Comparative analysis of aldosterone and renin assays for primary aldosteronism screening.

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.

METHODS: Validating an immunoassay to measure fecal glucocorticoid metabolites in yellow-bellied marmots.

Quantifying physiological stress in wild animals is essential for understanding their health, reproductive success, and survival in a variable environment. The yellow-bellied marmot (Marmota flaviventer) study at the Rocky Mountain Biological Laboratory near Crested Butte, Colorado, USA is the world's second longest study of free-living mammals. Historically, we used a validated corticosterone radioimmunoassay (RIA) to measure fecal glucocorticoid metabolites (FGMs) as a proxy for physiological stress. However, the costs and risks associated with working with radioisotopes drove us to consider a more sustainable method. Here we evaluate the suitability of two competitive corticosterone enzyme assays (EIA), one from Cayman Chemical Company (CCC) and one from Arbor Assays (AA), to measure marmot FGMs via their cross-reaction. The findings revealed that the AA EIA better matched the RIA in terms of accuracy across high and low FGM concentrations, had superior assay parameters, showed the highest correlations with RIA results and effectively captured the annual variations in FGM concentrations, thus demonstrating its reliability for use in longitudinal studies. We further analytically validated the AA EIA for FGMs and confirmed its efficacy and lack of matrix effects, thus establishing its suitability for ongoing and future studies of FGMs in marmots. The transition to the AA EIA from the RIA ensures continued data integrity while enhancing safety and environmental sustainability.

Evaluation of a Chemiluminescent Enzyme Immunoassay for the Detection of Prostaglandin E-Major Urinary Metabolite (PGE-MUM).

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.

The value of using ELISA to detect orexin-A in cerebrospinal fluid in the diagnosis of narcolepsy.

Orexin in cerebrospinal fluid (CSF) is a neuropeptide synthesized by a cluster of neurons in the lateral hypothalamus. It mainly functions to maintain arousal, regulate feeding, and participate in reward mechanisms. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) can detect CSF orexin. At present, RIA is widely used but is limited by various conditions, which is not conducive to its widespread development. We aimed to determine whether ELISA can replace RIA in detecting orexin in CSF. We investigated the results of 20 patients with central disorders of hypersomnolence, including 11 with narcolepsy type 1, 2 with narcolepsy type 2, 5 with idiopathic hypersomnia, and 2 with other causes of somnolence. RIA and ELISA were used to detect CSF orexin, and P values <.05 were considered to be significant. In the narcolepsy and non-narcolepsy type 1 groups, there was no correlation between the RIA and ELISA results (P > .05). In the narcolepsy type 1 group, the ELISA and RIA results were significantly different (P < .05), but this was not observed in the non-narcolepsy type 1 group (P > .05). The accuracy of ELISA to detect CSF orexin was lower than that of RIA (P < .05). ELISA cannot replace RIA in the measurement of CSF orexin, and RIA is recommended as the first choice when narcolepsy is suspected.

Impact of the transition from radioimmunoassay (RIA) to chemiluminescent enzyme immunoassay (CLEIA) for the measurement of plasma aldosterone concentration (PAC) on the diagnosis of primary aldosteronism (PA) via retrospective analyses in Okinawa, Japan.

