Double antibody radioimmunoassay

Blood glucose and insulin were analysed with a Cobas Modular system (Roche Diagnostics, Risch, Switzerland) at the Clinical Chemistry Laboratory, Sahlgrenska University Hospital (accredited in accordance with the International Standard ISO 15189:2007). CSF insulin was analyzed with a double-antibody radioimmunoassay (Linco Research) at the Department of Clinical Science, Lund University. Leptin and adiponectin were measured with ELISA kits (R&D Systems) in the Clinical Neurochemistry Laboratory at Sahlgrenska University Hospital Mölndal. ELISA plates were read on a Vmax plate reader, and concentrations were determined with Softmax software (Molecular Devices). Insulin was measured in undiluted samples and adiponectin in 100-fold diluted samples. For leptin analysis, CSF samples were diluted 2-fold and serum samples 100-fold. HOMA-IR was calculated as (fasting glucose × fasting insulin)/22.5 and HOMA-B as (20 × fasting insulin)/(fasting glucose − 3.5) [31 (link)]. GDF15 concentration was measured with Human GDF-15 Quantikine Elisa Kit (R&D Systems, Minneapolis, MN, USA). Serum GDF15 samples during and after pregnancy were diluted 1:64 and 1:4, respectively. CSF GDF15 samples were diluted 1:2. The intra- and inter-assay coefficients of variation (CVs) for GDF15 measurements were 1.7% and 7%, respectively.

After an overnight fast, participants were given a 3-h mixed meal challenge (500 kcal, 60% CHO, 20% fat, and 20% protein). Blood samples were collected at 0, 30, 60, 90, 120, and 180 min for plasma glucose, insulin, proinsulin, triglycerides, and glucagon measurements. Glucose was assessed using a glucose-oxidase method (GOD/PAP, Roche Diagnostics, Germany). Insulin was assessed by a chemiluminescent method (Roche Diagnostics, Germany). Proinsulin and glucagon were assessed by a double-antibody radioimmunoassay (Linco Research, USA). Free fatty acid fasting levels were assessed by a colorimetric assay (Wako, USA). Subjects were instructed to refrain from physical exercise, alcohol, and caffeine intake 24 h prior to the test.