Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library.

Single- domain antibodies (SdAbs) have been deployed in various biomedical applications in the recent past. However, there are no reports of their use in the immunoradiometric assays (IRMA) for thyroglobulin (Tg). Tg is the precursor molecule for the biosynthesis of thyroid hormones: thyroxine and triiodothyronine, which are essential for the regulation of normal metabolism in all vertebrates. Patients with differentiated thyroid cancer (DTC) require periodic monitoring of their serum thyroglobulin levels, as it serves as a prognostic marker for DTC. Here, we report a methodology to produce SdAbs against human-Tg, by a hybrid immunization/directed-evolution approach by displaying the SdAb gene-repertoire derived from a hyperimmune camel in the T7 phage display system. We have demonstrated the immunoreactivity of anti-Tg-SdAb (KT75) in immunoassays for thyroglobulin and measured its affinity by surface plasmon resonance (KD ~ 18 picomolar). Additionally, we have shown the quantitative-binding property of SdAb for the first time in IRMA for thyroglobulin. The serum Tg values obtained from SdAb-Tg-IRMA and in-house assay using murine anti-Tg-monoclonal antibody as tracer significantly correlated, r = 0.81, p < 0.05. Our results highlight the scope of using the T7 phage display system as an alternative for the conventional M13-phage to construct single-domain antibody display libraries.

Reference Ranges of Serum Insulin-Like Growth Factor-I and Insulin-Like Growth Factor Binding Protein-3: Results from a Multicenter Study in Healthy Korean Adults.

Insulin-like growth factor-I (IGF-I) plays a pivotal role in the diagnosis and treatment of growth hormone (GH) excess or deficiency. The GH study group of the Korean Endocrine Society aims to establish the Korean reference ranges of serum IGF-I and insulin-like growth factor binding protein-3 (IGFBP-3) and assess the relationship between IGF-I and IGFBP-3 and clinical parameters. Fasting serum was collected from healthy Korean adults at health promotion centers of five hospitals nationwide. Serum IGF-I and IGFBP-3 were measured via an immunoradiometric assay using a DSL kit (Diagnostic Systems Laboratories). Serum samples from 354 subjects (180 male, 174 female) were analyzed based on sex at 10-year intervals from 21 to 70 years. IGF-I levels were inversely correlated with age. After adjustment of age, the IGF-I/IGFBP-3 ratio was significantly negatively associated with blood pressure and free thyroxine and positively associated with weight, hemoglobin, creatinine, alanine transferase, fasting glucose, and thyroid stimulating hormone. Therefore, age- and sex-specific reference ranges of serum IGF-I and IGFBP-3 can be efficient in evaluating GH excess or deficiency in Korean population.

Determination of thyroglobulin levels by radioimmunoassay method in anti thyroglobulin positive differentiated thyroid patients: One center clinical experience.

It is very crucial to determine Tg accurately and precisely in thyroid cancer cases. Although there are many studies on the detection of Tg in thyroid cases in the literature, there are no sufficient clinical studies examining many cases with different features by using RIA methodology. Here, a radiometric and chromatographic method has been studied for the first time to eliminate the interference from anti-Tg positive patients. In this paper, radioimmunoassay (RIA) and immunoradiometric (IRMA) techniques were used for the analysis of 302 sera collected from patients for Tg and TgAb quantification. By the RIA technique, a reliable result was obtained by calculating the real Tg value quantitatively in 41 patients showing TgAb positivity out of 208 patients. Our findings show that the RIA assay is the most suitable approach for detection of changeable (low or undetectable) Tg value and metastases detected by post-therapeutic imaging in early-stage DTC cases showing preoperative and postoperative TgAb positivity. The new immunoradiometric method allows the real (%) Tg value to be reached in a part of TgAb-positive DTC. Even if TgAb positive in the metastatic and nonmetastatic DTC patient group. This allows the accurate clinical follow-up of patients.

An approach to computer analysis of the ligand binding assay data on example of radioligand assay data.

As a rule, receptor-ligand assay data are fitted by logistic functions (4PL model, 5PL model, Feldman's model). The preparation of the initial estimates for parameters of these functions is an important problem for processing receptor-ligand interaction data. This study represents a new mathematical approach to calculate the initial estimates more closely to the true values of parameters. The main idea of this approach is in using the modified linear least squares method for calculations of the parameters for the 4PL model and the Feldman's model. In this study, the convergence of model parameters to true values is verified for the simulated data with different statistical scatter. Also, the results of processing real data for the 4PL model and the Feldman's model are presented. A comparison is made of the parameter values calculated by the presented and a nonlinear method. The developed approach has demonstrated its efficiency in calculating the parameters of the complex Feldman"s models up to 4 ligands and 4 sites.

