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.

Comparison of Thyroglobulin Concentrations Measured by Two Immunoradiometric Assay

Circulating thyroglobulin measurements is a highly specific test in the management of patients affected by differentiated thyroid cancer after total thyroidectomy, followed by radioiodine ablation. The aim of our study was to compare two thyroglobulinimmunoradiometric assays (INEP, Serbia and Cisbio Bioassays, France). Study included 42 patients of both genders with DTC. The subjects were on suppres¬sive doses of levothyroxine and followed up. Results showed concordance between the two assay methods for determining serum thyroglobulin for 39 (92.85%) patients. Statistical analysis showed that there was a direct correlation between two IRMA tests, with a positive correlation coefficient r=0.613 (p 0.05). We concluded that there is good agreement between the two thyroglobulin assays compared in this study.

Megalin

Megalin is an endocytic receptor contributing to protein reabsorption. Impaired expression or trafficking of megalin increases urinary renin and allowed the detection of prorenin, which normally is absent in urine. Here, we investigated (pro)renin uptake by megalin, using both conditionally immortalized proximal tubule epithelial cells and Brown Norway Rat yolk sac cells (BN16). To distinguish binding and internalization, cells were incubated with recombinant human (pro)renin at 4°C and 37°C, respectively. (Pro)renin levels were assessed by immunoradiometric assay. At 4°C, BN16 cells bound 3× more prorenin than renin, suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3- to 4-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. No such differences were observed in conditionally immortalized proximal tubule epithelial cells, and M6P (mannose-6-phosphate) greatly reduced conditionally immortalized proximal tubule epithelial cells (pro)renin uptake, suggesting that these cells accumulate (pro)renin largely via M6P receptors. M6P did not affect (pro)renin uptake in BN16 cells. Yet, inhibiting megalin expression with siRNA greatly reduced (pro)renin binding and internalization by BN16 cells. Furthermore, treating BN16 cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin, while deleting the (pro)renin receptor affected the latter only. Exposing prorenin’s prosegment with the renin inhibitor aliskiren dramatically increased prorenin binding, while after prosegment cleavage with trypsin prorenin binding was identical to that of renin. In conclusion, megalin might function as an endocytic receptor for (pro)renin and displays a preference for prorenin. Megalin-mediated endocytosis requires the (pro)renin receptor.

Optimization of Thyroglobulin Coated Tube for Thyroglobulin IRMA Kit

<p>Immunoradiometric assay (IRMA) is a method of analysis based on immunological reactions of antigens-antibodies binding. This highly specific and sensitive method was used for in vitro diagnosis in small quantity of sample. Center for Radioisotope and Radiopharmaceutical Technology, BATAN has developed Thyroglobulin IRMA Kit using coated tube method that can determine thyroglobulin levels in microgram quantities. Coated tube was made with immobilisation of anti thyroglobulin into polistyrene tube. Development of IRMA kit performed through several steps including: optimization component of kit, optimization assay and kit validation. Optimization of coated tube involved selection and volume of solvent, using blocking and non-blocking agent, and volume of blocking agent. The optimum condition for coated tubes was found to be using 0.1M phosphate buffer pH 7.4 with coating volume of 500 μL, 3% BSA in 500 μL blocking agent 0.1M phosphate buffer pH 7.4, with maximum binding and non-specific binding (NSB) of 60.58 and 1.40%, respectively. The optimized coated tube was found to be stable up to 4 weeks.</p>