Porcine insulin elisa

Glucose-stimulated insulin release (GSIR) assay was performed on day 7 of tissue culture to evaluate in vitro islet secretion in response to glucose challenge [31 (link)]. For each sample, sets of three 100 IEQ were incubated at 37°C and 5% CO² in the following order of glucose media for 1 hour each: low glucose (2.8 mM; L1), high glucose (28 mM; H), low glucose (2.8 mM; L2), and high glucose plus 3-isobutyl-1-methylxanthine (28 mM + 0.1 mM IBMX; H+). The supernatant was collected after every hour for storage at -20°C until assessment. Insulin concentration in the supernatant was measured with a porcine insulin enzyme-linked immunosorbent assay kit (Porcine Insulin ELISA; cat#10-1200-01, Mercodia) and analyzed on a microplate reader (Infinite F200, Tecan and Magellan V7). The insulin concentration was normalized to the DNA content in each sample and expressed as pg of insulin/ng of DNA. The islet insulin secretion in high glucose media was divided by the insulin secreted in the first low glucose media to obtain the glucose-induced insulin stimulation index (SI).

Three arterial blood samples within the last 20 min before glucagon boluses were analysed for glucagon and insulin in all pigs. However, arterial blood samples, with the same intervals as glucose samples described above, were only collected from 8 pigs, in total after nine IP boluses and four SC boluses. Arterial blood samples were collected in empty syringes and immediately transferred to EDTA vacutainers (2 ml). The samples were stored in ice water for 10 min before they were centrifuged and the plasma transferred to Eppendorf tubes and stored at − 80 °C until analysis. Glucagon was analysed with Glucagon ELISA (10-1281-01 Mercodia, Uppsala, Sweden) and porcine insulin was analysed with Porcine Insulin ELISA (10-1200-01, Mercodia, Uppsala, Sweden). The assay ranges for the glucagon and Porcine Insulin ELISA kits were 2–172 pmol/L and 2.3–173 mU/L, respectively.
All glucagon samples were run in singles with a coefficient of variability (CV) < 10%. Inter-assay CV were 8%, 8% and 6% for 42.6, 14.7 and 4.98 pmol/L standards, respectively. This Glucagon ELISA kit cannot differentiate between endogenous and exogenous glucagon.

A glucose-stimulated insulin release (GSIR) assay was used to determine the insulin secretion of transplanted islets [30 (link)] in the following order of glucose media: low glucose (2.8 mM; L1), high glucose (28 mM; H), second low glucose (2.8 mM; L2), and high glucose plus 3-isobutyl-1-methylxanthine (28 mM + 0.1 mM IBMS; H+). The fluid medium in the Petri dish was continuously monitored to detect insulin concentrations at any given time. A standard porcine insulin enzyme-linked immunosorbent assay (Porcine Insulin ELISA, Mercodia, Winston Salem NC, USA) was used to quantify the insulin concentration from the supernatant of the medium and then analyzed on a microplate reader (Tecan Infinite F200, Tecan, Morrisville, NC, USA). As such, dividing the amount of insulin in the high glucose media over the first low glucose media was used to calculate the stimulation index (SI).

Blood glucose concentrations were measured with test strips (Accu-Chek, Roche Diagnostics, Basel, Switzerland; validated for porcine blood at the Department of Clinical Chemistry, SLU). Plasma insulin concentrations were determined by Porcine Insulin ELISA (10-1200-01, Mercodia, Uppsala, Sweden). All samples were run in duplicates with CV <10%. Inter-assay variations were 7%, 8% and 9% for 0.520, 0.161 and 0.043 μg/L standards, respectively. Results were converted from μg/L to pmol/L using a conversion factor of 174, as recommended by the manufacturer. Plasma glucagon concentrations were measured by Glucagon ELISA (10-1281-01 Mercodia, Uppsala, Sweden). All samples were run in duplicates with CV <10%. Inter-assay variations were 8%, 9% and 9% for 68.4, 24.7 and 8.47 pmol/L standards, respectively. Plasma concentrations of active GLP-1 were analysed by Glucagon-Like Peptide-1 (Active) ELISA (EGLP-35K, Merck Millipore, St Charles, Missouri, USA). This assay measures 100% of both GLP-1(7–36 amide) and GLP-1(7–37), but not the inactive peptides. All samples were run in duplicates with CV <10%. Inter-assay variations were 5% and 7% for 40.0 and 7.5 pmol/L standards, respectively.

Blood glucose concentrations were measured with test strips (Accu-Chek, Roche Diagnostics, Basel, Switzerland; validated for porcine blood at the Department of Clinical Chemistry, SLU). Plasma insulin concentrations were determined by Porcine Insulin ELISA (10-1200-01, Mercodia, Uppsala, Sweden). All samples were run in duplicates with coefficient of variation (CV) <10%, except one sample with CV 13.9%. Interassay variations were 4.7%, 5.9% and 1.7% for 55.4, 17.6 and 5.04 mU/L standards (Animal Insulin Control, 10-1221-01, Mercodia, Uppsala, Sweden), respectively. Results were converted from mU/L to pmol/L for porcine insulin by factor 6, as recommended by the manufacturer. GLP-1 concentrations in plasma were measured by radioimmunoassays after extraction of plasma with 70% ethanol (vol/vol, final concentration). Carboxy-terminal GLP-1 immunoreactivity was determined using antiserum 89390,15 (link) which has an absolute requirement for the intact amidated carboxy-terminus of GLP-1(7-36)amide and cross-reacts <0.01% with carboxy-terminally truncated fragments and 89% with GLP-1(9-36)amide, the primary metabolite of dipeptidyl-peptidase IV-mediated degradation. The sum of the two components (total GLP-1 concentration) reflects the rate of secretion of the L-cell. Sensitivity was <1 pmol/L, and intra-assay CV <5%.