Studying RNA-binding protein interactions with target mRNAs in eukaryotic cells: native ribonucleoprotein immunoprecipitation (RIP) assays.

Post-transcriptional regulation of mRNA can potently dictate protein expression patterns in eukaryotic cells. This mode of regulation occurs through cis-acting regulatory regions in the mRNA transcript that mediate direct interactions with trans-acting RNA-binding proteins (RBPs). This mRNA/protein interaction can be studied in numerous ways that range from in vitro to in vivo through messenger ribonucleoprotein immunoprecipitation (mRNP-IP or RIP) assays. This modified immunoprecipitation approach is an important and sensitive method to determine the regulation of gene expression by specific RBPs under different cellular stressors.

Highly specific detection of myostatin prodomain by an immunoradiometric sandwich assay in serum of healthy individuals and patients.

BACKGROUND: Myostatin is a muscle derived factor that functions as a negative regulator of skeletal muscle growth. Induction of myostatin expression was observed in rodent models of muscle wasting and in cachectic patients with cancer or pulmonary disease. Therefore, there is an increasing interest to use serum myostatin as a biomarker. METHODS: We established an immunoradiometric sandwich assay (IRMA), which uses a commercially available chicken polyclonal, affinity purified antibody directed against human myostatin prodomain. We determined the serum concentrations of myostatin prodomain in 249 healthy individuals as well as 169 patients with heart failure, 53 patients with cancer and 44 patients with chronic pulmonary disease. RESULTS: The IRMA had a detection limit of 0.7ng/ml, an intraassay imprecision of ≤14.1% and an interassay imprecision of ≤ 18.9%. The specificity of our assay was demonstrated by size exclusion chromatography, detection of myostatin by Western-blotting and a SMAD-dependent transcriptional-reporter assay in the signal-rich serum fractions, as well as lack of interference by unspecific substances like albumin, hemoglobin or lipids. Myostatin prodomain was stable at room temperature and resistant to freeze-thaw cycles. Apparently healthy individuals over the age of 55 had a median myostatin prodomain serum concentration of 3.9ng/ml (25(th)-75(th) percentiles, 2-7ng/ml) and we could not detect increased levels in patients with stable chronic heart failure or cancer related weight loss. In contrast, we found strongly elevated concentrations of myostatin prodomain (median 26.9ng/ml, 25(th)-75(th) percentiles, 7-100ng/ml) in the serum of underweight patients with chronic pulmonary disease. CONCLUSIONS: We established a highly specific IRMA for the quantification of myostatin prodomain concentration in human serum. Our assay could be useful to study myostatin as a biomarker for example in patients with chronic pulmonary disease, as we detected highly elevated myostatin prodomain serum levels in underweight individuals of this group.

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients.

BACKGROUND: Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence. METHODS: In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni. RESULTS: Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI-TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro. CONCLUSION: We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients. GENERAL SIGNIFICANCE: This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.

Comparison of intravenous versus intramuscular administration of corticotropin-releasing hormone in healthy cats.

BACKGROUND: Because of the lack of a current validated assay for feline endogenous adrenocorticotropic hormone (ACTH) in response to administration of currently available ovine corticotropin-releasing hormone (oCRH) preparations, a complete evaluation of the hypothalamic-pituitary-adrenal axis in cats has not been possible. OBJECTIVE: This study was undertaken to (1) determine the pituitary (ACTH) and adrenal (cortisol) response to both IV and IM administration of a currently available oCRH product in healthy cats, and (2) validate an endogenous ACTH assay for use in cats. ANIMALS: Seventeen healthy cats receiving oCRH (n = 11) or placebo (n = 6). METHODS: Prospective, randomized, placebo-controlled study. oCRH at 1 µg/kg or placebo was given either IM or IV. Endogenous cortisol and ACTH concentrations were evaluated after the injection. A comparison of IM versus IV and placebo versus treatment was made. RESULTS: The DiaSorin immunoradiometric assay (IRMA) assay for ACTH performed well, showing both parallelism and acceptable intra- and interassay coefficients of variation. There was a significant difference between groups (P = .025) and a significant difference between times (P = .025) when endogenous ACTH concentrations were compared after oCRH IV or IM. No significant differences were observed in cortisol concentrations comparing IV to IM oCRH. CONCLUSIONS: IM administration of oCRH results in significantly greater ACTH concentrations but not cortisol concentrations when compared with IV administration. Samples should be drawn before and at 60 minutes after the injection. The Diasorin IRMA is valid for feline endogenous ACTH measurements.

