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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., Competing Interests: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan (http://ffwk.fujifilm.co.jp) provided the authors an Accuraseed® automated chemiluminescent enzyme immunoanalyzer, and Accuraseed® Aldosterone and Accuraseed® Renin kits to perform this study. The company did not provide the authors grants, royalties or licenses, consulting fees, payment or honoraria, support for attending meeting and/or travel, or stock or stock options. There are no patents planned, issued, or pending in relation to this study. The company had no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Sharing the data of the present study is restricted by the Shizuoka General Hospital Research Ethical Committee following the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan as described in the Data Availability statement. The de-identified dataset of this study is available on request to Shizuoka General Hospital Research Ethical Committee, Email: chiken-sougou@shizuoka-pho.jp. All requests must include an analysis plan and the plan must be approved by this ethics committee. Except as described above, none of the authors have any competing interests associated with this research.

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.

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., (Copyright © 2011 Wiley-Liss, Inc.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

A tip washing system has been developed for use with disposable polypropylene micropipette tips of different sizes. The primary hardware components of the system are constructed from polyvinyl chloride (PVC) and Plexiglas. The system relies on a low concentration of detergent, gravity or aspirator-assisted feed and flush of minimal volumes of distilled/deionized water, and air or oven drying of the tips. Comparative analyses of new versus washed tips in FAO/IAEA radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) protocols indicated that disposable polypropylene micropipette tips of various sizes (e.g. 5-300 and 50-1000 microl) can be washed and reused at least ten times without introducing detectable variability into the results of either assay, if the tips are properly washed and dried using this system. Recovery cost of the system can be achieved after approximately eight reuses of 1000 tips, excluding technician time.

The main developments in the "ligand assay" field in which I have been involved are traced. These include the original development of "first generation" competitive assays relying on radiolabeled analyte markers; the development of the first "second generation", noncompetitive (ultrasensitive) methods, which rely on the use of labeled (monoclonal) antibodies and high specific activity nonisotopic labels (leading to the transformation of the immunodiagnostic field in the 1980s); and the development of the first "third generation" miniaturized, chip-based, microarray methods, which permit the simultaneous ultrasensitive measurement of many analytes in the same small sample. The latter--applicable both to immunoassay and to DNA/RNA analysis--are likely to revolutionize the diagnostic and pharmaceutical fields in the next decade.

The aim of this study was to analyze the prevalence of every RAST (Radio-Allergo-Sorbent-Test) class in terms of total IgE serum level, and to establish the existing relationship between the total IgE level and the probability to obtain a definite specific IgE concentration. The analyzed sample was 352 total IgE values obtained by means of RIA in 611 patients of both sexes and ages from 5 to 15 years [corrected], and the RAST results of every one of them. As mathematical model, Logit was used. It was established a mathematical function [p = 1/(1 + K(-1).IgE-beta)], which allows to calculate the probability to obtain a definite RAST class of allergy for a definite total IgE level. The propounded model establishes that, for a plasmatic IgE level lower or equal to 17.0 kU/l (11.5-23.3), the probability to obtain a specific IgE concentration lower than 0.35 kU/l (absence of allergy) is 0.95. For a total IgE level of 23.32 kU/l (16.43-30.91) there is a probability of 0.95 that RAST result to be negative or slightly positive. Finally, for a total IgE level of 62.6 kU/l (49.20-76.55) specific IgE levels of classes 0 or 1 or 2 would be obtained. The mathematical model allows to predict the most probably RAST class of allergy result once known the total IgE level. It makes easier the allergological diagnostic and facilitates, in some cases, to omit the RAST, with the resulting saving of time and money.

