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Current threshold values for primary aldosteronism (PA) diagnostic testing are based on measuring aldosterone (PAC) using immunoassays. Quantification of PAC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) yields lower values. To compare aldosterone measurement by radioimmunoassay (RIA) with LC-MS/MS and evaluate performances of proposed LC-MS/MS-specific cutoffs for PA screening and confirmatory testing. Forty-one patients underwent aldosterone/renin ratio (ARR) testing to screen for, and fludrocortisone suppression testing (FST) to confirm or exclude, PA. Renin (DRC) was measured by chemiluminescent immunoassay. Median serum PACLC-MS/MS was 27.8% lower (P < 0.05) than plasma PACRIA in 164 pairs of FST samples. A positive correlation (Spearman coefficient, 0.894, P < 0.01; Pearson r coefficient, 0.861, P < 0.01) was observed between the two assays. Thirty-seven patients showed consistent FST diagnoses (29 positive, 8 negative), whereas four showed inconsistent FSTs by the two assays. Good agreement (κ coefficient, 0.736; P < 0.01) was observed between the current FST diagnostic PACRIA cutoff of 165 pmol/L and the proposed PACLC-MS/MS cutoff of 133 pmol/L. Among 37 patients with consistent FST results, no differences were observed in sensitivity (89.7% vs 93.1%) or specificity (87.5% vs 87.5%) for PA screening between the current ARR cutoff of 70 pmol/mU (PACRIA/DRC) and the proposed cutoff of 55 pmol/mU (PACLC-MS/MS/DRC). Adjustment of the current cutoffs for PA diagnostic testing is necessary if PAC is measured by LC-MS/MS. Our preliminary results suggest that the proposed LC-MS/MS cutoffs for ARR and FST perform as well as current RIA cutoffs.

Background and Purpose Acetylcholine receptor antibody (AChR‐Ab) detection is crucial in myasthenia gravis (MG) diagnosis and, currently, the radioimmunoassay (RIA) is the gold standard. However, RIA may detect AChR‐Ab against nonpathogenic intracellular epitopes. In this study, we performed fixed cell‐based assay (F‐CBA) in RIA‐AChR‐Ab positive subjects without MG symptoms, to assess whether F‐CBA could show a higher specificity compared to RIA in detecting pathogenic Abs. Methods We reviewed medical records of patients referred to our MG outpatient clinic because of RIA‐AChR‐Ab detection. MG diagnosis was based on clinical examination, electrophysiology and Ab detection. AChR‐Abs were tested by RIA in the whole cohort. Serum samples from RIA‐positive asymptomatic subjects were retested by F‐CBA. Results Of 605 subjects who tested RIA‐AChR‐Ab positive, MG diagnosis was confirmed in 599. Six subjects were RIA‐AChR‐Ab positive although they had never had MG symptoms; in four of these subjects AChR‐Abs were not detected by F‐CBA, whereas the remaining two (both non‐MG thymoma cases) were positive also by F‐CBA. Conclusions RIA false positivity for AChR‐Ab is very rare. Previous literature has demonstrated that F‐CBA has higher sensitivity than RIA for MG, especially in ocular cases. Our preliminary results show that, in rare instances, F‐CBA may be more specific than RIA for MG diagnosis.

Abstract Context Serum thyroglobulin (Tg) measured by immunometric assay (IMA) is prone to underestimation due to Tg autoantibody (TgAb) interference, often prompting reflex Tg measurement by liquid chromatography/tandem mass spectrometry (MS) or radioimmunoassay (RIA). Objective IMA, MS, and RIA methodologies were used to measure serum Tg in TgAb-negative (TgAb−) and TgAb-positive (TgAb+) patients with either distant metastatic differentiated thyroid cancer (DTC) or hyperthyroidism (HY)—patients in whom a detectable serum Tg would be expected. Results When TgAb was absent, all methodologies detected Tg in the sera of all DTC and HY patients and reported appropriate Tg trends and treatment responses for DTC patients with progressive distant metastatic disease, albeit with high between-method variability (> 30% coefficient of variability). When TgAb was present, all methodologies reported lower serum Tg levels for both DTC and HY groups vs their respective TgAb− group. No Tg was detected by IMA or MS in ∼50% TgAb+ DTC patients (6% had no Tg detected by RIA). Surprisingly, 5% of TgAb+ HY patients also had no Tg detected by IMA or MS. The inverse log TgAb/log Tg correlations seen for the TgAb+ HY patient group with all methods suggested the presence of a TgAb-associated serum Tg-lowering effect. Conclusion (i) Between-method Tg variability necessitates method continuity when monitoring the Tg trends of TgAb− DTC patients. (ii) The presence and concentration of TgAb appeared to have a lowering effect on serum Tg measured by all methodologies (IMA, MS, and RIA). (iii) Since the reliability of Tg measured in the presence of TgAb is often uncertain, the TgAb trend (measured by the same method) may be a useful surrogate DTC tumor marker.