Valores de referência para níveis séricos do fator de crescimento semelhante à insulina tipo I (IGF-I) numa população adulta do Estado do Rio de Janeiro, Brasil / Reference ranges for serum levels of insulin-like growth Factor I (IGF-I) in an adult population of Rio de Janeiro State, Brazil

O nível sérico do Fator de crescimento semelhante à insulina tipo I (IGF-I) é fundamental para auxiliar no dignóstico e controle terapêutico dos transtornos relacionados à secreção do Hormônio de Crescimento (GH), bem como no diagnóstico e seguimento de outras doenças. Estabelecer valores de referência para as dosagens séricas de IGF-I por um ensaio imunoquimioluminométrico (ICMA), utilizando o sistema automatizado Immulite 2000/Diagnostic Products Corporation (DPC), e por um ensaio imunoradiométrico (IRMA), utilizando o kit comercial ACTIVE IGF-I/Diagnostic System Laboratories (DSL)-5600, numa população brasileira adulta da cidade do Rio de Janeiro. Este estudo, aprovado pelo Comitê de Ética do Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti, Rio de Janeiro, Brasil, incluiu amostras de 484 indivíduos saudáveis (251 homens e 233 mulheres) com idades entre 18 e 70 anos. As amostras foram estudadas por ICMA- Immulite 2000/DPC and IRMA- ACTIVE IGF-I/DSL-5600. Para análise dos dados foram utilizados modelos específicos para idade e sexo, após transformação dos dados de IGF-I. Foi observada uma lenta diminuição dos níveis de IGF-I com a idade usando ambos os ensaios. Os níveis de IGF-I foram signicativamente (p=0,0181) mais elevados em mulheres do que em homens, quando as amostras foram analisadas usando ICMA. Não houve diferença significativa dos níveis de IGF-I entre homens e mulheres quando as amostras foram analisadas usando IRMA. Este estudo estabeleceu valores de referência de IGF-I específicos para idade e sexo, determinados com o sistema automatizado ICMA-Immulite 2000/DPC, e valores de referência de IGF-I específicos para idade, determinados com o kit comercial IRMA- ACTIVE IGF-I/DSL-5600, em uma população adulta brasileira, da cidade do Rio de Janeiro

Serum level of insulin-like growth factor I (IGF-I) is fundamental in order to aid in the diagnosis and follow-up of growth hormone (GH)-related disorders, as well as in the diagnosis and follow-up of other diseases. The aim of this investigation was to determine reference values for IGF-I using an automated immunochemiluminometric assay (ICMA) system Immulite 2000/Diagnostic Products Corporation (DPC); and an immunoradiometric assay (IRMA), using the commercial kit ACTIVE IGF-I/Diagnostic System Laboratories (DSL)-5600, in an adult Brazilian population of Rio de Janeiro city. The study, approved by the Ethical Committee of the Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti, Rio de Janeiro, Brazil, included samples of blood taken from 484 healthy subjects (251men, 233 women) aged from 18 up to 70. The samples were analyzed by ICMA- Immulite 2000/DPC and IRMA- ACTIVE IGF-I/DSL-5600. For statistical analysis, age and sex-specific models were fitted after transformation of IGF-I values. In adulthood, a slow age-dependent decrease was found, using both assays. IGF-I in women were significantly (p=0,0181) higher than in men when samples were analayzed using ICMA.There was no significant difference between men and women IGF-I values when samples were analayzed using IRMA. The present study established age- and sex specific IGF-I reference values, determined with the automated system: ICMA-Immulite 2000/DPC and age-specific IGF-I reference values determined with the IRMA- ACTIVE IGF-I/DSL-5600, in an adult Brazilian population of Rio de Janeiro city

The hook effect in calcitonin immunoradiometric assay: a case report.