We evaluated a novel assay for the measurement of 1,25 dihydroxyvitamin D (1,25 (OH)2D). Immunoextraction of 1,25(OH)2D is performed using a mini column containing a solid-phase monoclonal antibody followed by radioimmunoassay (RIA) using an 125I-labelled 1,25(OH)2D derivative tracer and Sac-cell separation. The mean recovery of 1,25(OH)2D3 was 101%, linearity was excellent, inter- and intra-assay coefficients of variation were 9, 8 and 13% and 11, 10 and 14% at low, medium and high concentrations of 1,25 (OH)2D3, respectively. The cross-reactivity of vitamin D metabolites was < 0.0015% for 25-hydroxyvitamin D3, 24, 25 dihydroxyvitamin D3 and dihydrotachysterol and 0.54% for 1 alpha calcidol. 1,25 dihydroxyvitamin D2 cross-reactivity was 79%. The detection limit of the assay was 5 pmol/L. Comparison with a commercial radio receptor assay (RRA) and an in-house RIA gave regression equations of y = 0.94x + 11.8 (r = 0.98) and y = 0.91x-1.7 (r = 0.95), respectively, with no major discrepancies between the methods in all patient groups studied. Plasma concentrations of 1,25(OH)2D obtained with the assay were as follows: normal, unsupplemented subjects: mean 88, range 48-155 pmol/L, n = 68, patients with chronic renal failure: mean 11, range 3-36 pmol/L, n = 27, primary hyperparathyroidism: mean 198, range 130-299 pmol/L, n = 23, Paget's disease: mean 92, range 42-149 pmol/L, n = 24, osteomalacia: mean 43, range 27-61 pmol/L, n = 9. A minimum sample volume of 300 microL is required, the hands-on time is significantly less than other commercial assays and the measuring procedure is gamma counting rather than scintillation counting. The assay offers several advantages over previous methods and should allow more laboratories to offer measurement of 1,25(OH)2D as part of their repertoire.

Objective: Test performance of two automated progesterone assays available on the immunoassay analysers Abbott AxSYM and Technicon immuno 1, respectively, was evaluated in comparison with the radioimmunoassay Progesterone MAIA., Methods: For assessment of test performance imprecision, functional sensitivity and linearity of dilution was examined. Correlation with the manual radioimmunoassay was assessed using 122 serum samples over the range 0-110 nmol/L., Results: Imprecision studies revealed for the AxSYM Progesterone within-run CV's of 1.8-6.4% and day-to-day CV's of 3.5-9.7% (concentration range 2.3-75 nmol/L); Immuno 1 Progesterone: within-run CV's 1.0-7.3%, day-to-day CV's 2.3-7.7% (concentration range 1.2-60 nmol/L). The functional sensitivity was < 1.7 nmol/L for the AxSYM Progesterone and < 1.1 nmol/L for the Immuno 1 Progesterone. With the AxSYM Progesterone the mean recovery after dilution from five samples was 102% (89-107%), from one sample only 69-80% was recovered; with the Immuno 1 Progesterone the mean recovery was 95% (80-105%). Despite of a quite good overall correlation (coefficients 0.972 and 0.981) the relationship of both assays to the Progesterone MAIA significantly deviate from linearity with a considerably higher slope within the lower concentration range. The relationship between the automated assays was linear over the entire concentration range (Immuno = 1.207 * AxSYM + 1; r = 0.986). The time to first result was 20 min for the AxSYM Progesterone, 45 min for the Immuno 1 Progesterone and 90 min for the Progesterone MAIA., Conclusion: The evaluated progesterone assays both exhibit an excellent precision and a high degree of sensitivity. They offer a rapid and flexible method for progesterone determination which may be especially useful for the monitoring of ovarian stimulation during in-vitro fertilization.

An original solid phase method for direct radioimmunoassay of the antipsychotic savoxepine (CGP 19,486 A) in plasma has been developed which does not require the extraction of the parent drug with organic solvents. The assay showed good reproducibility over the working concentration range 1.9-30.6 nmol/l with intra- and inter-assay coefficients of variation < or = 16%. The procedure, which requires only small volumes of plasma (10 microliters), is simple to handle and well suited for routine analysis. The method allowed to investigate the pharmacokinetics of savoxepine in schizophrenic patients given low oral doses of the drug.

