Comparisons of plasma aldosterone and renin data between an automated chemiluminescent immunoanalyzer and conventional radioimmunoassays in the screening and diagnosis of primary aldosteronism.

Determining values of plasma renin activity (PRA) or plasma active renin concentration (ARC), plasma aldosterone concentration (PAC), and aldosterone-to-renin ratio (ARR) is essential to diagnose primary aldosteronism (PA), but it takes several days with conventional radioimmunoassays (RIAs). Chemiluminescent enzyme immunoassays for PAC and ARC using the Accuraseed® immunoanalyzer facilitated the determination, but relations between Accuraseed® immunoanalyzer-based and RIA-based values in samples of PA confirmatory tests and adrenal venous sampling remained to be elucidated. We addressed this issue in the present study. This is a prospective, cross-sectional study. ARC and PAC values were measured by the Accuraseed® immunoanalyzer in samples, in which PRA and PAC values had been measured by the PRA-FR® RIA and SPAC®-S Aldosterone kits, respectively. The relations between Accuraseed® immunoanalyzer-based and RIA-based values were investigated with regression analyses. The optimal cutoff of Accuraseed® immunoanalyzer-based ARR for PA screening was determined by the receiver operating characteristic analysis. After log-log transformations, linear relations with high coefficients of determination were observed between Accuraseed® immunoanalyzer-based and RIA-based data of renin and aldosterone. Following the PA guidelines of Japan Endocrine Society, Accuraseed® immunoanalyzer-based cutoffs were calculated from the regression equations: the basal PAC for PA screening >12 ng/dL, PAC for the saline infusion test >8.2 ng/dL, ARC for the furosemide-upright test <15 pg/mL, and ARR for the captopril challenge test >3.09 ng/dL per pg/mL. The optimal cutoff of Accuraseed® immunoanalyzer-based ARR for PA screening was >2.43 ng/dL over pg/mL not to overlook bilateral PA patients. The present study provided conversion formulas between Accuraseed® immunoanalyzer-based and RIA-based values of renin, aldosterone, and ARR, not only in basal samples but also in samples of PA confirmatory tests and adrenal venous sampling. Although validation studies are awaited, the present study will become priming water of harmonization of renin and aldosterone immunoassays.

[Comparative study of the serum measurement of PTH on Roche Cobas e411® versus the Abbott Architect ci8200®]. / Étude comparative du dosage de la PTH sérique sur Architect ci8200® versus Cobas e411®.

Parathormone (PTH) is the main hormone of phosphocalcic homeostasis. It is synthesized and secreted by the parathyroid glands. PTH has become a routine test in the medical biology laboratory. However, its measurement presents analytical difficulties with the various marketed kits. The aim of this work is to present the results of a comparative study between the PTH measurment on Abbott architect ci8200 and on Roche's Cobas e411 automaton. It is a prospective study carried out for 252 hospitalized patients in the various departments of the University Hospital Center Mohammed VI of Oujda. The "intact" PTH tests were performed on two automata: Abbott Architect ci8200 and Roche Cobas e411. The first uses chemiluminescent microparticle immunoassay. The second uses electrochemiluminiscence sandwich enzyme immunoassay. The agreement of the results between the different techniques was evaluated using the Bland-Altman difference diagram and the Passing-Bablok and Deming regression line (MedCalc software version 14.8.1.0®). By analyzing the diagram of Bland-Altman, we note that the average bias between both methods is of the order of 193.9 pg/mL. As for the equation of the right of Passing-Bablok, it is: Y(Architect) = 3.11 X (Cobas) - 12.26. In conclusion, our study shows a great discrepancy between the results of the PTH assay on the Architect ci8200 versus the Cobas e411, hence the biologist's indisputable role in the control and evaluation of the kits marketed through the various validation tests.

Measurement of testosterone in infant fecal samples.

OBJECTIVES: This study reports the validation of a noninvasive method for repeated assessment of testosterone from infant fecal samples. METHODS: Fecal samples were collected from cotton diaper liners, subjected to methanol extraction, and assayed using a modified commercial testosterone RIA kit. RESULTS: Method validity was supported by a recovery near 100%, a sensitivity of 1.23 pg/ml, and inter- and intra-assay coefficients of variations less than 10 and 15%, respectively. Testosterone was detected in all samples from male and female infants aged 2 weeks to 15 months. CONCLUSIONS: Fecal assessment is supported as a novel, non-invasive tool for studying testosterone during early human development.

