Conventional Radioimmunoassay Research Articles

Investigating the cut-off values of captopril challenge test for primary aldosteronism using the novel chemiluminescent enzyme immunoassay method: a retrospective cohort study

The measurement evolution enabled more accurate evaluation of aldosterone production in hypertensive patients. However, the cut-off values for novel assays have been not sufficiently validated. The present study was undertaken to validate the novel chemiluminescent enzyme immunoassay for aldosterone in conjunction with other methods. Moreover, we also aimed to establish a new cut-off value for primary aldosteronism in the captopril challenge test using the novel assay. First, we collected 390 plasma samples, in which aldosterone levels measured using liquid chromatography-mass spectrometry ranged between 0.18 and 1346 ng/dL. The novel chemiluminescent enzyme immunoassay showed identical correlation of plasma aldosterone with liquid chromatography-mass spectrometry, in contrast to conventional radioimmunoassay. Further, we enrolled 299 and 39 patients with primary aldosteronism and essential hypertension, respectively. Plasma aldosterone concentrations measured using the novel assay were lower than those measured by radioimmunoassay, which resulted in decreased aldosterone-to-renin ratios. Subsequently, positive results of the captopril challenge test based on radioimmunoassay turned into “negative” based on the novel assay in 45% patients with primary aldosteronism, using the conventional cut-off value (aldosterone-to-renin activity ratio > 20 ng/dL per ng/mL/h). Receiver operating characteristic curve analysis demonstrated that aldosterone-to-renin activity ratios > 8.2 ng/dL per ng/mL/h in the novel assay was compatible with the conventional diagnosis (sensitivity, 0.874; specificity, 0.980). Our study indicates the great measurement accuracy of the novel chemiluminescent enzyme immunoassay for aldosterone, and the importance of measurement-adjusted cut-offs in the diagnosis of primary aldosteronism.

Performance evaluation of the EUROIMMUN anti‐acetylcholine receptor enzyme‐linked immunoassay

AbstractObjectivesAnti‐acetylcholine receptor antibodies (AChR Ab) are one of the pathogenic autoantibodies in myasthenia gravis (MG), and they are specifically mentioned in the Japanese clinical guidelines for MG. In the serological diagnosis and measurement of autoantibodies as an adjunct marker for MG, the radioimmunoassay (RIA) method has been conventionally used as the gold standard. However, there have been several worldwide concerns regarding the use of radioisotopes. In this study, we evaluated the performance of the EUROIMMUN AChR Ab enzyme‐linked immunosorbent assay (ELISA) in comparison with RIA.MethodsELISA reproducibility and dilution linearity were analyzed using AChR Ab‐positive samples. Sera from 50 patients with suspected MG and 50 healthy donors were used to assess the correlation and qualitative agreement between the two methods.ResultsThe ELISA showed good interassay precision and dilution linearity. The correlation coefficient, sensitivity, specificity and overall agreement of the ELISA in comparison with the conventional RIA amounted to 0.87, 98%, 92% and 95% (kappa = 0.895), respectively. Analysis of the discordant results suggested that the ELISA might be more capable of detecting low AChR Ab reactivity in patients with suspected MG.ConclusionsThe AchR Ab ELISA provides a reliable tool for the quantification of AchR Ab, supporting the diagnosis of MG. As a non‐isotopic assay with good precision, shorter handling time and high correlation with the conventional RIA, the AChR ELISA could be a useful alternative in the laboratory routine.