The automation and validation of the HPLC-radioimmunoassay (RIA) method for the determination of lacidipine are reported. The solid-phase extraction step was automated by the introduction of the ASPEC system. A two-column system was adopted for the HPLC purification. The RIA was converted from heterogeneous to homogeneous by the scintillation proximity assay system and automated using an automatic dilution system. All characteristics in terms of accuracy, precision, specificity, and linearity resulted similar to the manual version. The quantification limit was set to 40 pg/ml. The new version of the method increased the number of samples assayed per month two- to three-fold.

The immunosorbent obtained through a covalent immobilization of antibodies on a polyacrolein latex was studied in comparison with the sorbents based on microcrystalline cellulose and with the surface of polystyrene test tubes in a two-center radioimmunoassay for ferritin. It was shown that the polyacrolein latex provides a covalent binding of up to 75 mg protein per g dry polymer with an immobilization efficiency of 50-70% and a high effective association constant of immobilized antibodies (Ka = 4.5 x 10(9) M-1). The same parameters for the sorbent based on microcrystalline cellulose were 33 mg/g, 25-35%, and 4.3 x 10(8) M-1, respectively. In the radioimmunoassay for ferritin, the immunosorbents based on polyacrolein latex and cellulose ensured a nearly the same analytical sensitivity at the level of (0.7-0.9) x 10(-13) M ferritin and pointed to the possibility of eliminating the "hook-effect". Compared to the immunosorbent based on polystyrene test tubes, the advantages of the polyacrolein latex are the decrease in the minimum detectable concentration (increase in analytical sensitivity), the extension of the dynamical range of analysis, and the absence of the "hook-effect".

Objective: To automate the radioimmunoassay (RIA) of steroids in a diagnostic clinical laboratory., Setting: The Laboratory of Pediatric Endocrinology, Cornell Medical Center-New York Hospital., Practice Description: A tertiary-care center supporting the diagnosis and treatment of pediatric patients with endocrine disorders. PRODUCT COMPARISON: The Packard MultiPROBE Model 100 Automated Liquid Handling System (Packard Instrument Company, Meriden, CT 06450) was compared with the assays typically used to assist in the diagnosis of steroidogenic disorders in children: extraction of biologic fluids, celite partition chromatography, and RIA., Main Outcome Measurement: The comparative efficiency, cost-effectiveness, specificity, and sensitivity of the two methods., Result: The automated RIAs correlated (r = 0.963) with the established manual assays for steroids. The specificity and sensitivity of the assays were uniformly maintained after automation., Conclusion: The automation of steroid RIAs has made the method a highly efficient and cost-effective one for diagnostic clinical laboratories.

Previous assays for cytidine 3', 5'-cyclic monophosphate (cyclic CMP) have been criticized as being ambiguous. Here a modified RIA protocol, in which the production of assay components has been optimized and a novel trilayer chromatography column separation introduced which successfully separates cyclic CMP from compounds, endogenous to living tissues, which cross-react with anti-cyclic CMP sera, is described. The assay is capable of assaying cyclic CMP between 0.1 and 5 pmol, can be increased in sensitivity by means of an additional acetylation step, and enables the separation of cyclic CMP, cyclic AMP and cyclic GMP so that all three can be estimated in a single sample.

A variant of the solid-phase radioimmunoassay (RIA) has been developed for the detection of specific immunoglobulins in laboratory animals infected with Pseudomonas pseudomallei. The proposed method has advantages over the indirect hemagglutination test and the enzyme immunoassay in its sensitivity and specificity. The newly developed RIA variant, based on group-specific antigens 6 + d, makes it possible to classify the strain causing the disease with the Asian or Australian serovar of P. pseudomallei according to the composition of detected immunoglobulins.