The importance of using the optimal plasticware and glassware in studies involving peptides.

The unpredictable nature of peptide binding to surfaces requires optimization of experimental containers to be used. To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research, we tested the recovery of radiolabeled (125)I endocrine peptides under different conditions and provide guidelines for determining the surfaces to use for other peptides. (125)I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin-releasing factor, glucagon-like peptide-1 [GLP-1], insulin, leptin, nesfatin-1, and peptide YY), representing a wide spectrum in net charge, size, end group, and modification, were incubated for 48 h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides were incubated with bovine serum albumin (BSA, 1%) with or without subsequent lyophilization. Recovery of (125)I-labeled peptides was determined by gamma counting. Important differences in (125)I-labeled peptide binding capacities to various types of surfaces exist. Siliconization decreased, whereas the addition of BSA improved recovery from surfaces tested. Lyophilizing solutions containing (125)I-labeled peptides and BSA in the tubes best suited for individual peptides rendered more than 89% recovery for all peptides. Ghrelin specifically displaced (125)I-ghrelin from borosilicate glass, whereas GLP-1 and Fmoc-arginine did not. Choosing the appropriate experimental container avoids unpredictable peptide loss that results in inaccurate measurements and false conclusions.

The effects of anti-insulin antibodies and cross-reactivity with human recombinant insulin analogues in the E170 insulin immunometric assay.

BACKGROUND: Insulin assays are affected by varying degrees of interference from anti-insulin antibodies (IAs) and by cross-reactivity with recombinant insulin analogues. We evaluated the usefulness of the E170 insulin assay by assessing IA effects and cross-reactivity with 2 analogues. METHODS: Sera were obtained from 59 type 2 diabetes patients receiving continuous subcutaneous insulin infusion and 18 healthy controls. Insulin levels were determined using an E170 analyzer. To investigate the effects of IAs, we performed IA radioimmunoassays, and analyzed the differences between directly measured insulin (direct insulin) and polyethylene glycol (PEG)-treated insulins (free, IA-unbound; total, IA-bound and unbound insulin). We performed in-vitro cross-reactivity tests with insulin aspart and insulin glulisine. RESULTS: In IA-positive patients, E170 free insulin levels measured using the E170 analyzer were significantly lower than the direct insulin levels. The mean value of the direct/free insulin ratio and IA-bound insulin, which were calculated as the difference between total and free insulin, increased significantly as endogenous IA levels increased. The E170 insulin assay showed low cross-reactivities with both analogues (< 0.7%). CONCLUSIONS: IAs interfered with E170 insulin assay, and the extent of interference correlated with the IA levels, which may be attributable to the increase in IA-bound insulin, and not to an error in the assay. The E170 insulin assay may measure only endogenous insulin since cross-reactivity is low. Our results suggest that the measurement of free insulin after PEG pre-treatment could be useful for beta cell function assessment in diabetic patients undergoing insulin therapy.

Migraine models.

Migraine is a high prevalence disorder which affects a significant proportion of the general population, especially women during their central and more productive time of the life, thus causing severe disability. The genetic basis of the disease is unknown and the mechanism is poorly understood. The possibility that following a perturbation in the central nervous system, and particularly in the brainstem, trigeminal neurons become hyperexcitable and produce an uncontrolled release of sensory neuropeptides which eventually results in arterial vasodilatation and neuronal sensitization, has been gaining credit from studies in experimental animals and humans. In particular, experimental and clinical data with antagonists of the calcitonin gene-related peptide (CGRP) propose this molecule and its receptor as a major target for migraine treatment.

Performance of a fully automated quantitative neopterin measurement assay in a routine voluntary blood donation setting.

BACKGROUND: Acute virus infections are indicated by increased serum neopterin concentrations. Testing of blood donations for increased neopterin was introduced in the Austrian Tyrol in 1986 and throughout Austria in 1994 to improve the safety of blood transfusions. Radioimmunoassay (RIA) was the first available test, and the 98th percentile for neopterin concentrations from 76,500 donations was defined as the cut-off threshold, excluding donations with neopterin >or=10 nmol/L. When ELISAs were introduced on fully automated test systems to measure neopterin concentrations, results were generally higher and the cut-off was adjusted up to 12 nmol/L. METHODS: In early 2006, new equipment (Hamilton Microlab Star pipettor + Dade Behring ELISA Processor III, BEP III) became available which monitors sample and reagent volumes for each uptake and dispense, and takes the viscosity of the fluids into account. Using this system, the performance of neopterin determinations was evaluated over 21 months and compared to earlier data. RESULTS: When the new apparatuses were introduced, an immediate decrease in mean neopterin concentrations and the percentage of samples with neopterin concentrations >or=10 nmol/L was observed. The 55,516 measurements performed in 2007 showed a mean value of 5.5 nmol/L. Neopterin in 680 donations (1.23%) were >or=10 nmol/L. CONCLUSIONS: The new Hamilton diluter increased the accuracy of the neopterin test procedure. Results are now identical with those of RIA and ELISA when run in a semi-automated manner. Differences in pipetting performance of standards and samples are considered as one possible explanation for the earlier discrepancies seen with the automated apparatus.

