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
Manufactured by Mercodia
Sourced in Sweden
The Ultrasensitive ELISA is a laboratory equipment designed for highly sensitive and precise measurement of analytes in biological samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) technique to quantify the presence and concentration of specific target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Ultrasensitive elisa kit, by Mercodia (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Clariostar monochromator microplate reader, by BMG Labtech (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Luminex assay, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Triton x 100, by Integra LifeSciences (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Eclia, by Roche (1 mentions)
Nycocard, by Axis-Shield (1 mentions)
11 protocols using ultrasensitive elisa
1
Fasting Serum C-Peptide Measurement
2
Quantifying Insulin Residue on Catheter Membranes
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Catheter membranes from seven of the eleven subjects were analyzed for insulin residue (Fig. 1 ). The frozen membranes were thawed at room temperature. 50 µL phosphate buffered saline (PBS) (Medicago AB, Uppsala, Sweden) with 0.05% Tween 20 (Bio-Rad, California, USA) was added to each vial. After an incubation overnight at 4 °C, the vials were placed on a plate shaker with occasional mixing at room temperature for four hours to extract the adsorbed proteins from the membranes. This method has previously been used for detection of proteins adsorbed to the catheter membrane in surgically treated intracerebral hemorrhage patients19 (link). Insulin concentration was then measured using ultrasensitive ELISA (Mercodia AB, Uppsala, Sweden) and a Clariostar Monochromator Microplate reader with a 450 nm filter (BMG Labtech, Ortenberg, Germany).
3
Quantification of Plasma Glucagon and Insulin
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4
Autoantibody and C-peptide Measurement Protocol
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IA–2A, ZnT8A, and GADA were measured from serum with use of Islet Autoantibody Standardization Program (IASP)-validated ELISA kits (KRONUS Inc., Star, ID) according to modified protocols as previously described (23 (link)). When necessary, sera were titrated to fall within the assay’s dynamic range. C-peptide was measured from serum by ultrasensitive ELISA (lower detection limit 2.5 pmol/L; Mercodia, Uppsala, Sweden), according to the manufacturer’s instructions.
5
Comprehensive Biomarker Analysis in Diabetes
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Non-fasting blood samples were obtained during regular follow-up visits to respective diabetes services. Hemoglobin A1c (HbA1c), serum creatinine, blood glucose, glutamic acid decarboxylase antibodies (GADA; detection limit 5 IU/mL) and islet antigen-2 antibodies (IA-2A; detection limit 7.5 kU/L) were analyzed according to standards and routine at the Department of Clinical Chemistry at Uppsala University Hospital. The laboratory is certified by a Swedish government authority (Swedac). To analyze C peptide concentrations, plasma was separated from samples of EDTA blood and stored frozen at −80°C until analysis. Plasma C peptide concentrations were analyzed by Mercodia (Uppsala, Sweden) using an ultrasensitive ELISA (catalog no 10-1141-01; Mercodia). The assay was calibrated against the international reference reagent for C peptide, C peptide 84/510, which is the WHO standard and is categorized by the US Food and Drug Administration as class I in vitro diagnostic device. The detection limit of the assay is set to 1.17 pmol/L, with interassay and intra-assay coefficients of variation of 5.5% and 3.8% at 37 pmol/L.
6
Blood Biochemistry Profile Analysis
7
Islet Protein Extraction and Quantification
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Blood was collected and allowed to clot for 2 h at room temperature. Serum was isolated by centrifugation at 10,000g for 10 min and frozen. Islets (n = 6–8 per group) were isolated, and tissue was resuspended in 1 × PBS (vol/vol) with 0.1% Triton-X 100 (vol/vol; Integra), 1% Protease Inhibitor Cocktail (vol/vol; Sigma), and 1% Phosphatase Inhibitor Cocktail (vol/vol; Sigma). Tissues were homogenized and centrifuged for 10 min at 10,000g. Lysate samples were normalized to total protein concentration as measured by BCA Assay (Pierce). Insulin was measured by Ultra Sensitive ELISA (Mercodia), and C-peptide was measured by ELISA (ALPCO). Cytokine and chemokine panels were measured by Luminex Assay (EMD Millipore).
8
Fasting Blood Plasma Biomarkers
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Participants were asked to fast overnight (for those attending in the morning) or for a minimum of six hours for those attending after lunch. Blood plasma samples (EDTA) were immediately spun and frozen at − 80 °C. Measurements were assayed shortly (3–9 months) after samples were taken with no previous freeze-thaw cycles. Insulin was measured by an ultrasensitive ELISA (Mercodia, Uppsala, Sweden) automated microparticle enzyme immunoassay that does not cross-react with pro-insulin. Its sensitivity was 0.07 mu/L and inter and intra-assay CVs were < 6%. Electrochemiluminescence immunoassays (ECLIA) (Roche Diagnostics, Lewes, UK) were used to measure plasma concentrations of βCTX (detection limit 0.01 ng/mL). Inter- and intra-assay coefficients of variation (CVs) were < 6% across the working range.
9
Fasting Glucose and Insulin Measurements
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We used the biochemical measurements of fasting plasma glucose (FPG) and fasting insulin (FI). Fasting samples were taken at 0900 after a 10 -h fast (water only). Blood samples were immediately spun, frozen and stored at −80 °C and measurements were assayed within 3–9 months of the samples being taken with no previous freeze-thaw cycles. FPG and FI were measured by an ultrasensitive ELISA (Mercodia, Uppsala, Sweden) automated microparticle enzyme immunoassay. Its sensitivity was 0.07 mU/L, and inter- and intra-assay coefficients of variation were <6%. Insulin resistance was calculated as a continuous measure from FPG and FI by using the computerized, updated version of the Homeostatic Model Assessment (HOMA2) for Insulin Resistance (Levy et al., 1998 (link)). The algorithm generates a relatively precise measurement of IR taking into account variations in hepatic and peripheral glucose resistance, increases in the insulin secretion curve for plasma glucose concentrations above 10 mmol/L (180 mg/dL) and the contribution of circulating proinsulin (Levy et al., 1998 (link)).
10
Ultrasensitive ELISA for C-peptide Quantification
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Plasma (EDTA) was stored (-80ºC) until analysis by ultrasensitive ELISA (Mercodia, Sweden) with a detection limit of 1.25 pmol/L (0.0038 ng/mL) as per the manufacturer’s instructions. Undetectable levels were expressed as ¼ of the assay’s lowest calibration as per manufacturer’s instructions (Tech. note 34–0144). Intra and inter-assay coefficients of variation were 3.2% and 5.8% respectively. C-peptide levels from CON were quantified in the same ELISA at 20-fold dilution, with the analyst masked to sample identity. Each assay plate contained samples from individuals with and without diabetes.
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