Serum myoglobin (Mb) is an important biological marker in the early diagnosis of the acute myocardial infarction (AMI). In a previous paper we showed that RPHA, which consists in the agglutination of anti-Mb antibody--coated erythrocytes by solutions containing Mb, is a simple, rapid and highly sensitive assay (approx. 1 ng/ml). 42 sera from AMI patients and 20 sera from healthy subjects were investigated by RPHA. The geometric mean (x/divided by SD) of serum Mb titers in AMI patients was 362 x/divided by 3.94, in the range of 64-8192 (the reciprocal dilutions) and in normal sera was 19 x/divided by 1.36, in the range of 16-32. The difference between the mean values was statistically significant at p < 0.001. Subsequently, serum Mb concentration was determined by RPHA and RIA in 51 AMI sera prelevated at various stages of the disease. A high degree of correlation was found between the two methods. Log Y (RPHA titers) = -0.9174 + 1.5879 log X (RIA); ESE: +/- 0.1947; r = 0.9601. In four AMI patients successive determinations were made at every 6 hours: serum Mb curves determined by RPHA and RIA had a parallel evolution and this was an additional argument for the validation of RPHA. Being quite simple and easy of execution RPHA is the most suitable method by which an early AMI diagnosis can be made.

Increased concentrations of serum alpha-fetoprotein (AFP) occur in several physiological (pregnancy) and pathological (liver regeneration, tumour formation etc.) conditions. Measurement of serum AFP levels may assist in the detection of fetal abnormalities during pregnancy, in the detection of renewed tumour growth and metastases in cases of AFP producing tumours and in monitoring some diseases of the liver. In this paper some results are given concerning the follow-up of some patients with tumours of the testis and the possibilities of determining the source of elevated serum AFP (regenerating hepatocytes, malignant liver cells, hepatic metastases from other tissues) in patients with liver diseases.

With the aim of improving the sensitivity and simplifying the analytical procedure, changes have been made in the test for determining alpha-fetoprotein (AFP) by radioimmunoassay, using a locally developed kit first produced about ten years ago. Close correlation between values obtained for serum AFP using the old and new procedures was obtained (r = 0.977), but the mean value for healthy nonpregnant adults was found to be 2.5 micrograms AFP/l with the new test. This is about half the level obtained earlier and confirms the recent findings of other authors. Besides reducing the time and equipment necessary for performing the test, the modified procedure enables more precise distinction between normal and elevated concentrations of AFP in patient serum.

By taking advantage of a newly available microplate counter for radioactivity and the organic solvent-resistant, pigmented microplates, we have successfully established radioimmunoassays (RIA) for both CA-GI and CA-BR on microplate for routine clinical use. In the process of assay development, we found that both pigmented PicoPlate, made of acrylonitrile, and polystyrene Microlite 2 can be coated with antialpha fetoprotein (AFP) and antinerve growth factor (NGF) and used for setting up immunoassays for AFP and nerve growth factors. There were no problems following a test format of either competitive binding or sandwich design. Microlites 2 is recommended over PicoPlate because Microlites 2 is made of polystyrene, which is less expensive and separable into 8-well strips or even single wells. Single-well separation allows for the use of regular gamma counters in case Topcount is unavailable. We also found that the sensitivity of these tests was not significantly affected even though Topcount counts the weaker beta emissions. Similar dose-response curves could also be generated between original Biomira tube assays and assays using PicoPlate or Microlite 2 coated with protein antigens CA-Br and CA-GI. Excellent correlations were also obtained between the microplate assays and the Biomira tube assays for CA-GI and CA-Br using groups of serum specimens from cancer patients. We recommend the development of various RIAs on the microplate: it requires less reagents and less sample handling by the technologists and it can be essentially automated.

To assess the role of sulfidopeptide leukotrienes in the pathogenesis of exercise-induced asthma (EIA), the urinary levels of leukotriene E4 (LTE4), a metabolite of LTC4 and LTD4, were measured by RIA before and after exercise in 13 children with EIA and 10 healthy children. Mass spectrometry was used to confirm the presence of LTE4 in urine and the specificity of the RIA. There was no significant difference in the urinary LTE4 levels before exercise between the children with asthma and healthy children (109 [21 to 265] versus 122 [45 to 156] pg/mg of creatinine; median and range). Urinary LTE4 levels increased significantly after exercise in the children with EIA (from 109 [21 to 265] to 196 [40 to 655] pg/mg of creatinine; median and range; p less than 0.05) but not in the healthy children. The children with asthma demonstrated no significant correlation between the LTE4 level after exercise and the degree of bronchoconstriction, as revealed by the maximal percent fall in the peak expiratory flow rate. Taken together with a recent study that pretreatment with a potent and selective LTD4 antagonist markedly attenuated EIA, our findings suggest that sulfidopeptide leukotrienes may play some role in the pathogenesis of this type of asthma with other factors also being involved in determining the overall airway response.