UNLABELLED: The hook effect, which has long been detected and documented for immunoradiometric assays (IRMA) such as those measuring prolactin or thyroglobulin, occurs when the serum antigen level is extremely high, thus inducing a bias in the methodology of measurement. RESULTS: We report the case of an 80-year-old man with confirmed medullary thyroid carcinoma (MTC). In the case reported here, the clinical status of the patient contrasts with his tumor antigen, serum calcitonin (CT), concentrations. The measured increased CT concentrations revealed the presence of a hook effect. This phenomenon occurs due to an excess of antigen during the one-step IRMA where the signal antibodies, bound to the non-captured antigens, are washed out during the measurement, inducing the loss of signal. Aiming to prevent the "hook effect", successive dilutions of the same sample of serum were done. CONCLUSIONS: Previous studies have shown when one-step IRMA reveals high concentrations of a tumor serum antigen (i.e. prolactin or thyroglobulin), a two-step IRMA or a systematic 1:10 dilution of the serum sample prevents the formation of the "hook effect". In our case report, the CT "hook effect" formation was prevented by performing serial dilutions of the serum sample.

Development of a bioassay system for human growth hormone determination with close correlation to immunoassay.

Serum growth hormone (GH) level is measured largely through immunoassays in clinical practice. However, a few cases with bioinactive and immunoreactive GH have also been reported. We describe here a new bioassay system for GH determination using the BaF/GM cell line, which proliferates in a dose-dependent manner on hGH addition; cell proliferation was blocked by anti-hGH antibody. This bioassay had the lowest detection limit (∼0.02 ng/ml) reported thus far and the highest specificity for GH. The bioassay results were compared with those of an immunoradiometric assay across 163 patient samples in various endocrine states. A close correlation (the ratio of bioactivity/immunoreactivity was 1.04 ± 0.33, mean ± SD) was observed between bioactivity and immunoreactivity in these samples. The newly developed system is a specific, sensitive, easy, and fast bioassay system for GH determination; we consider it useful for evaluating GH bioactivity in various endocrine states.

Reference values for serum levels of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in Korean children and adolescents.

OBJECTIVE: Measurements of serum insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3) are utilized in the diagnostic work-up and clinical management of children with growth disorders. We designed this study to establish the reference values of serum IGF-I and IGFBP-3 levels according to age, sex and pubertal stage in Korean children and adolescents. METHODS: For the study, 1378 healthy Korean children and adolescents aged 0 to 17 years (722 boys, 656 girls) were randomly selected. Blood samples were collected, and the stored sera were assayed for IGF-I and IGFBP-3 using immunoradiometric assay (IRMA, Immunotech). The R 2.8.1 program (Bell Laboratories) was used to generate reference percentile curves for IGF-I and IGFBP-3 according to age, sex, and pubertal stage RESULTS: Serum IGFBP-3 level was higher in girls compared to that in boys of the same ages throughout the pubertal period, whereas IGF-I was only higher for girls younger than 13 years of age. Serum levels of IGF-I and IGFBP-3 increased steadily with age in the prepubertal stage, followed by a progressive decline thereafter. Peak levels of serum IGF-I and IGFBP-3 were observed two years earlier in girls compared to those in boys (13 vs. 15 years of age, respectively). Serum IGF-I and IGFBP-3 concentrations were highest at Tanner stage IV in boys and girls, with a subsequent decline. CONCLUSIONS: Our reference value model based on age, sex, and pubertal stage can improve the diagnostic utility of IGF-1 and IGFBP-3 levels in the evaluation and management of Korean children and adolescents with growth disorders.

Multiplexed immunoassay of thyroglobulin autoantibodies in patients with differentiated thyroid carcinoma.

BACKGROUND: Thyroglobulin (Tg) antibodies (TgAb) screening is recommended in patients with differentiated thyroid carcinoma (DTC) to validate Tg measurements. We compared a new multiplexed and 2 conventional TgAb immunoassays in patients with DTC and healthy controls. METHODS: TgAb were measured by 2 conventional automated immunoassays and a multiplexed immunoassay in sera from 163 patients with DTC and 64 controls. RESULTS: No significant differences were found between the different assays when the suggested manufacturer cutoffs were used. The positive rate of multiplexed assay, but not of conventional assays, significantly increased when limits of detection were used as cutoff. CONCLUSION: Further technical evaluations are necessary to explain the very high rate of detectable TgAb values in both patients with DTC and controls by the multiplexed assay. For the moment, we suggest caution in using such methods in the current clinical practice.

Standardisation of a two-site PTH immunoradiometric assay using various solid phase formats.