FRI136 Adrenal Venous Sampling Criteria For Chemiluminescent Enzyme Immunoassay

Abstract Disclosure: K. Nakai: None. Y. Tsurutani: None. K. Irie: None. K. Teruyama: Employee; Self; Fujirebio Inc. J. Saito: None. M. Omura: None. T. Nishikawa: None. Objective: In primary aldosteronism (PA), plasma aldosterone concentration (PAC) was measured by radioimmunoassay (RIA), but the supply of the RIA kit was discontinued in March 2021 in Japan. The Japan Endocrine Society's 2021 Clinical Practice Guideline for the Diagnosis and Management of PA recommends that PAC measured by chemiluminescent enzyme immunoassay (CLEIA), which yields standardized LC-MS/MS compliant measurements, be used for clinical judgment. Because CLEIA shows a lower PAC than RIA, various cutoff values for PA need to be revised. Although conversion formulas for RIA and CLEIA for various kits have been provided, these conversions were studied mainly using samples representing aldosterone concentrations in peripheral blood; thus, it is unclear whether the same conversion formula can be used for samples obtained by adrenal venous sampling (AVS). In this study, we examined PAC conversion in AVS samples and the criteria for AVS when measured using CLEIA. Methods: PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) from 63 patients with PA, were measured using RIA and CLEIA methods. The established conversion formula for PAC in peripheral blood samples was PAC (CLEIA) = 0.852 × PAC (RIA) − 36.0. PAC and aldosterone/cortisol ratio (A/C ratio) conversion formulas were determined between the two assays for AVS samples. Moreover, the CLEIA conversion value, which corresponds to the AVS criteria of conventional RIA, was obtained. Results: The median [interquartile range] PAC (CLEIA) in the samples was 9823.3 [3659.8-20600.5]. PAC measured using CLEIA was significantly correlated with RIA, and the correlation coefficient was 0.971. The PAC conversion formula determined was PAC (CLEIA) = PAC (RIA) × 0.7723 - 1199 pg/mL. PAC at 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. The correlation coefficient for the A/C ratio was 0.997, and the conversion formula was A/C (CLEIA) = A/C (RIA) × 0.7864 - 2.427. Discussion: Samples obtained from AVS had a much higher level of PAC than those obtained from peripheral blood. The PAC conversion formula for samples in AVS has a very large intercept compared with those in peripheral blood. Thus, the results of AVS tested by CLEIA should be interpreted with care because of the differences in the conversion formula. A different cut-off value may also be needed when evaluating the lateralization ratio because of the large intercept. Conclusion: PAC conversion formulas for AVS samples using CLEIA and RIA methods were obtained. The study outcomes are useful for interpreting AVS results using the CLEIA. Presentation: Friday, June 16, 2023

Impact of a change to a novel chemiluminescent immunoassay for measuring plasma aldosterone on the diagnosis of primary aldosteronism.

In Japan, the standard method for measuring plasma aldosterone concentration (PAC) for primary aldosteronism (PA) diagnosis was changed from radioimmunoassay (RIA) to a novel chemiluminescent enzyme immunoassay (CLEIA). The purpose of this study is to simulate the possible impact of the change on PA diagnosis. This retrospective study assessed 2,289 PA patients. PACs measured by conventional RIA were transformed to estimated PACs (CLEIA) as follows: RIA (pg/mL) = 1.174 × CLEIA (pg/mL) + 42.3. We applied the estimated PAC (CLEIA) to the conventional cut-off of aldosterone-to-renin activity ratio ≥200 for screening and captopril challenge test (CCT) and PAC ≥60 pg/mL for saline infusion test (SIT). Application of the estimated PAC to screening and confirmatory tests decreased the number of PA diagnoses by 36% (743/2,065) on CCT and 52% (578/1,104) on SIT (discrepant cases). Among the discrepant cases, 87% (548/628) of CCT and 87% (452/522) of SIT were bilateral on adrenal venous sampling (AVS). Surgically treatable aldosterone-producing adenomas (APAs) were observed in 6% (36/579) and 5% (23/472) of discrepant cases on CCT and SIT, respectively; most were characterized by hypokalemia and/or adrenal nodule on CT imaging. Application of the PAC measured by the novel CLEIA to conventional cut-offs decreases the number of PA diagnoses. Although most discrepant cases were bilateral on AVS, there are some APA cases that were characterized by hypokalemia and/or adrenal tumor on CT. Further studies which evaluate PACs measured by both RIA and CLEIA for each patient are needed to identify new cut-offs for PAC measured by CLEIA.