A radioimmune assay (RIA) for IgG anti-granulocyte antibodies (AGA) was developed which utilized 96 well microfilter plates, required less than 50 microliters plasma, and yielded reproducible autologous determinations on patients with absolute granulocyte counts as low as 300 cells/microliter. Granulocytes were isolated by a single density ficoll-hypaque gradient procedure with red blood cell (RBC) removal by lysis and washing. Anti-granulocyte IgG coating the cells was detected by the addition of 125I labeled anti-human IgG. Six patients with autoimmune granulocytopenia and three multiply transfused patients with febrile transfusion reactions believed to be secondary to allogeneic granulocyte antibodies had increased granulocyte IgG. Plasma containing anti-a and/or anti-B titers greater than or equal to 512 gave positive results when tested against ABO incompatible granulocytes. This technique was capable of detecting antibodies which were reactive in granulocyte agglutination and immunofluorescence tests as demonstrated with known anti-NA-1, anti-NB-1, and anti-Mart sera. Comparison with granulocyte agglutination test (GAT) using clinical criteria for immune granulocytopenia demonstrated concordance with 19 negative samples and 8 positive samples, 1 false negative and 3 false positives were observed using the agglutination method. The multimeric and monomeric IgG fractions of G200 purified Cohn Fraction II were nonreactive by both RIA and GAT methods. However, granulocyte activation by phorbol myristate acetate (PMA) produced false positives in 5 out of 5 cases by GAT but 0 out of 4 by RIA. This RIA assay is not effected by nonimmune granulocyte activation and is more reproducible when compared to existing techniques.

A sensitive, specific and accurate homologous radioimmunoassay (RIA) for ovine follicle stimulating hormone (oFSH) has been developed, using a [125I]oFSH tracer and a polyclonal rabbit anti-OFSH-serum at a final dilution of 1:224,000. The separation of free and antibody-bound tracer is based on the double antibody solid phase system. The assay was found to be specific for oFSH; cross-reactivity with oLH, oPrl and oGH was lower than 0.2%. The detection limit was 7 pg per tube. Inter- and intra- assay coefficients of variation (CV) were 3.7-8.5% and 6.6%, respectively. The use of a few selfmade appliances in combination with a well-considered time-schedule enabled the processing of three thousand blood plasma samples in triplicate within two weeks. The particular advantage of the method described here is the fast, easy and safe separation of free and antibody-bound tracer with minimal handling of radioactive tubes.

Measurements of human growth hormone (hGH) plasma levels using mono or polyclonal antibodies based on kits are often highly variable depending upon the kit used. We compared six kits commercially available: one based on polyclonal antiserum, five based on monoclonal antisera. We measured 695 sera obtained in short stature children (GH deficiency or normal GH secretory) and adults (normal, hypopituitarism or acromegaly). Our results confirm the variability between the assays. The maximal variation was obtained with SBhGH kit which showed mean results 1.7 fold higher than those obtained with hGH Coatria. Using the OMS 80.205 standard with the six kits, we found a good correlation between the six kits indicating that the standard used in each case was not responsible for the observed variations. This suggest that the different antibodies used in these kits may recognize different isoforms of circulating hGH. In addition variations in reagents used in the different kits may also explain these discrepancies. The variations observed using these six commercially available kits may have important clinical repercussion and especially may impede the diagnosis in hGH deficiency in children.

