Serum specimens were obtained upon arrival the morning of the 25-year exam in 185 participants who were instructed to fast overnight. Participants who reported a history of pancreas transplantation (n=12) were excluded from these analyses. Serum c-peptide levels were measured in duplicate by Mercodia ultra-sensitive ELISA (Uppsala, Sweden) using the ultrasensitive ELISA kits (10–1141-01) which have a lower detection limit of 1.15 pmol/L and an intra-assay coefficient of variation (CV) of 4.5%.
Ultrasensitive elisa kit
Manufactured by Mercodia
Sourced in Sweden
The Ultrasensitive ELISA kit is a laboratory equipment designed to perform enzyme-linked immunosorbent assays (ELISA) with high sensitivity. The kit provides the necessary reagents and materials to detect and quantify target analytes in biological samples with enhanced detection capabilities.
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11 protocols using ultrasensitive elisa kit
1
Fasting Serum C-Peptide Measurement
2
Glucose Tolerance Testing in Diabetic Mice
3
Implantable Glucose Monitoring and SC-β Cell Transplantation
4
Glucose Tolerance Testing in Diabetic Mice
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All animal protocols were approved by the IACUC at Baylor College of Medicine. NOD female mice were purchased from Jackson Labs (Bar Harbor, ME) at 8 weeks of age and followed weekly with non-fasting blood glucose (One touch glucometer, LifeScan) and body weight measurements between 9–10AM on regular chow (2020x Teklad). If the blood glucose was greater than 250 mg/dl, it was retested the following day. Diabetes was defined as two consecutive blood glucose measurements >250 mg/dl. Diabetic mice were treated with HDAd vectors by tail vein injection within 48 hours after the diagnosis of diabetes. Glucose tolerance testing was performed, as described before17 (link), at 4 weeks after treatment in 6 hour fasted mice with an intra-peritoneal injection of 1.5 gm/kg body weight of D-glucose. Insulin was assayed using the Mercodia Ultrasensitive ELISA kits.
5
Glucose-Stimulated C-Peptide Secretion
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Cell were pre-incubated for 1 h in Krebs–Ringer buffer (KRB), followed by incubation for 1 h in KBR containing 2.8 mM glucose followed by 1 h incubation in KRB containing 16.7 mM glucose followed by 30 min in KRB containing 16.7 mM glucose and 30 mM KCl. Human C-peptide levels were quantified using an ultrasensitive ELISA kit (Mercodia; cross-reactivity with insulin and pro-insulin, 0.0006% and 1.8%, respectively).
6
Measuring C-peptide in Pancreatic Cells
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Cells were pre-incubated for one hour in Krebs–Ringer buffer (KRB), followed by incubation for two hours in KRB containing 0.5 mM 1-isobutyl 3-methylxanthine (IBMX) and 16.7 mM glucose. C-peptide content was determined in acidic alcohol cell extract. Human C-peptide was quantified using an ultrasensitive ELISA kit (Mercodia, Uppsala, Sweden; sensitivity 1.5 pmol/L; cross-reactivity with insulin and proinsulin 0.0006% and 1.8%, respectively) according to the manufacturer’s protocol.
7
Ultrasensitive Insulin Quantification
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A total of 100 μL of blood was collected in heparinized tubes. Plasma was separated and insulin was measured in duplicate for each data point using an ultrasensitive ELISA kit (Mercodia AB, Uppsala, Sweden).
8
Measuring Human C-Peptide Secretion in Mice
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Cells were pre-incubated for 1 hour in Krebs–Ringer buffer (KRB), followed by incubation for 2 hours in KRB containing 0.5 mM 1-isobutyl 3-methylxanthine (IBMX) and 16.7 mM glucose. C-peptide content was determined in acidic alcohol cell extract. Human C-peptide was quantified using an ultrasensitive ELISA kit (Mercodia, Uppsala, Sweden; sensitivity 1.5 pmol/L; crossreactivity with insulin and proinsulin 0.0006% and 1.8%, respectively) according to the manufacturer’s protocol. 1–2 × 106 cells were injected with a 30-gauge needle under the kidney capsule of >12 week-old nonobese diabetic severe combined immunodeficient IL2Rγ-/- (NOD-SCID-gamma, NSG) mice. Human C-peptide was quantified in serum samples obtained from the facial vein of fed mice. Serum obtained from untreated mice was used as control.
9
Intraperitoneal Glucose Tolerance and Insulin Tolerance Tests
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An intraperitoneal (i.p.) glucose tolerance test (IPGTT) was performed at 12 weeks of feeding intervention. Animals were fasted overnight before the experiments. Mice were administered glucose as a single bolus i.p. injection at 1 g/kg of body weight. Blood samples for plasma glucose measurement were collected from the tail vein 30 min before and 30, 60, 90, and 120 min after glucose administration. Glucose was measured using an Accu-Chek® glucometer (Roche Diabetes Care, Meylan, France). The plasma (5 µL sample) insulin concentration was determined 30 min before and 15 min and 60 min after glucose loading using an ultrasensitive ELISA kit (#10-1247, Mercodia AB, Uppsala, Sweden).
An insulin tolerance test (ITT) was performed at 14 weeks of feeding intervention in the same mouse groups that were tested for IPGTT. Animals were fasted 5 h before the experiment and received a single i.p. bolus injection of insulin at 0.5 U/kg of body weight. Blood was collected from the tail vein 30 min before and 15, 30, 60, 90, and 120 min after insulin administration. Glucose was measured using the Accu-Chek® glucometer (Roche Diabetes Care, Meylan, France).
An insulin tolerance test (ITT) was performed at 14 weeks of feeding intervention in the same mouse groups that were tested for IPGTT. Animals were fasted 5 h before the experiment and received a single i.p. bolus injection of insulin at 0.5 U/kg of body weight. Blood was collected from the tail vein 30 min before and 15, 30, 60, 90, and 120 min after insulin administration. Glucose was measured using the Accu-Chek® glucometer (Roche Diabetes Care, Meylan, France).
10
Plasma Biomarkers in Blood Samples
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Venous blood samples (~6 mL) were collected into EDTA tubes, stored on ice and then centrifuged at 4°C and 1006 × g for 15 minutes. Aliquots of plasma were then stored at -70°C and later analysed for glucose (Glucose Oxidase kit; Instrumentation Laboratories, Cheshire, UK), NEFA (Randox, London, UK) and lactate (Randox, London, UK) using an ILAB 650 Clinical Chemistry Analyzer (Instrumentation Laboratory, Warrington, UK) and insulin using a commercially available ultrasensitive ELISA kit (Mercodia AB, Uppsala, Sweden). Area under the curve (AUC) was calculated between time points 'baseline' and 60-min of SS.
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