In Japan, the traditional method for measuring plasma aldosterone concentration (PAC) was radioimmunoassay (RIA), which had several challenges, including poor traceability of certified reference materials and reduced detection sensitivity at low concentrations. To overcome these issues, a chemiluminescent enzyme immunoassay (CLEIA) for PAC measurement was introduced in April 2021 and the Japan Endocrine Society published new guidelines for primary aldosteronism (PA). This study aimed to evaluate the impact of the transition from RIA to CLEIA for PAC measurement on PA diagnosis. Data from 190 patients admitted to the Second Department of Internal Medicine, University of the Ryukyus Hospital, between April 2012 and March 2021 were analyzed. Patients who were diagnosed with PA underwent adrenal venous sampling. The PAC measured by RIA (PAC(RIA)) was converted to the estimated PAC measured by CLEIA (ePAC(CLEIA)) using a conversion formula. The present study evaluated the discordance rates in diagnoses based on screening (SC), captopril challenge test (CCT), saline infusion test (SIT), and diagnosis of PA between results judged by PAC(RIA) according to the previous guidelines and those judged by ePAC(CLEIA) according to the new guidelines. The results revealed discordant diagnosis rates of 6.4% for SC and 10.1% for CCT, with no discordance for SIT. The discordant diagnosis rate for PA was 3.7%. Our study reveals the challenges in establishing appropriate diagnostic criteria for PA using PAC(CLEIA) and highlights the demand for further research on provisionally positive categories.

Adrenal venous sampling criteria for chemiluminescent enzyme immunoassay as a preferable alternative to radioimmunoassay in primary aldosteronism.

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats.

Identifying xenobiotics that interrupt the thyroid axis has significant public health implications, given that thyroid hormones are required for brain development. As such, some developmental and reproductive toxicology (DART) studies now require or recommend serum total thyroxine (T4) measurements in pregnant, lactating, and developing rats. However, serum T4 concentrations are normally low in the fetus and pup which makes quantification difficult. These challenges can be circumvented by technologies like mass spectrometry, but these approaches are expensive and not always widely available. To demonstrate the feasibility of measuring T4 using a commercially available assay, we examine technical replicates of rat serum samples measured both by liquid chromatography mass spectrometry (LC/MS/MS) and radioimmunoassay (RIA). These samples were obtained from rats on gestational day 20 (dams and fetuses) or postnatal day 5 (pups), following maternal exposure to the goitrogen propylthiouracil (0-3 ppm) to incrementally decrease T4. We show that with assay modification, it is possible to measure serum T4 using low sample volumes (25-50 µL) by an RIA, including in the GD20 fetus exposed to propylthiouracil. This proof-of-concept study demonstrates the technical feasibility of measuring serum T4 in DART studies.

Thyroglobulin immunoassay with a fully automated pretreatment process provides accurate thyroglobulin values in anti-thyroglobulin antibody positive specimens.

OBJECTIVES: Human thyroglobulin (Tg) is widely used as a tumor marker for recurrence and metastasis of differentiated thyroid cancer (DTC). Currently, serum Tg values are measured using second-generation sandwich immunoassays (2nd-IMA). However, interference by endogenous autoantibodies to thyroglobulin (TgAbs) can lead to false-negative results or falsely low Tg values. Here, we describe a new Tg assay using the immunoassay for total antigen including complex via pretreatment (iTACT) method to prevent TgAb interference and compare it with 2nd-IMA. METHODS: Tg values were evaluated by three assays: iTACT Tg, Elecsys Tg-II, which is a 2nd-IMA, and LC-MS/MS (Liquid chromatography tandem-mass spectrometry). The ratio of Tg values between each assay was then compared to the Tg value by LC-MS/MS and TgAb titer. Tg immunoreactivity was analyzed by size-exclusion chromatography. RESULTS: Correlation between iTACT Tg and LC-MS/MS using TgAb-positive specimens was good: Passing-Bablok regression with iTACT Tg = 1.084 × LC-MS/MS + 0.831. Correlation between 2nd-IMA and LC-MS/MS showed a relatively lower slope: 2nd-IMA = 0.747 × LC-MS/MS - 0.518. Thus, Tg values determined by iTACT Tg are equivalent to those of LC-MS/MS regardless of TgAb titer, whereas 2nd-IMA gave lower Tg values due to TgAb interference. Tg-TgAb complexes of various molecular weights were verified by size-exclusion chromatography. Tg values measured by 2nd-IMA fluctuated depending on the molecular weight of the Tg-TgAb complexes, whereas iTACT Tg accurately quantified Tg values regardless of the size of the Tg-TgAb complexes. CONCLUSION: Tg values in TgAb-positive specimens were accurately determined by iTACT Tg. TgAb-positive specimens contain Tg-TgAb complexes of various molecular weights that interfere with Tg value determination by 2nd-IMA, whereas iTACT Tg is unaffected by the presence of Tg-TgAb complexes.