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen–antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [ 1 ], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

Abstract Background Many diagnostic tests for insulin dysregulation use reference intervals established with an insulin radioimmunoassay (RIA) that is no longer available. A chemiluminescent immunoassay (CLIA) is commonly used for the measurement of serum insulin concentration in clinical practice but requires further validation, especially at clinically relevant reference intervals. Objectives To evaluate the CLIA for measurement of equine insulin and compare it to the previously validated, but now unavailable RIA. Samples Equine serum samples (n = 78) from clinical and experimental studies. Methods In this experimental study, performance of the CLIA was evaluated using standard variables, including comparison with the RIA. Continuous and binary outcomes were analyzed. Results The CLIA showed good intra-assay (coefficient of variation [CV], 1.8–2.4%) and interassay (CV, 3–7.1%) precision. Acceptable recovery on dilution (100 ± 10%) was achieved only at dilutions <1:1. Recovery on addition was acceptable. Comparison of the CLIA and RIA showed strong positive correlation (r = 0.91–0.98), with fixed and proportional bias. At 3 diagnostic cutoffs, sensitivity of CLIA compared with RIA ranged from 67 to 100% and specificity from 96 to 100%. Conclusions and Clinical Importance The CLIA is a highly repeatable assay which is suitable for within- and between-horse comparisons. Dilution of high concentration samples should be performed with charcoal-stripped serum (CSS) and at the lowest dilution factor possible. At concentrations commonly used for diagnosis of insulin dysregulation (≤100 μIU/mL), results from the CLIA tend to be lower than from the RIA and should be interpreted accordingly. Further standardization of equine insulin assays is required.

The transition from radioimmunoassay (RIA) to chemiluminescent enzyme immunoassay (CLEIA) for plasma aldosterone concentration (PAC) assays has raised concerns over its impact on primary aldosteronism (PA) diagnosis. This study investigated the correlation between PAC and renin values using RIA, CLEIA, and liquid chromatography/mass spectrometry/mass spectrometry (LC–MS/MS), established cutoff values for PA diagnosis using the aldosterone-to-renin ratio (ARR) with PAC_CLEIA, and assessed the differences in PAC values by measuring weak mineralocorticoids (WMs). This retrospective study evaluated 312 serum PAC samples using RIA, CLEIA, and LC–MS/MS, and analyzed 315 plasma renin samples. Method correlations were assessed through Passing-Bablok regression. Receiver operating characteristic curves determined ARR cutoffs for PA diagnosis. WMs were quantified to evaluate their impact on ΔPAC (RIA-LC–MS/MS) through multiple regression analysis. PAC_CLEIA and PAC_LC-MS/MS values were highly correlated. ARRs derived from PAC_RIAs demonstrated more false positives and lower specificity than ARRs using PAC_CLEIA or PAC_LC-MS/MS. WMs significantly influenced ΔPAC in both the PA and non-PA groups. ARRs using PAC_CLEIA are valuable for determining PA cutoffs in clinical practice. The transition to PAC using CLEIA may enhance PA detection rates. WMs were found to interfere with PAC measurements in the RIA method, affecting outcomes.