Comparative evaluation of various solid phases for the development of coated tube assays for the estimation of progesterone in human serum, bovine serum and bovine milk.

Immobilization of progesterone antibody using three polystyrene surfaces and two progesterone radiotracers for use in the development of a coated tube assay for the evaluation of progesterone levels in human serum, bovine serum and bovine milk was studied. The selection of the solid phase and the tracers were based on the maximum binding, non-specific binding, sensitivity and percentage recovery. Amongst the polystyrene tubes studied, streptavidin coated tubes showed the acceptable assay features such as low non-specific binding (0.5-1.0%), adequate sensitivity (0.13-0.16 ng/ml) and recovery (85-115%) for all the three sample matrices, human serum, bovine serum and bovine milk.

An evaluation of automated methods for measurement of serum 25-hydroxyvitamin D.

OBJECTIVES: To compare two new automated assays with the well-established reference method, DiaSorin radioimmunoassay (RIA), for quantitation of serum total 25-hydroxyvitamin D [25(OH)D]. METHODS: 25(OH)D from human sera (n=158) was measured using DiaSorin RIA and two automated platforms, DiaSorin "LIAISON 25 OH Vitamin D TOTAL", and Roche Modular "Vitamin D3 (25-OH)". Methods were compared by regression and Bland-Altman analyses. RESULTS: DiaSorin LIAISON demonstrated a stronger correlation (r=0.918) and better agreement (bias=-0.88 nmol/L) with DiaSorin RIA than the Roche Modular assay (r=0.871, bias=-2.55 nmol/L). Precision ranges (CV%) for the RIA, LIAISON, and Roche Modular assays, respectively, were: within run (6.8-12.9%, 2.8-8.1%, and 1.9-5.5%), and total precision (7.4-14.5%, 7.3-17.5%, and 7.6-14.5%). CONCLUSION: DiaSorin LIAISON displayed the best correlation and agreement with DiaSorin RIA. The DiaSorin LIAISON 25 OH Vitamin D TOTAL assay is an accurate and precise automated tool for serum total 25(OH)D determination.

Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: application in GPCR-mediated adenylate cyclase assays.

Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

Measurement of salivary cortisol--effects of replacing polyester with cotton and switching antibody.

Stable performance between-runs are essential in longitudinal studies and when different studies are being compared. However, changes in analytical kits and laboratory material occur and have the potential to threaten analytical stability. In the present case, we examined how salivary cortisol measurements in our laboratory were affected by: 1) changes in the tampon material and 2) changes in the antibody of the analytical kit. In study 1, saliva from healthy subjects (n = 19) was split and spiked to Salivette polyester and cotton tampons, respectively, and treated as ordinary samples before being analysed for cortisol using a Spectria RIA kit for cortisol. In study 2, 68 anonymous saliva samples were analysed with the Spectria Cortisol RIA kit both before and after the manufacturer changed the antibody. The change from polyester to cotton tampons reduced the measured concentration of salivary cortisol by 62 %. A difference of 12 % between the two runs with different antibodies could not be attributed to differences in storage or in thawing and freezing of samples. To conclude, both a change in the material of the Salivette used for collecting saliva samples as well as a change of antibody in a kit can have a major impact on measurements, as illustrated here for concentrations of cortisol in saliva. It is therefore recommended always to check that the analysis stays in statistical control in one's own laboratory when changes are made, even if the manufacturer reports that the changes should have no effects.

Automated, high-resolution cellular retention and uptake studies in vitro.

