Brain-derived neurotrophic factor (BDNF) protein levels were measured in serum samples using the Emax Immuno Assay system from Promega (Madison, WI, USA). IGF-I (nmol/l) was assayed centrally by chemiluminescence immunoassay of EDTA plasma on the Liaison autoanalyzer (DiaSorin, S.p.A., Italy). Both measures have previously been found to be associated with depression with melancholic features in this sample (Patas et al., 2014 ).
Liaison autoanalyzer
Manufactured by DiaSorin
Sourced in Italy, United States
The Liaison autoanalyzer is a diagnostic instrument designed for automated immunoassay testing. It is capable of performing a variety of laboratory tests, including those for infectious diseases, hormones, and other analytes. The Liaison autoanalyzer is intended to provide efficient and reliable results for clinical laboratories.
Automatically generated - may contain errors
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Immulite, by Siemens (3 mentions)
Vitros 5 1 fs chemistry auto analyzer, by Ortho Clinical Diagnostics (2 mentions)
Emax immunoassay system, by Promega (1 mentions)
Bn 2 system analyzer, by Siemens (1 mentions)
Cobas c6000 analyzer, by Roche (1 mentions)
Radioimmunoassay kit, by Merck Group (1 mentions)
Au480 autoanalyzer, by Beckman Coulter (1 mentions)
14 protocols using liaison autoanalyzer
1
Measuring Serum BDNF and IGF-I Levels
2
Vitamin D Levels Assessment Protocol
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Serum vitamin 25(OH)D levels were estimated using an ELISA immunoassay [63 (link)–65 ]. A direct competitive chemiluminescence immunoassay with a Liaison auto-analyzer (Liaison, DiaSorin, Turin, Italy) was used to estimate the total 25-hydroxyvitamin (25(OH)D3) concentrations. According to the manufacturer's instructions, serum concentrations of <10 ng/mL were defined as severe VitD deficiency, serum concentrations of <30 ng/mL were defined as VitD insufficiency, and a range between 30 and 100 ng/mL was considered normal [66 (link)–68 (link)].
3
Comprehensive Vitamin D Biomarker Assessment
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Each participant underwent basic anthropometric measurements. Blood samples were also collected in tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant (for DNA) and plain tubes containing no additives (no clot activators, anticoagulants, preservatives or separator material) for serum from all participants. The following parameters were measured in all samples, as serum total serum 25(OH)D and intact PTH quantified by chemiluminescence immunoassay (CLIA), using a LIAISON auto-analyzer (DiaSorin Inc., Stillwater, MN, USA), directly measured free 25 (OH)D by immunoassay using ELISA kit (KAPF1991, Future Diagnostics Solutions B.V., Wijchen, Netherlands), VDBP by quantitative sandwich enzyme immunoassay technique using Quantikine® ELISA (DVDBP0B, R&D Systems, Minneapolis, MN, USA). Serum albumin, Ca, PO4, magnesium (Mg), lipid profile and blood glucose were measured by the colorimetric method using a VITROS 250 Clinical Chemistry auto-analyzer (Ortho-Clinical Diagnostics Inc., Rochester, NY, USA).