BACKGROUND & OBJECTIVES: Estimation of parathyroid hormone (PTH) levels is important in the management of metabolic bone disorders. Here we describe a simple, sensitive and specific second generation immunoradiometric assay (IRMA) to detect intact PTH levels using different solid phase matrices. Different methods for immobilization of antibodies have also been evaluated. METHODS: Experiments were carried out with physical adsorption of antibodies, covalent coupling using 2 per cent glutaraldehyde and N,N`carbonyldiimidazole. In all cases, antibodies raised against C-terminal were used as solid phase agent. Detector antibodies were N terminal antibodies that were radio-iodinated with [125] I followed by gel purification. Several of the antibodies coupled to various solid phase matrices were incubated with PTH standards and the detector antibody as well as the commercially available tracer from DiaSorin kit to identify a suitable match pair. RESULTS: The best pair was polyclonal C-terminal PTH antibody along with the kit tracer from DiaSorin with regards to antibody coated to magnetic cellulose particles. Among the various antibodies and the solid phases evaluated, the best assay was obtained with the matched pair of antibodies (70×G67 and 70×G68) from Fitzgerald immobilized on polystyrene tubes. The polyclonal antibody against C-terminal PTH was chosen as the capture antibody and [125] I labelled polyclonal antibody against N-terminal PTH as the tracer. The sample values obtained in the antibody coated tubes were comparable to those obtained using a commercial kit. INTERPRETATION & CONCLUSIONS: The results indicated the feasibility of adopting this system for further development into a PTH IRMA for regular production as there is no indigenous kit available for intact PTH.

[Inappropriately undetectable thyroglobulin after exclusion of interference from thyroglobulin antibodies]. / Discordances entre différents immunodosages de la thyroglobuline en l'absence d'anticorps anti-thyroglobuline.

Thyroglobuline (Tg) is a large molecule of high molecular weight mainly indicated in monitoring differentiated thyroid cancer (DTC), its measurement remains difficult. We report the case of a patient who underwent total thyroidectomy for a poorly differentiated thyroid insular carcinoma. Despite several (131)I treatments, a progressive elevation of serum Tg is observed. A control performed in another laboratory using an immunoradiometric assay (IRMA Cisbio) returns an undetectable value (< 0,2 mg/L). A new sample was simultaneously sent to different laboratories. Three nonisotopic immunometric assays showed high values of Tg while the IRMA assay, considered the gold standard, gave a result below the detection threshold. The absence of Tg antibodies and of anti-mouse antibodies was confirmed. The IRMA Kit manufacturer agreed to carry out an expertise. After changing their detection antibody, the presence of a high Tg was demonstrated, in agreement with non-isotopic techniques. The expertise conclusion was a lack of detection by the IRMA Tg assay. This incident was notified to AFSSAPS by the manufacturer.

Evaluation of a direct prorenin assay making use of a monoclonal antibody directed against residues 32-39 of the prosegment.

BACKGROUND: Prorenin is an early marker of microvascular complications in diabetes. However, it can only be measured indirectly (following its conversion to renin), with a renin immunoradiometric assay (IRMA). Unfortunately, treatment with a renin inhibitor interferes with this assay, because renin inhibitors induce a conformational change in prorenin, thereby allowing its detection as renin. METHODS: We evaluated Molecular Innovation's new direct prorenin ELISA, which makes use of an antibody that recognizes an epitope near prorenin's putative cleavage site (R 43 L 44), thus no longer requiring prorenin activation. Plasma samples of 41 diabetic individuals treated with aliskiren (renin inhibitor) or irbesartan were tested. Semi-purified recombinant prorenin was used as standard, because the ELISA standard yielded approximately 10-fold lower values in the renin IRMA following its conversion to renin. RESULTS: The ELISA detected prorenin levels that were identical to those determined by the IRMA in untreated and irbesartan-treated individuals. Yet, it yielded higher prorenin levels in aliskiren-treated individuals. Aliskiren, at levels reached in plasma during treatment, did not interfere with the ELISA, but allowed the detection of up to 20-30% of prorenin as renin in the IRMA, thereby resulting in a significant overestimation of renin and an underestimation of prorenin. The ELISA rendered results within 2 h and did not require a pretreatment period of several days to convert prorenin to renin. CONCLUSION: The new direct assay allows rapid prorenin detection, is not hampered by aliskiren when used at clinically relevant doses, and might be used to identify diabetic patients developing retinopathy and/or nephropathy.

Immunoradiometric assay for human serum amyloid P component.