This report describes an automated method for the measurements of cellular retention and uptake of radiolabeled proteins interacting with cell-surface receptors on intact cancer cells. A complete uptake and retention measurement was performed in one cell dish using a rotating radioimmunoassay (RIA) principle. Compared to common manual measurements, rotating RIA saved both labor time and reagents and provided real-time binding traces with superior time-resolution. The rotating RIA retention profiles for different interactions agreed with retention times reported in the literature.

Development of isotopic and non-isotopic microwell based immunoassays for hCG using 125I and biotin labeled hCG.

Isotopic and non-isotopic immunoassays of hCG, based on the principle of competitive inhibition, using micro-well as solid support and 125I and biotin as labels for hCG, have been developed. In both the assays, rabbit polyclonal antibody was immobilized onto micro-wells. In the non-isotopic assay, to the hCG antibody coated micro-wells, 50 microL of standard or samples along with 100 microL of biotinylated hCG were incubated for 1 hour at 37 degrees C. After incubation, wells were washed and 100 microL of streptavidin-HRP conjugate was added to each well and incubated again for a half hour at 37 degrees C. Bound enzyme activity was measured using tertramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. In the isotopic assay, to the hCG antibody coated micro-wells, 50 microL of standard or samples along with 100 microL of 125I-hCG were incubated for 1 hour at 37 degrees C. The bound radioactivity was measured using a gamma counter. The sensitivities of the non-isotopic and isotopic assays were 0.12 IU/mL and 0.1 IU/mL, respectively. The intra- and inter-assay CVs for both the assays were less than 12.3%. There was a good correlation between the developed non-isotopic and isotopic immunoassays (r = 0.97, n = 20).

Development of coated tubes RIA for serum T3 (tri-iodothyronine) for production scale.

A coating procedure that could provide immobilization of antibodies, with increased binding capacity, that is cost effective, simple, robust, and appropriate for production scale application, is described. This coating approach of T3 antibodies to the polystyrene tubes has been systematically investigated to determine its utility for the development of coated tube Radioimmunoassay (RIA) for T3 in human serum. Further, the results obtained by the developed coating procedure are found to be comparable with those obtained by the "gold standard," the liquid phase RIA for T3. The coating procedure is completed in three major steps, each step involving an overnight incubation. The normal rabbit gamma-globulins are physically adsorbed onto the polystyrene tubes and incubated. After washing, a second antibody (goat anti-rabbit antiserum) is added and incubated. To this antigen specific antibody is added (T3 antibody produced in rabbit) and further incubated. Finally, the non-specific sites on the tubes are saturated by the blocking solution. The concentration of normal rabbit globulin, titers of second antibody and T3 antibody, and time required for coating are optimized to arrive at a suitable coating protocol. The coated tubes were evaluated for precision, reproducibility, and stability. Various parameters such as total reaction volume, incubation time and temperature, total number and volume of washings, concentration of 8-anilino-1-naphthalene sulfonic acid (ANS), and quantity of tracer per tube are optimized to arrive at a suitable standard curve. The optimized assay is validated for the quality control parameters such as intra- and inter-assay variations, recovery, and parallelism. The developed coated tubes assay had an assay range of 0.3-4.8 ng/mL with a sensitivity of 0.3 ng/mL at 90% B/B0. Batch to batch variation in coating was < 10%. The coated tubes were stable up to 1 year, which is adequate for production scale.

Comparative evaluation of D-dimer assays for exclusion of deep venous thrombosis in symptomatic outpatients.

Diagnostic work-up of patients sent to the hospital for diagnosing of deep venous thrombosis (DVT) often combines the determination of D-dimer and the application of a clinical probability score. To fulfill diagnostic as well as economic needs, D-dimer assays should exhibit a high negative predictive value (NPV) as well as reasonable specificity. In this study, we evaluated both a latex-enhanced immunoassay for use on routine coagulation analyzers and a radial partition immunoassay (RPIA) for use on a point-of-care analyzer. Samples included were from 344 outpatients with suspected deep venous thrombosis. Among them, 100 had deep venous thrombosis. Results obtained for both assays show a good efficiency for exclusion of deep venous thrombosis with well-acceptable specificity.

Evaluation of circulating calcitonin: analytical aspects.