4
Glycosylated Hemoglobin and Insulin Resistance
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Glycosylated hemoglobin (HbA1c) was determined using a VITROS 5,1 FS chemistry auto-analyzer (Ortho-Clinical Diagnostics Inc., Rochester, NY, USA). Fasting glucose in serum was measured by means of a colorimetric method, using a VITROS 250 Clinical Chemistry Auto-analyzer (Ortho-Clinical Diagnostics Inc., Rochester, NY, USA). The intra and inter-assay CV for HbA1c and fasting glucose samples were both < 5%. Insulin and c-peptide (a consequent product produced when insulin is secreted) were measured in serum by a sandwich CLIA using a LIAISON autoanalyzer (DiaSorin Inc, Stillwater, MN, USA). The intra and inter-assay CV for insulin and C-peptide serum samples were both < 6%. Fasting insulin and Homeostasis Model Assessment 2 (HOMA2) [21] were measured for all women, with the exception of those on insulin therapy, due to the in uence of insulin intake on these measures. HOMA2 was used to estimate insulin resistance and β-cell function. HOMA2 indices [22] (HOMA2-IR and HOMA2-%β) were calculated from fasting glucose, fasting insulin and fasting c-peptide in a steady-state condition (fasting glucose: 3-25 mmol/L, fasting insulin: 2.88-43.16 mIU/L and fasting c-peptide: 0.6-10.5 µU/ml) using an updated computer HOMA calculator software (version 2.2.3) issued by University of Oxford Diabetes Trials Unit, available at https://www.dtu.ox.ac.uk/homacalculator/ .
5
Genetic Factors in Vitamin D Deficiency
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All participants answered a questionnaire (filled by the researcher), which requested information including socio-demographic data, medical history, drug history, and lifestyle history. Each participant underwent basic anthropometric and blood pressure measurements. Multi-generation pedigree was carefully made for each family by interviewing the family and documenting the family history of vitD deficiency. Fasting blood samples of all members of the family and from 100 unrelated controls were collected. Total serum 25(OH)D and intact PTH were measured by chemiluminescence immunoassay (CLIA), using a LIAISON auto-analyzer (DiaSorin Inc., Stillwater, MN, United States); free 25 (OH)D was directly measured by immunoassay using ELISA kit (KAPF1991, Future Diagnostics Solutions B.V., Wijchen, Netherlands); and VDBP was measured by quantitative sandwich enzyme immunoassay using Quantikine® ELISA (DVDBP0B, R&D Systems, Minneapolis, MN, United States). Serum albumin, Ca, PO4, magnesium (Mg), lipid profile, blood glucose, and renal and liver function were all measured by the colorimetric method using a VITROS 250 Clinical Chemistry auto-analyzer (Ortho-Clinical Diagnostics Inc., Rochester, NY, United States).
6
Biomarker Profiling for Clinical Assessment
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Glucose, triglycerides, total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using the α-ketoglutarate reaction; gamma-glutamyltransferase (GGT) levels with the L-gamma-glutamyl-3-carboxy-4-nitroanilide rate method. Albumin concentration was determined with a Alb2 kit on a Cobas C6000 analyzer (Roche Diagnostics, Milan, Italy). High sensitivity C reactive protein (hsCRP) levels were measured by automated instrument (CardioPhase hsCRP, Milan, Italy). The erythrocyte sedimentation rate (ESR) was measured automatically by the stopped-flow technique in a capillary microphotometer (Alifax Test 1 System Polverara, Italy). An automated nephelometric technology using the BN II System analyzer (Siemens Healthcare, Italy) was employed to measure plasma fibrinogen concentration. Plasma insulin concentration was measured with a chemiluminescence-based assay (Immulite, Siemens, Italy), and total serum IGF-1 levels were determined by one-step sandwich chemiluminescence immunoassay (CLIA) after prior separation of IGF-I from binding proteins on the Liaison autoanalyzer (DiaSorin, Saluggia, Italy).
7
Measurement of Plasma IGF-I Levels
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Fasting EDTA plasma samples were collected in the morning during the baseline measurement, and kept frozen at −80 °C until assaying. As described earlier17 (link), IGF-I (nmol/l) was assayed centrally by chemiluminescence immunoassay on the Liaison autoanalyzer (DiaSorin, S.p.A., Italy), with intra-assay and interassay coefficient of variation of 8.3 and 11.1%, respectively. IGF-I was studied as a continuous measure. However, as both low and high IGF-I levels might be associated with poor health outcomes, sex- and age-specific quintiles of IGF-I were also computed.