Human serum amyloid P component (SAP) is of increasing interest for its possible pathogenic role in amyloidosis and Alzheimer's disease, and as a therapeutic target in these conditions. We have developed and validated a robust and reproducible immunoradiometric assay (IRMA) for human SAP in serum, plasma and cerebrospinal fluid, and characterized the notable stability of human SAP immunoreactivity during storage of undiluted serum at 4°C and 37°C as well as frozen at -30°C. SAP values were also stable after repeated freeze thawing of highly diluted serum samples. The 100 fold dynamic range of the assay, 0.5-50 µg/L, encompassed all values seen in blood and cerebrospinal fluid, when tested at suitable dilutions, from both normal healthy individuals and patients, including subjects receiving the SAP-depleting drug, CPHPC. Furthermore by comparing the IRMA values in the presence and absence of calcium, the new assay revealed interference due to the binding of CPHPC by SAP, which was markedly enhanced in heparinized plasma. It is therefore essential that SAP assays in samples from patients on CPHPC be conducted in the absence of free calcium, in order to completely abrogate interference and determine the actual total SAP concentration. Estimates by the IRMA of SAP concentration in 49 serum samples from amyloidosis patients corresponded closely with those obtained by the established standard electro-immunoassay method and by a newly developed commercial ELISA kit (Hycult Biotechnology).

Novel electrochemiluminescence immunoassay exclusively for full-length parathyroid hormone during treatment with cinacalcet for secondary hyperparathyroidism.

A novel, electrochemiluminescence immunoassay that exclusively measures full-length parathyroid hormone (PTH), called Elecsys PTH (1-84) assay, is currently under development for clinical use. We measured serum PTH levels using this novel assay, as well as the Elecsys Intact PTH assay and the Whole PTH immunoradiometric assay, in 53 hemodialysis patients who participated in a 52-week clinical trial of cinacalcet. At baseline, serum PTH (1-84) levels measured with the Elecsys PTH (1-84) assay and those with the Whole PTH assay were comparable, and both values were significantly lower than Elecsys Intact PTH levels. After 52 weeks of cinacalcet treatment, Elecsys PTH (1-84) levels and Whole PTH levels decreased significantly by 56% and 60% from baseline, respectively. These results indicate that the Elecsys PTH (1-84) assay provides comparable data to the Whole PTH assay for monitoring parathyroid function in patients receiving hemodialysis. Introduction of this novel automated immunoassay would provide more widespread measurements of full-length PTH (1-84) in clinical practice.

The clinical utility of two renin mass methods to detect primary hyperaldosteronism compared with renin activity.

BACKGROUND: Primary hyperaldosteronism (PHA) is characterized by a raised plasma aldosterone concentration (PAC) with suppressed plasma renin activity (PRA). We evaluated two renin mass methods for PHA detection compared with the PAC:PRA ratio. METHODS: Samples from patients attending a specialist hypertensive clinic were analysed by Liaison automated chemiluminescent immunoassay and Diagnostic Systems Laboratories (DSL) immunoradiometric assay (IRMA) for renin mass; I(-125) radioimmunoassay of angiotensin I generated from endogenous angiotensinogen for PRA; Siemens Coat-a-count radioimmunoassay for PAC. Subjects included those on ß-blockers which suppress renin, causing an equivalent biochemical picture to PHA. Aldosterone/renin ratios (ARR) were calculated for PRA, DSL and Liaison methods. The first 100 subjects were used to identify cut-off ratios ensuring maximum specificity at 100% sensitivity for PHA detection. This cut-off was retested in a subsequent population (n = 43). RESULTS: A Liaison renin of 5 ng/L separated PRAs of ≤0.5 from ≥0.6 pmol/mL/h. The DSL method had greater scatter. In population 1 (18 PHA), cut-off ratios of >118 pmol/ng (Liaison) and >60 pmol/ng (DSL) gave specificities of 58.5% and 61%, respectively, with 100% sensitivity. If criteria for PHA included PAC ≥350 pmol/L and excluded ß-blocked subjects, specificity increased to 95.1% and 90% for Liaison and DSL, respectively. In population 2 (6 PHA), specificities for Liaison and DSL ARRs were 86.4% and 78.3%. Using the ratio with PAC and ß-blocker criteria, specificities for Liaison and DSL were 97.3% and 86.5%, respectively. CONCLUSIONS: The Liaison ARR used with PAC and ß-blocker criteria provided an automatable alternative to identify the same patients as the PAC:PRA ratio.