Background Exact analysis of equine insulin in blood samples is the key element for assessing insulin resistance or insulin dysregulation in horses. However, previous studies indicated marked differences in insulin concentrations obtained from sample analyses with different immunoassays. Most assays used in veterinary medicine are originally designed for use in human diagnostics and are based on antibodies directed against human insulin, although amino acid sequences between equine and human insulin differ. Species-specific assays are being used more frequently and seem to provide advantages compared to human-specific assays. The aim of this study was to compare three immunoassays, one porcine-specific insulin enzyme-linked immunosorbent assay (ELISA), advertised to be specific for equine insulin, one porcine-specific insulin radioimmunoassay (RIA) and one human-specific insulin chemiluminescence immunoassay (CLIA), all three widely used in veterinary laboratories for the analysis of equine insulin. Furthermore, we tested their clinical applicability in assessing insulin resistance and dysregulation by analysis of basal blood and blood samples obtained during a dynamic diagnostic stimulation test (OGT) with elevated insulin concentrations. Results Insulin values obtained from the ELISA, RIA and CLIA, investigated for analyses of basal blood samples differed significantly between all three assays. Analyses of samples obtained during dynamic diagnostic stimulation testing with consecutively higher insulin concentrations revealed significantly ( p  < 0.001) lower insulin concentrations supplied by the CLIA compared to the ELISA. However, values measured by ELISA were intermediate and not different to those measured by RIA. Calculated recovery upon dilution, as a marker for assay accuracy in diluted samples, was 98 ± 4 % for ELISA, 160 ± 41 % for RIA and 101 ± 11 % for CLIA. Conclusions Our results indicate that insulin concentrations of one sample measured by different methods vary greatly and should be interpreted carefully. Consideration of the immunoassay method and reliable assay-specific reference ranges are of particular importance especially in clinical cases where small changes in insulin levels can cause false classification in terms of insulin sensitivity of horses and ponies.

There has been an unprecedented interest in the modulatory effects of intranasal oxytocin on human social cognition and behaviour, however as yet no study has actually demonstrated that this modality of administration increases concentrations of the peptide in the brain as well as blood in humans. Here using combined blood and cerebrospinal fluid (CSF) sampling in subjects receiving either 24 IU of oxytocin (n = 11) or placebo (n = 4) we have shown that oxytocin levels significantly increased in both plasma and CSF. However, whereas oxytocin plasma concentrations peaked at 15 min after intranasal administration and decreased after 75 min, CSF concentrations took up to 75 min to reach a significant level. Moreover, there was no correlation (r = <0.10) between oxytocin plasma and CSF concentrations. Together, these data provide crucial insights into the plasma and CSF kinetics of intranasally administered oxytocin.

Background We developed a fully automated quantitative immunoassay for the detection of prostaglandin E‐major urinary metabolite (PGE‐MUM). In this study, we evaluated the analytical performance of this assay. Methods Sensitivity, within‐run reproducibility, correlation with radioimmunoassay (RIA), cross‐reactivity, dilution linearity, spike recovery performance, analyte stability, and effects of coexisting substances were evaluated. The assay was also used to measure PGE‐MUM in 211 healthy people. Results The limit of detection and quantification were 1.0 and 1.3 ng/mL, respectively. When the assay was performed six times in a single run, the coefficient of variation ranged from 1.4% to 2.2%. The coefficient of correlation with a preceding RIA method was 0.970 with a correlation slope of 0.88. There was no cross‐reactivity with PGE‐MUM analogs. Linearity of dilution was confirmed at up to 16‐fold dilution with assay results within 100 ± 20% of the theoretical values calculated based on the undiluted sample. Spike recovery was good and ranged from 94% to 101%. Analyte stability was tested by storing samples at 25°C for 6 days, 10°C for 1 month, and by performing up to five freeze–thaw cycles. Assay results were all within 100 ± 10%, the values measured before storage and before the freeze–thaw process. Assay results in healthy people ranged from 3.1 to 162.7 ng/mL (mean: 35.8 ng/mL). After correction for creatinine, the 95% confidence interval was 8.68–42.25 μg/g creatinine. Conclusion The assay precisely detects PGE‐MUM. We developed a chemiluminescent enzyme immunoassay (CLEIA), which can detect prostaglandin E‐major urinary metabolite (PGE‐MUM) with automated procedure in ~30 min. Here, we evaluated its analytical performance. This assay can precisely detect PGE‐MUM with good reproducibility and showed a good correlation with preceding radioimmunoassay (RIA).