DiaSorin RIA Research Articles

Screening and confirmation of recombinant human erythropoietin and darbepoietin-α in spiked plasma samples from drug-free horses

Methods for screening and confirmation of erythropoiesis stimulating glycopeptides (ESGs) such as endogenous equine erythropoietin (eEPO), recombinant human erythropoietin (rhEPO) and darbepoietin-α (DAR) have been described. Four rhEPO immunoassay kits (R&D ELISA, Diagnostic Systems Limited (DSL) ELISA, chemiluminescent IMMULITE (CHEM), and DiaSorin RIA kits) were evaluated for eEPO, rhEPO and DAR screening in basal and spiked horse samples. The R&D-ELISA detected rhEPO and DAR, but did not detect eEPO. DSL-ELISA, CHEM and DS-RIA kits detected all three ESGs. The immunoassay kits did not differentiate rhEPO and DAR. Deglycosylation of the ESGs increased the detection limits of the immunoassays. DAR, rhEPO and eEPO were extracted from positive or spiked samples by using the Affi-gel and/or immunoaffinity columns. The R&D antibodies did not retain eEPO but retained rhEPO and DAR. The antibodies from DS and DSL effectively retained all three ESGs. The isolated ESGs were confirmed by using either gel electrophoresis or MALDI-TOF-MS methods. DAR and rhEPO extracted from horse plasma were hydrolyzed using trypsin and the fragments were analyzed by the MALDI-TOF-MS for their amino acid sequence. DAR and rhEPO could be differentiated according to their intact mass-ions ( m/ z 29,000 for rhEPO and m/ z 36,000 for DAR) or trypsin-hydrolysis products ( m/ z 2689 and 2359 for rhEPO and m/ z 2696 and 2293 for DAR).

Measurement of 25-Hydroxyvitamin D by the Nichols ADVANTAGE, DiaSorin LIAISON, DiaSorin RIA, and Liquid Chromatography–Tandem Mass Spectrometry

Vitamin D [ergocalciferol (D2) and cholocalciferol (D3)] is 25-hydroxylated by the liver and subsequently hydroxylated by the kidney to form 1,25-vitamin D, the active form of the vitamin. 25-Hydroxyvitamin D (25-OH-vitamin D) and 1,25-dihydroxyvitamin D have important effects on calcium absorption, bone calcium balance, and renal excretion of calcium and phosphorus (1). Assessment of the monohydroxy form of vitamin D is important for identifying insufficient endogenous synthesis disorders that impair gastrointestinal absorption and to identify renal or hepatic abnormalities (2). Here we report on 25-OH-vitamin D as measured by the Nichols ADVANTAGE® 25-OH Vitamin D, DiaSorin LIAISON® 25-OH Vitamin D, DiaSorin 25-OH Vitamin D RIA, …

Assay-Specific Decision Limits for Two New Automated Parathyroid Hormone and 25-Hydroxyvitamin D Assays

The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P <0.001). Regression and Bland-Altman analyses suggested that the LIAISON 25OHD assay reads lower than the DiaSorin RIA at low concentrations but higher at high concentrations. However, the cutoff (50 nmol/L) used in our laboratories to define vitamin D insufficiency with the DiaSorin RIA is applicable to the LIAISON 25OHD assay. In 927 healthy individuals, the 3rd-97th percentile intervals were 3-80 ng/L and 13-151 nmol/L for the LIAISON PTH and 25OHD concentrations, respectively. However, 506 individuals (54.6%) were vitamin D-insufficient; we therefore considered only the 421 individuals with a LIAISON 25OHD >50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.