Assessment of the Androstenedione Assay on a Roche Analytical Platform and Comparison with Different Methods.

BACKGROUND: D4-androstenedione (D4ASD) is an intermediate hormone of androgen biosynthesis by the gonads and the adrenal glands. The interest in D4ASD concentration assessment resides in diagnostics of androgenic hyperproduction pathologies. Currently, many D4ASD quantification methods are available on the market including immunological methods that remain problematic due to the possible cross-reactivity with endogenous or exogenous steroids. METHODS: Recently Roche® launched a new fully automated instrument for the measurement of D4ASD concentration. In this paper, the criteria of analytical performance (repeatability and intermediate precision) of the D4ASD Roche® assay were assessed and compared with 2 different methods including a radioimmunoassay (RIA) as well as a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. RESULTS: Repeatability and intermediate precision of the D4ASD Roche® were acceptable according to the prede-fined RICOS standard (CV ≤ 7.9%) and the assay showed a good correlation with other assays considering the 95% CI obtained for the slope and the y-intercept. CONCLUSIONS: This method demonstrates acceptable criteria of analytical performance with an intermediate imprecision and a trueness within the fixed acceptance limits.

Validation and method comparison for a point-of-care lateral flow assay measuring equine whole blood insulin concentrations.

The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from <139 to >695 pmol/L (<20 to >100 µIU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139-278 pmol/L; 20-40 µIU/mL), intermediate (278-417 pmol/L; 40-60 µIU/mL), and high (>417 pmol/L; >60 µIU/mL) concentrations, respectively. For 10 replicates at each level (3 assay lots), inter-assay CVs were 15.9%, 11.0%, and 11.7%, respectively. In the weighted linear regression of 5 measured insulin concentrations against expected concentrations, R2 = 0.98, slope = 1.02, and y-intercept = 14.4 pmol/L (2.08 µIU/mL). The Spearman correlation coefficient (rs) was 0.90 (95% CI: 0.85-0.94) between the WRT and RIA; the WRT = f(RIA) Passing-Bablok regression yielded the fit, y = 1.005x + 24.3 pmol/L (3.50 µIU/mL). The WRT result averaged 10.4% higher than the RIA result, with targeted bias of 25.9, 26.1, and 26.7 pmol/L (3.74, 3.76, and 3.84 µIU/mL) for cutoffs used to diagnose insulin dysregulation of 312, 347, and 451 pmol/L (45, 50, and 65 µIU/mL). Assay clinical sensitivities, specificities, and accuracies determined at the 3 selected clinical cutoffs and using the RIA as gold standard were 87-95%, 92-96%, and 91-95%, respectively (n = 99 samples). Observed total error was 28.4-30.4%. The WRT had acceptable precision, excellent linearity, and good association with the RIA.

Comparing the clinical significance and antigen specificity of insulinoma-associated antigen-2 autoantibodies between radioimmunoassay and enzyme-linked immunosorbent assay in Japanese patients with type 1 diabetes.