BACKGROUND AND AIM: Calcitonin (hCT) is a useful serum marker for the diagnosis and monitoring of medullary thyroid carcinoma (MTC). However, hCT values provided by different methods may differ, leading to difficulties in the interpretation of hCT results. In this study we compared four immunoradiometric (IRMA) and radioimmunometric (RIA) assays for hCT determination. MATERIAL AND METHODS: hCT was measured in 35 patients by means of the following commercially available IRMA or RIA kits: CT US (Biosource), IRMA hCT (Schering-CIS bio international), ultra-sensitive calcitonin (DSL), and calcitonin assay (Scantibodies). A comparison of the distribution of the hCT values measured by the tested IRMA-RIAs and a correlation analysis were performed. RESULTS: The hCT values were widely dispersed and the classification of the patients according to the hCT cutoff value varied depending on the assay used. CONCLUSION: Despite efforts to develop new, highly specific antibodies, the evaluation of this marker is still flawed by analytical inaccuracy. hCT values are widely dispersed depending on the method used for marker measurement; as a consequence, patient classification according to the hCT cutoff value is still dependent on the assay used.

Mini-RIA: adaptation of conventional 125I-labeled radioimmunoassay to a 96-tube format.

Radioimmunoassay (RIA) employing iodinated ligands represents a popular measurement method for small molecules due to its excellent sensitivity and specificity. Yet performing RIAs of large numbers of tubes remains a tedious laboratory chore due to the need to individually handle tubes multiple times. We here present a method in which conventional 125I-labeled RIA ([125I] RIA) is adapted to a microtiter plate format, termed mini-RIA. Tubes are handled in batch for centrifugation or during the separation of antibody-bound ligand from free ligand. A simple draining device for batch decantation of free ligand from 96 minitubes is used. Optimal conditions for the mini-RIA were established using two workup methods-double-antibody immunoprecipitation and direct polyethylene glycol precipitation. Use of the mini-RIA method was found to result in a considerable savings in assay times; in addition, the sensitivity of the mini-RIA was improved over conventional RIA. The mini-RIA is particularly useful for assay of large numbers of samples derived from chromatographic methods, since aliquots can be transferred directly from the fraction collector into the minitubes using multiple channel pipettors. Because the method is flexible with regard to assay workup, we predict that most conventional [125I] RIAs can be adapted to the mini-RIA format.

Improved antibody coating protocol using a second antibody antiserum. Application to total thyroxin immunoassay.

A complete antibody coating protocol for the preparation of dry antibody coated tubes is presented. This protocol is based on a recently described antibody immobilization principle. We modify this immobilization principle in order to improve and simplify the coating procedure. In addition, we propose a drying procedure that provides long-term storage stability of the antibody coated tubes. According to the modified protocol, polystyrene plastic tubes are first coated with rabbit gamma-globulins. The tubes are incubated with a sheep anti-rabbitIgG antiserum dilution. After incubation, antigen-specific antibody antiserum raised in rabbits is added directly into the tubes containing the sheep anti-rabbit IgG antiserum solution (difference from the original protocol). Finally, the tubes are washed, blocked, and dried following the drying procedure developed. The suitability of the modified protocol for the development of immunoassays requiring high loading of antibody was exemplified through the development of a RIA for total thyroxin. The estimated assay characteristics (detection limit 4 microg/L, dynamic range up to 210 microg/L, within-run CV 2.7-5.7%, between-run CV 5.1-7.3%, recovery 84.4-112%, cross-reactivity for T3 1.9%) were comparable with those provided by commercially available RIA kits for the determination of thyroxin.

[Freely programmable automatic radioimmunoanalysis on the STRATEC ST 300 analyzer]. / Volne programovatelná automatická radioimunoanalýza na prístroji STRATEC SR 300.

We compared the free programmable automatic radioimmunoassay on analyser STRATEC SR 300 to the manual performance of RIA and IRMA. One-step and two-step methods were evaluated. Manual delivery proved to be faster than the automatic one. The speed of dispensing depends on the volume of the dose sample and of the reagent, on the adjustment of rinsing volumes of the needle, washing volumes among separate doses and also on the size of the sample set. The total efficiency of the manual and the automatic analysis is also affected by other operations, especially by washing test tubes and the administration of reports. For a set of 100 analyses, the manual process is from 20 to 90 minutes longer than the automatic one, depending on the analytical method used. Thus, the automatic analysis proved to be significantly faster. Nor the comparison of isotope and non-isotope immunoassays showed any relevant qualitative or quantitative differences in analytical response. At the same time the prices of radioimmunoassays are more favourable, and in addition to that, a big choice of analytical sets is available which are not dependent on the user's instrumentation. However, this does not concern very small series, or separate tests where size of calibration is important. Moreover, the turn-around-time of RIA and IRMA is in these cases longer than when automatic non-isotope methods are used.