PS-C22-3: VALIDATION AND CLINICAL EVALUATION OF DIAGNOSTIC ABILITY OF NOVEL CHEMILUMINESCENT ENZYME IMMUNOASSAY OF PLASMA ALDOSTERONE CONCENTRATION.

Plasma aldosterone concentration has been usually measured by radioimmunoassay and chemiluminescent enzyme immunoassay which have demonstrated lack of high specificity and variability in assay performances. Liquid chromatography-tandem mass spectrometry is considered to be ideal measurement method of plasma aldosterone, however it takes relatively high cost and measurement time. We recently developed a novel non-competitive chemiluminescent enzyme immunoassay for plasma aldosterone concentrations by the fully automated assay system. We performed clinical validation of diagnostic ability of the new assay-based screening of 344 hypertensive patients including 133 patients with primary aldosterone producing adenoma, 100 patients with bilateral hyperaldosteronism, and 111 patients with essential hypertension. Passing-bablok analysis revealed that the new measurement of plasma aldosterone concentration was significantly correlated and almost equivalent to liquid chromatography-tandem mass spectrometry measurement of aldosterone; the regression slope and intercept were 0.962 and -0.043, and the correlation coefficient was 0.962, which is more similar than radioimmunoassay especially in lower range (< 10 ng/dL). Bland-Altman plot analysis with the mass-spectrometry measurement also demonstrated that both bias and limits of agreement with 95% confidence interval of the automated aldosterone assay were smaller than those of the radioimmunoassay, suggesting smaller systemic errors in the novel measurement. The lowest plasma aldosterone concentration and ratio of aldosterone-over-renin concentrations in aldosterone producing adenoma group were 6.0 ng/dL and 16.3 ng/dL per pg/mL/hr, respectively. We suggest if aldosterone-over-renin concentrations is over 15 ng/dL per pg/mL/hr, the patient should be performed confirmatory test for primary aldosteronism. The novel chemiluminescent enzyme immunoassay is clinically reliable alternative for conventional radioimmunoassay and liquid chromatography-tandem mass spectrometry to provide better throughput and cost-effectiveness in diagnosis of primary aldosteronism from larger number of hypertensive patients.

Assessment of Two Different Glucagon Assays in Healthy Individuals and Type 1 and Type 2 Diabetes Patients.

Methods for glucagon analysis suffered in the past from lack of specificity and a narrow sensitivity range, which has led to inaccurate results and to the suggestion that type 1 diabetes (T1D) and type 2 diabetes (T2D) patients have elevated fasting glucagon levels. However, the availability of more specific and more sensitive methods to detect intact glucagon has shown that actual glucagon levels are lower than previously assumed. This study aimed to characterize fasting plasma glucagon levels in healthy individuals and T1D and T2D patients with two different glucagon assays. The study included 20 healthy individuals, 20 T1D and 20 T2D patients. Blood was collected under fasting conditions. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) and a conventional radioimmunoassay (RIA) were used. A significant difference in fasting glucagon levels between healthy individuals and T2D was observed by ELISA, but not by RIA. ELISA also yielded lower glucagon levels in healthy individuals than in T1D and T2D patients which RIA did not. RIA produced significantly (p = 0.0001) higher overall median glucagon values than ELISA in a pooled analysis. These results underline the notion that the choice of selective laboratory methods is highly relevant for mechanistic endocrine research.

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.

Lateral flow immunogold assay as a rapid detection tool for screening of congenital hypothyroidism

ABSTRACT Congenital hypothyroidism (CH) is one of the most common preventable causes of mental retardation. The majority of infants are diagnosed after detection through newborn screening programs using thyroid-stimulating hormone (TSH) test. A rapid immunochromatographic lateral flow assay based on monoclonal antibodies (MAbs) colloidal gold nanoparticles was developed in a sandwich format for the detection of TSH. Two MAbs binding distinct TSH epitopes are used; one is conjugated to the detection reagent while the other is immobilized at the test line on the membrane. The colloidal gold was prepared by the reduction of gold salt coupled with MAbs and this optimal concentration was determined by spectrophotometry method. The sensitivity of our developed lateral flow immunoassay was determined using 5, 10, 15, 25 and 50 μUI/mL of TSH. The color intensity of the test line was directly proportional to the TSH concentration and the visual limit of detection was 10 μUI/mL. Twenty samples of umbilical cord serum were analyzed by the developed strips and the intensity of the signal was in agreement with the results obtained by the conventional radioimmunoassay method. The results suggest that this rapid test can be used in initial screening for congenital hypothyroidism especially in rural areas.