The radioimmunologic assays appeared to be the most convenient methods for determination of atrial natriuretic peptide (ANP) concentration in blood plasma. The purpose of this work was to compare the control quality parameters of two radioimmunologic methods for the ANP determination in human plasma: the methods described by Pruszczyński et al. and the method with the use of Amersham ready-made set (kit). The comparison of the two methods was performed by analyzing 10 series of ANP determinations carried out by Pruszczyński method, and 15 series by means of the kit. Both methods appeared to fulfil the criteria of satisfactory quality for RIA determinations. The second purpose of this study was to define the utility of the RIA method for the determination of ANP in clinical practice, taking into the consideration some factors influencing or modifying plasma ANP levels. Three groups of persons of were studied: 15 healthy subjects, 16 patients with acromegaly and 47 patients with the impairment of renal function. The ANP level in plasma was determined in these 3 groups of persons. It has been demonstrated that the change of body position from upright to supine caused the increase of the ANP concentration in plasma. It has also been found that the decrease in the intravascular fluid volume induced by furosemide administration resulted in the reduction of ANP concentration in plasma. The enlarged intravascular fluid volume, accompanying acromegaly and probably present in persons with the impairment of kidneys, was associated with the increased ANP concentrations in plasma.

A composite filter pad formed by embedding activated carbon powder in fibrous matrix was applied for removal of hormones from human serum. Enhanced adsorption was demonstrated through direct comparison between the stacked filter pads and the powered carbon packed in a column both having an equal weight of carbon. In addition, the composite structure offers the advantage of eliminating the carbon fines mixed in the filtrant, avoiding the flow channeling, and providing favorable rate of adsorption. The adsorption of both thyroid hormones T3 (triiodothyronine) and T4 (thyroxine), and steroid hormone, cortisol, by the carbon filter were studied. The effect of pH on hormone adsorption by activated carbon was verified as an important environmental factor, minimum adsorption occurs at the neutral pH. It was found that T4 carrying one more iodine atom than T3 is less effectively adsorped by carbon adsorption from the serum under competitive condition due most likely to their size differences. The filter pad containing 70% activated carbon as adsorbent was fabricated in disk form and stacked in a column for processing bulk volume of biological fluid. Such a device provides an effective means for removal of hormones from biological solutions.

Radio-immuno-assay estimations of urinary hCG were carried out with pharmacia Kit beta hCG Riact on 36 women who had received embryo transfers. The analysis was carried in order to try and find how and at what moment in time it is possible to diagnose very early a developing pregnancy after embryo transfer. In this work we have found that the mean level of urinary hCG was significantly higher (P less than 0.001) in women who became pregnant (group B) than in those who did not become pregnant (group A). The difference in mean hormone levels in these two groups became more marked in the following days. The time doubling of the hCG level varied between 1.21 and 3.24 days in group B. By the eighth day the level of urinary hCG was less in group B than in group A, probably due to rapid renal breakdown of hCG. In summary these results show that the level of urinary hCG estimated from the 11th day onwards after an embryo transfer repeated two or three days later makes it possible to detect early pregnancies.

Three immunoradiometric assay (IRMA) kits (Pharmacia, bioMérieux and Cis-ELISA) and one competitive radioimmunoassay (RIA) kit (Cis-SB) designed for routine hGH quantitation were compared. Reproducibility was better with the IRMAs than with the RIA, especially when hGH levels were low (less than 3 mUI/l). This substantial advantage was responsible for a decrease in the qualitative detection threshold from 1.22 mUI/l for the RIA to approximately 0.08 mUI/l for the IRMAs. Analysis of accuracy showed that the Cis-ELSA kit overestimated recovery of added hGH (1st IRP 66/217) and demonstrated a marked influence of matrix effects with Cis-ELSA and Cis-SB. Pharmacia and bioMérieux kits were more accurate and showed less sensitivity to matrix effects. hGH concentrations obtained with the four kits were determined in 113 normal or abnormal sera. Despite the above-mentioned differences in accuracy, the three IRMAs yielded comparable results. Concentrations measured using the RIA were higher than those obtained with the other kits. Two sera were submitted to gel filtration chromatography. hGH assays in the fractions obtained showed that the immunologic systems used in the kits display different levels of immunoreactivity towards the circulating oligomeric and dimeric forms of hGH that they recognize. These data suggest that normal reference values should be established for each kit.