8
Comprehensive Metabolic Profiling of Participants
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Subjects underwent anthropometrical evaluation and venous blood samples were drawn in the morning, after an overnight fast, using vacutainer tubes for laboratory determinations. Height (m) was measured to the closest centimetre, weight (kg) to the closest g, and BMI was calculated as the ratio of weight in kg to the square of height in m. Waist circumference was taken at umbilical level to the closest centimetre. Blood pressure was measured using a standard sphygmomanometer in the sitting position, as the average of the last two of three consecutive measurements obtained at 3 min intervals. After a 12-h fast, a 75-g OGTT was performed with 0, 30, 60, 90 and 120 min sampling for plasma glucose.
Glucose, triglycerides, total and high density lipoprotein (HDL) cholesterol concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). Glucagon was measured using a radioimmunoassay kit (Millipore Corporation, Billerica, MA, USA). Plasma insulin concentration was measured with a chemiluminescence-based assay (Immulite®, Siemens Healthcare GmbH, Erlangen, Germany), and total serum IGF-1 was assayed by one-step sandwich chemiluminescence immunoassay (CLIA) after prior separation of IGF-1 from binding proteins on the Liaison® autoanalyzer (DiaSorin, Saluggia, Italy).
Glucose, triglycerides, total and high density lipoprotein (HDL) cholesterol concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). Glucagon was measured using a radioimmunoassay kit (Millipore Corporation, Billerica, MA, USA). Plasma insulin concentration was measured with a chemiluminescence-based assay (Immulite®, Siemens Healthcare GmbH, Erlangen, Germany), and total serum IGF-1 was assayed by one-step sandwich chemiluminescence immunoassay (CLIA) after prior separation of IGF-1 from binding proteins on the Liaison® autoanalyzer (DiaSorin, Saluggia, Italy).
9
Comprehensive Vitamin D Biomarker Assessment
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Measurement of total serum 25(OH) D was done by chemiluminescence immunoassay, using a LIAISON auto-analyzer (DiaSorin Inc., Stillwater, MN, USA) while directly measured free 25 (OH) D was measured by immunoassay using ELISA kit (KAPF1991, Future Diagnostics Solutions B.V., Wijchen, Netherlands). Vitamin-D binding protein (VDBP) was assessed by quantitative sandwich enzyme immunoassay using Quantikine® ELISA (DVDBP0B, R&D Systems, Minneapolis, MN, USA). Quantification of albumin, calcium, phosphate, magnesium, lipid profile, blood glucose, renal and liver function in serum was done through the colorimetric method using a VITROS 250 Clinical Chemistry auto-analyzer (Ortho-Clinical Diagnostics Inc., Rochester, NY, USA).
10
Plasma Kisspeptin and Metabolic Markers
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Plasma kisspeptin concentration was measured with KISS 1 (112–121) Amide/Kisspeptin 10/Measin [45–54] Amide (Human) EIA KIT (EK-048-56, Phoenix Pharmaceuticals, Inc. Burlingame, CA, USA). Intra-assay variation was <10%; inter-assay variation was <15%, with minimum detectable concentration = 0.05 ng/ml.
Glucose, triglycerides, total and high density lipoprotein (HDL) cholesterol concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). Plasma insulin concentration was measured with a chemiluminescence-based assay (Immulite®, Siemens Healthcare GmbH, Erlangen, Germany), and total serum IGF-1 was assayed by one-step sandwich chemiluminescence immunoassay (CLIA) after prior separation of IGF-1 from binding proteins on the Liaison® autoanalyzer (DiaSorin, Saluggia, Italy).
Glucose, triglycerides, total and high density lipoprotein (HDL) cholesterol concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). Plasma insulin concentration was measured with a chemiluminescence-based assay (Immulite®, Siemens Healthcare GmbH, Erlangen, Germany), and total serum IGF-1 was assayed by one-step sandwich chemiluminescence immunoassay (CLIA) after prior separation of IGF-1 from binding proteins on the Liaison® autoanalyzer (DiaSorin, Saluggia, Italy).
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