AIMS/INTRODUCTION: This study aimed to investigate the clinical significance and antigen specificity of autoantibodies to insulinoma-associated antigen-2 (IA-2A) by radioimmunoassay (RIA; IA-2A-RIA) and enzyme-linked immunosorbent assay (ELISA; IA-2A-ELISA) in Japanese patients with type 1 diabetes. MATERIALS AND METHODS: A total of 338 type 1 diabetic patients were enrolled, including 38 fulminant type 1 diabetes, 168 acute-onset type 1 diabetes and 137 slowly-progressive type 1 diabetes (SPIDDM). The concordance, correlation of autoantibody titer, and the relationship between IA-2A and progression to the insulin-deficient state were examined. Also, competitive assay was used to examine the antigen specificity. RESULTS: The prevalence of IA-2A-ELISA was 4-5% lower than that of IA-2A-RIA in both the acute-onset type 1 diabetes and SPIDDM, but the diagnostic sensitivities of both subtypes, when measured in combination with glutamic acid decarboxylase autoantibody, were comparable. The diagnosis of type 1 diabetes using either the RIA or ELISA methods showed substantial agreement with the exponential correlation of autoantibody titers detected by RIA and ELISA. Among the SPIDDM patients, the fasting C-peptide for IA-2A-positive cases by ELISA, but not the RIA method, was significantly lower than in the negative cases (P < 0.05). Furthermore, IA-2A-ELISA proved superior to the RIA method in predicting the progression to insulin deficiency in SPIDDM. Competitive analysis showed that even sera with discrepant results by RIA and ELISA have IA-2-specific autoantibodies. CONCLUSION: These results suggest that IA-2A-ELISA is a reliable marker not only for the diagnosis of type 1 diabetes, but also for the prediction of future insulin dependency; that is, detection of IA-2A-ELISA helps identify a subtype of SPIDDM patients who would likely progress onto insulin-deficient state.

Method Performance Verification of Anti-GAD65 and Anti-Insulin antibody Assays.

BACKGROUND: The aim of this study was to evaluate the performance of chemiluminescence immunoassays for anti-GAD65 and anti-insulin antibodies following user verification guidelines. METHODS: The analytical performance of anti-GAD65 and anti-insulin antibodies using a MAGLUMI 2000 analyzer was verified following user verification guidelines by the Clinical and Laboratory Standards Institute. RESULTS: Performance specifications including precision, linearity, carry-over, cutoffs for positive results, reference intervals, and comparability with pre-existing commercially available radioimmunoassays using patient specimens and certified reference material were verified (coefficients of variation for precision of anti-GAD65 and anti-insulin antibodies were 2.6% and 3.4%, respectively). Comparability assessed using clinical serum specimens showed overall agreement with radioimmunoassay of 87.2% (95% confidence interval 74.8% - 94.0%) for the anti-GAD65 antibody assay and 85.4% (95% confidence interval 71.6% - 93.1%) for the anti-insulin antibody assay. CONCLUSIONS: The results of this study verified the analytical performance of MAGLUMI anti-GAD65 and anti-insulin antibody assays for clinical use.

Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification.

STUDY OBJECTIVES: The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cutoff of 110 pg/mL, and the inter- and intra-assay variability. METHODS: Raw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established and defined as the lowest converted concentration with a CV <25%; the conversion is performed using a harmonization sample which is internationally used to minimize variation between RIAs. RESULTS: The mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n = 41, range 1.6%-17.0%), which was significantly higher than in RIAs with higher specific activity (n = 36; median 3.2%, range 0.4%-11.6%, p = .013). The CV around the 110 pg/mL cutoff was <7%. CONCLUSIONS: Hypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cut-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as "undetectable" when measured with the Phoenix Pharmaceuticals RIA. CLINICAL TRIAL INFORMATION: This study is not registered in a clinical trial register, as it has a retrospective database design.

Effect of iodinated radiographic contrast media on radioimmunoassay for measuring thyroid hormones.

