the ratio of insulin secretion value to INS⁺ cell rate. Cells were detected by flow cytometry to evaluate the ratio of INS⁺ cells.