Novel chemiluminescent immunoassay to measure plasma aldosterone and plasma active renin concentrations for the diagnosis of primary aldosteronism

Determination of plasma aldosterone concentrations (PAC) and plasma active renin concentrations (ARC) is essential for the diagnosis of primary aldosteronism (PA). In Japan, although PAC and ARC are measured by radioimmunoassay and immunoradiometric assay, respectively, non-radioisotopic methods with better detection sensitivity, measurement accuracy, and technical simplicity are needed. We developed two-site sandwich chemiluminescent enzyme immunoassays (CLEIAs) to measure both PAC and ARC using monoclonal antibodies immobilized onto ferrite particles. The results of both assays are obtained simultaneously from a single plasma sample within 30 min using a fully automated system. The novel CLEIAs were validated using plasma samples from patients with PA (n = 52) and essential hypertension (n = 23). The PAC determined by the CLEIA was significantly correlated with that measured by liquid chromatography/mass spectrometry or conventional radioimmunoassay. The ARC determined by the CLEIA was significantly correlated with that measured by immunoradiometric assay. The limits of detection of the CLEIAs for PAC and ARC were 0.1 ng/dl and 0.04 pg/ml, respectively, which were better than those of conventional methods (PAC: 2.5 ng/dl; ARC: 5 pg/ml). The PAC and PAC/ARC ratio (ARR) were significantly higher, and the ARC significantly lower, in patients with PA than in those with essential hypertension. An ARR cut-off of 1.31 ng/dl per pg/ml showed a sensitivity of 96.2% and specificity of 78.3% for PA screening. The newly developed CLEIAs for measuring PAC and ARC could provide a clinically powerful alternative to conventional methods used for hypertension screening in clinical practice.

Development of novel lab-on-a-chip platform for high-throughput radioimmunoassay

Radioimmunoassay (RIA) is an extremely specific and a highly sensitive type of immunoassay, but the long incubation time and generation of radioactive wastes limit the use of RIA. To complement these disadvantages of RIA, we suggest an advanced type of RIA based on a lab-on-a-chip (LOC) platform: μ-RIA. We designed a microfluidic chip for RIA and optimized the procedures of μ-RIA analysis, including surface modification, immunoreaction time, and washing. Based on the optimized conditions, we conducted a radioimmunoassay on the μ-RIA platform using a commercial RIA kit. With the μ-RIA, 5 min are adequate for analysis. The amount of reagent consumption is significantly reduced compared with conventional RIA. The standard curve with R2 = 0.9951 shows that we can quantitatively evaluate the amount of antigen present in unknown samples. We show the applicability of μ-RIA for the analysis of biomolecules and the potential of μ-RIA to be a novel platform for high-throughput analysis.

Do metabolites account for higher serum steroid hormone levels measured by RIA compared to mass spectrometry?

Higher circulating estradiol levels are generally obtained using conventional radioimmunoassays (RIA) compared to liquid chromatography/tandem mass spectrometry (LC-MS/MS) assays, and this has been attributed to the presence of estradiol metabolites that cross-react with the antibody used in the RIA. This study aimed to determine which estradiol metabolites may contribute to this effect. LC-MS/MS analysis was performed on 70 serum samples from premenopausal women, after purification by extraction and Celite column partition chromatography as would be used prior to conventional RIA for estradiol. The metabolites estrone and 2-methoxyestradiol accounted for 6.9% and 2.1% of the estradiol fractions in the purified samples overall, but the extent of contamination with these metabolites was greater in the samples containing <50 pg/mL estradiol (14.6% and 3.8% respectively) than in those containing >50 pg/mL estradiol. However, since these metabolites have a < 1% cross-reactivity to the antibody in the estradiol RIA, this level of contamination is too small to account for the differences between RIA and LC-MS/MS measurements of estradiol.