Radioimmunoassay (RIA) is one of the most routine laboratory tests for diagnosing thyroid disease. Patients might receive iodine in the form of intravenous iodinated radiographic contrast media (IRCM) before testing of serum thyroxin (T4) or triiodothyronine (T3) concentration by RIA. The objective was to determine the effect of IRCM on T4 and T3 hormone tests in normal, hypothyroid, and hyperthyroid hormone conditions by RIA. IRCMs (0, 2.5, 5 and 10 mgI/mL) used in this study were iopromide and iodixanol. RIA was determined by commercial T4 RIA kit and T3 RIA kits. The method suggested by the manufacturer was followed. Normal, hypothyroid, and hyperthyroid hormones condition were 1.2 ng/mL, 0.2 ng/mL and 2.2 ng/mL for T3 hormone concentration and 70 ng/mL, 30 ng/mL and 140 ng/mL for T4 hormone concentration, respectively. %Bound values were compared between IRCM-incubated groups and non-incubated group. The data showed that iopromide-incubated groups did not statistically significant change %bound values of T3 and T4 hormone tests in normal, hypothyroid, and hyperthyroid conditions, compared to the non-incubated group. In the same way, %bound values of T3 and T4 hormone tests in iodixanol-incubated groups did not change at all conditions when compared to the non-incubated group. This finding suggested that iodinated radiographic contrast media was unlikely to result in significant problems with radioimmunoassay for measuring T3 and T4 thyroid hormones.

Variability in testosterone measurement between radioimmunoassay (RIA), chemiluminescence assay (CLIA) and liquid chromatography-tandem mass spectrometry (MS) among prostate cancer patients on androgen deprivation therapy (ADT).

INTRODUCTION & AIMS: Monitoring testosterone (T) levels is recommended to assess the effectiveness of androgen deprivation therapy (ADT) in advanced prostate cancer. T levels below 20 ng/dL have been associated with better outcomes. Three main measures for T exist including radioimmunoassay (RIA), chemiluminescence assay (CLIA) and mass spectrometry (MS). While CLIA and RIA are ubiquitous, MS is regarded as the reference standard. We set out to determine the discordance of T measurements amongst men on ADT. METHODS: A retrospective review of men with prostate cancer on ADT for ≥3 months was conducted. Serum samples were split in triplicate. Observational data was reported and T measurements were compared analyzing for variability looking for categorical concordance. Over and under-estimation rates were calculated. RESULTS: Ninety-five patients were included with a mean age of 70 (50-92) years. Mean ADT duration was 24.1 (3-144) months. Ninety-five percent of patients had T ≤20ng/dL by MS and CLIA as compared to only 80% by RIA. After subdividing into T categories of ≤20, 20 to 50 and ≥50 ng/dL concordance analysis showed that 4.3% and 18.9% of T measured by MS would have a different category result when remeasured by CLIA (Kappa 0.84) or RIA (Kappa 0.50) respectively. CLIA and RIA overestimated T in 66.7% of patients with T <20 ng/dL measured by MS. Conversely CLIA and RIA underestimated T in only 4.4% of cases with T >20 ng/dL measured by MS. CONCLUSIONS: There is significant variability in T measured with RIA, CLIA and MS. CLIA and RIA overestimated T levels in majority of patients leaving a concern of misdiagnosing truly castrate patients as being inadequately treated.

Effective purification of human chorionic gonadotropin and production of highly specific polyclonal anti-ßHCG as a component of radioimmunoassay kit.

This study aimed to purify human chorionic gonadotropin (HCG) from the urine of pregnant women with high biological activity (10811 IU/mg) and purity (98.2%), by simple capturing of HCG using DEAE Sepharose FF and polishing using Sephacryl S200 HR. The HCG obtained was characterized by SDS-PAGE and dissociated into alpha and beta subunits using the urea treatment method. The ßHCG subunits were injected into rabbits for the production of highly specific polyclonal anti-ßHCG antisera. The polyclonal anti-ßHCG was locally produced in rabbits and assessed for binding titer (1/10000), displacement (84.8%), and specificity (98.8%). Purified HCG along with locally prepared polyclonal anti-ßHCG antisera were used as basic components of the in-house Radioimmunoassay system for quantitative estimation of HCG in human serum.

Comparison and commutability study between standardized liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and chemiluminescent enzyme immunoassay for aldosterone measurement in blood.

A commutability confirmation test for the blood aldosterone measurement was performed on liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) as a designated comparison method (DCM) and four chemiluminescent enzyme immunoassay (CLEIA) measurement procedures based on metrological traceability. A conventional radioimmunoassay (RIA) and two measurement procedures of CLEIA which obtains RIA equivalent values were also compared. The relationship between the DCM value and the CLEIA value with respect to 120 pg/mL of the RIA value, which is the screening criterion of primary aldosteronism (PA) was clarified. For the correlation test, 75 samples of patient serum and plasma were used. Regression analysis revealed that the standardized LC-MS/MS and four CLEIA measurement procedures were in good agreement. This is the effect of measurement specificity and calibration using by certified reference material (CRM). The median of the LC-MS/MS corresponding to 120 pg/mL of RIA was 48.5 pg/mL. In the mean of standardized four CLEIA values corresponding to the 48.5 pg/mL of LC-MS/MS value was 47.51 pg/mL and the standard deviation (SD) was 2.93 pg/mL. However, the correlation between the RIA value and the RIA equivalent of the two measurement procedures by CLEIA differed depending on the measurement procedure. This is due to the influence of RIA measurement performance. Standardized CLEIA measurements are suitable for routine measurement procedure. When converting the LC-MS/MS equivalent value by the standardized CLEIA to the conventional RIA value, it is necessary to use the conversion formula.

Inhibition of endogenous ouabain by atrial natriuretic peptide is a guanylyl cyclase independent effect.

BACKGROUND: Endogenous ouabain (EO) and atrial natriuretic peptide (ANP) are important in regulation of sodium and fluid balance. There is indirect evidence that ANP may be involved in the regulation of endogenous cardenolides. METHODS: H295R are human adrenocortical cells known to release EO. Cells were treated with ANP at physiologic concentrations or vehicle (0.1% DMSO), with or without guanylyl cyclase inhibitor 1,2,4 oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cyclic guanosine monophosphate (cGMP), the intracellular second messenger of ANP, was measured by a chemiluminescent immunoassay and EO was measured by radioimmunoassay of C18 extracted samples. RESULTS: EO secretion is inhibited by ANP treatment, with the most prolonged inhibition (90 min vs ≤ 60 min) occurring at physiologic ANP concentrations (50 pg/mL). Inhibition of guanylyl cyclase with ODQ, also reduces EO secretion. The inhibitory effects on EO release in response to cotreatment with ANP and ODQ appeared to be additive. CONCLUSIONS: ANP inhibits basal EO secretion, and it is unlikely that this is mediated through ANP-A or ANP-B receptors (the most common natriuretic peptide receptors) or their cGMP second messenger; the underlying mechanisms involved are not revealed in the current studies. The role of ANP in the control of EO synthesis and secretion in vivo requires further investigation.

Imaging alloreactive T cells provides early warning of organ transplant rejection.

Diagnosis of organ transplant rejection relies upon biopsy approaches to confirm alloreactive T cell infiltration in the graft. Immune molecular monitoring is under investigation to screen for rejection, though these techniques have suffered from low specificity and lack of spatial information. ImmunoPET utilizing antibodies conjugated to radioisotopes has the potential to improve early and accurate detection of graft rejection. ImmunoPET is capable of noninvasively visualizing the dynamic distribution of cells expressing specific immune markers in the entire body over time. In this work, we identify and characterize OX40 as a surrogate biomarker for alloreactive T cells in organ transplant rejection and monitor its expression by utilizing immunoPET. In a dual murine heart transplant model that has both syngeneic and allogeneic hearts engrafted in bilateral ear pinna on the recipients, OX40 immunoPET clearly depicted alloreactive T cells in the allograft and draining lymph node that were not observed in their respective isograft counterparts. OX40 immunoPET signals also reflected the subject's immunosuppression level with tacrolimus in this study. OX40 immunoPET is a promising approach that may bridge molecular monitoring and morphological assessment for improved transplant rejection diagnosis.

A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin.

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 ß cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by ß cells and searching for new modulators of insulin secretion.