OP.6B.06] SIMULTANEOUS MEASUREMENT OF ALDOSTERONE AND RENIN CONCENTRATIONS IN TEN MINUTES COULD CHANGE THE CLINICAL ASSESSMENT OF HYPERTENSIVE PATIENTS

Objective: The measurement of plasma aldosterone (PAC) and renin concentration (ARC) or activity (PRA) is useful for selecting antihypertensive agents as well as diagnosing primary aldosteronism (PA) in hypertensive patients. However, it takes several days to get results when measured by radioimmunoassay and development of more rapid assay has been long expected. In the present study, we characterized recently developed fully-automated chemiluminescent enzyme immunoassay (CLEIA) for PAC and ARC, which can be measured simultaneously in 10 minutes and 20 seconds, and clinical validation of their diagnostic abilities for detecting PA patients from hypertensive patients was also performed in this study. Design and method: We performed clinical validation of diagnostic ability of this newly developed assay-based screening of 125 patients with primary aldosteronism (75 aldosteronoma (APA) and 50 bilateral hyperplasia (BHA)) from 97 patients with essential hypertension (EH).The newly developed assays of both PAC and ARC adopted antibody-immobilized magnetic particles, called MAGRAPID with abilities of quick aggregations and dispersions. Results: Results of this novel measurements were significantly correlated with both radioimmunoassay measurements (PAC, ARC and PRA) and liquid chromatography-tandem mass spectrometry measurements (PAC). The analytical sensitivity of this particular novel ARC measurement was 0.1 pg/mL, which was better than that of radioimmunoassay (2.0 pg/mL). The ARC values measured by CLEIA were below than 2.0 pg/mL in 28.8% of all patients (52.0%, 44.0% and 3.1% in those with unilateral APA, BHA and EH, respectively). Using Bland-Altman plot analysis with the mass-spectrometry measurement, both bias and limits of agreement with 95% confidence interval of the automated PAC assay were smaller than those of the radioimmunoassay, indicating smaller systemic errors in the novel measurement. The ratio of PAC-over-ARC of 11.2 (ng/dL per pg/mL) provided 80.8% sensitivity and 94.9% specificity as a cut-off for differentiating primary aldosteronism from essential hypertension. Conclusions: This novel measurement is expected to be clinically reliable alternatives for conventional radioimmunoassay and to provide better throughput and cost-effectiveness in diagnosis of hyperaldosteronism from larger number of hypertensive patients in clinical settings.

Rapid Screening of Primary Aldosteronism by a Novel Chemiluminescent Immunoassay.

Measurement of plasma aldosterone and renin concentration, or activity, is useful for selecting antihypertensive agents and detecting hyperaldosteronism in hypertensive patients. However, it takes several days to get results when measured by radioimmunoassay and development of more rapid assays has been long expected. We have developed chemiluminescent enzyme immunoassays enabling the simultaneous measurement of both aldosterone and renin concentrations in 10 minutes by a fully automated assay using antibody-immobilized magnetic particles with quick aggregation and dispersion. We performed clinical validation of diagnostic ability of this newly developed assay-based screening of 125 patients with primary aldosteronism from 97 patients with essential hypertension. Results of this novel assay significantly correlated with the results of radioimmunoassay (aldosterone, active renin concentration, and renin activity) and liquid chromatography-tandem mass spectrometry (aldosterone). The analytic sensitivity of this particularly novel active renin assay was 0.1 pg/mL, which was better than that of radioimmunoassay (2.0 pg/mL). The ratio of aldosterone-to-renin concentrations of 6.0 (ng/dL per pg/mL) provided 92.0% sensitivity and 76.3% specificity as a cutoff for differentiating primary aldosteronism from essential hypertension. This novel measurement is expected to be a clinically reliable alternative for conventional radioimmunoassay and to provide better throughput and cost effectiveness in diagnosis of hyperaldosteronism from larger numbers of hypertensive patients in clinical settings.

Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC-MS/MS for clinical laboratory applications.

The quantification of serum 25-hydroxyvitamin D [25(OH)D] as an indicator of vitaminD status is currently primarily conducted by immunoassays, yet LC-MS/MS would allow more accurate determination. Furthermore, LC-MS/MS would allow simultaneous measurement of multiple analytes. The aim of this study was to develop and validate an LC-MS/MS method to simultaneously measure four vitaminD metabolites (25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3) in serum for clinical laboratory applications. Serum samples were first prepared in a 96-well supported liquid extraction plate and the eluate was derivatized using the Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), which rapidly and quantitatively reacts with the s-cis-diene structure of vitaminD metabolites. The derivatized samples were subjected to LC-MS/MS, ionized by electrospray ionization (positive-ion mode), and detected by selected reaction monitoring. The lower limits of quantification for 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 were 0.091, 0.020, 0.013, and 0.024ng/mL, respectively. The accuracy values and the extraction recoveries for these four metabolites were satisfactory. Serum 25(OH)D levels determined by our LC-MS/MS were compared with those obtained by conventional radioimmunoassay (RIA) that cannot distinguish 25(OH)D3 and 25(OH)D2. The values obtained by the RIA method exhibited a mean bias of about 8.35ng/mL, most likely as a result of cross reaction of the antibody with low-abundance metabolites, including 24,25(OH)2D3. Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze-thaw cycles, and the presence of anticoagulants, had no significant effects on these determinations. This high-throughput LC-MS/MS simultaneous assay of the four vitaminD metabolites 25(OH)D3, 3-epi-25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 required as little as 20μL serum. This method will aid further understanding of low-abundance vitaminD metabolites, as well as the accurate determination of 25(OH)D3 and 25(OH)D2.

Postabsorptive hyperglucagonemia in patients with type2 diabetes mellitus analyzed with a novel enzyme-linked immunosorbent assay.

Aims/introductionThe aims of the present study were to investigate the performance of a novel sandwich enzyme‐linked immunosorbent assay (ELISA) for measuring glucagon (1–29) with monoclonal antibodies against both the C‐ and N‐terminal regions of glucagon (1–29), and to analyze the differences in plasma levels and responses of glucagon (1–29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus.Materials and MethodsThe cross‐reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75‐g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1–29) concentration was measured using three types of kit.ResultsThe ELISA kit clearly had the lowest cross‐reactivity against miniglucagon (19–29) and glicentin (1–61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1–29) levels measured by the ELISA kit after glucose loading were significantly higher at all time‐points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1–29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups.ConclusionsThe novel sandwich ELISA accurately determines plasma glucagon (1–29) concentrations with much less cross‐reactivity against other proglucagon fragments than conventional RIA kits.

Endogenous sex steroid hormones and glucose in a South-Asian population without diabetes: the Metabolic Syndrome and Atherosclerosis in South-Asians Living in America pilot study.

To examine the associations between endogenous sex steroid hormones (oestradiol, testosterone and sex hormone-binding globulin) with diabetes risk in a South-Asian population living in the USA. We used data from the Metabolic Syndrome and Atherosclerosis in South-Asians Living in America pilot study. The analytical sample included 60 women and 45 men of Asian Indian origin living in the San Francisco Bay Area, who were free from diabetes and cardiovascular disease and did not use exogenous sex steroids. Sex steroid hormone levels were assessed by validated conventional radioimmunoassays, and visceral and hepatic adiposity were assessed by computed tomography. We used multivariable regression to examine the association between endogenous sex steroid hormone levels (log-transformed) and fasting glucose and 2-h glucose levels in a series of sex-stratified models adjusted for age, waist circumference, visceral and hepatic adiposity, and insulin resistance. In age-adjusted models, lower levels of sex hormone-binding globulin (β=-0.18, 95% CI -0.30, -0.06) and higher levels of free testosterone (β=0.14, 95% CI 0.02, 0.26) were associated with elevated fasting glucose levels in South-Asian women, whereas lower levels of sex hormone-binding globulin (β=-0.14, 95% CI -0.26, -0.02) and lower levels of total testosterone (β=-0.12, 95% CI -0.24, 0.00) were associated with elevated fasting glucose levels in South-Asian men. Adjustment for waist circumference, visceral adiposity and insulin resistance attenuated most of these associations, while adjustment for hepatic adiposity strengthened some of the observed associations. Similar results were found for 2-h glucose levels. Results were consistent with previous research, which suggests that endogenous sex steroid hormones are a risk factor for diabetes across multiple race/ethnic groups. Additional studies are needed to determine whether visceral fat is a mediator or confounder of associations between sex steroid hormone and glucose levels.

Direct chemiluminescent enzyme immunoassay for atrial natriuretic peptide in mammalian plasma using a PEGylated antibody

Atrial natriuretic peptide (ANP) is a peptide hormone that is synthesized and secreted by cardiac tissues and plays a pivotal role in maintaining cardiovascular homeostasis. Clinically, ANP is used as a marker of cardiovascular diseases, including heart failure. Although multiple ANP assays are currently available, a more sensitive assay is required for the direct measurement of plasma ANP where there is limited plasma availability, especially in mouse experiments. In the current study, we developed a plate-based sandwich chemiluminescent enzyme immunoassay for the measurement of plasma ANP in rats and mice without the need for prior extraction. To minimize nonspecific binding, we performed a single-step PEGylation procedure targeting the immobilized antibody, which markedly improved the assay’s sensitivity and linearity. The linear range was 0.1 to 250pM, and the minimum detection limit was 0.13pM, 5-fold lower than the lowest value of the commercially available kits. ANP was directly measured in plasma samples without detectable cross-reactivity with B- and C-type natriuretic peptides. The accuracy of the assay was confirmed by spike recovery tests and dilution tests and by comparison with a conventional radioimmunoassay. Based on the species cross-reactivity, this assay can be used to measure human ANP.

Renal hemodynamics in the anesthetized giraffe

In the giraffe, the brain is located some two meters above the heart most of the time and the central mean arterial blood pressure is twice that of mammals of similar mass. Consequently, the heart perfuses the kidney at a mean pressure of some 200 mmHg. The renal capsule is thick and non‐translucent.Nine giraffes (mean body mass 493 kg) were anaesthetized and instrumented. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were determined by conventional clearance techniques using continuous infusions of inulin and p‐aminohippurate. Renal interstitial hydrostatic pressure (RIHP) was measured by a 22G needle inserted in the renal parenchyma. The intrarenal artery resistance index (AI) was determined by Doppler ultrasound. Hormone immunoactivities in plasma were measured by conventional radioimmunoassays.GFR was 342 ± 99 ml/min and ERPF 1252 ± 305 ml/min. AI was median 0.27 (max. 0.49, min. 0) and RIHP 142 ± 6 mmHg. Plasma renin concentration was 17 ± 4 mIU/l, peptide levels were 6.3 ± 0.9 pg/ml for AngII, 135 ± 23 pg/ml for atrial natriuretic peptide, and highly variable for vasopressin.The data show low values of GFR, ERPF and resistance index, yet the RIHP is &gt;20 fold higher than in other mammals. The results suggest that a strong renal capsule allows for a high RIHP reducing the net arterial driving pressure and effectively protecting the kidney against organ damage.

Chemiluminescence Immunoassay Analyzer Control System Based on S3C2410A

Chemiluminescence immunoassay (CLIA) is the combination of chemiluminescence system and immunoassay. CLIA is established as one of the best alternatives to conventional radioimmunoassay for the quantitation of low concentrations of analytes in complex samples. A high-performance CLIA control system based on the embedded microprocessor S3C2410A is presented in this paper. The system principle, the overall structure of the system, the control structure of the key functional blocks and the flow chart of the software are given. The hardware drivers and the application software are developed on the basis of S3C2410A. The experimental results show that the presented system is feasible in the clinical diagnosis and can be used in the automatic chemiluminescence immunoassay analyzer.

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile