Background: Fibroblast growth factor-23 (FGF-23) is a bone secreted hormone that regulates phosphate homeostasis and calcitriol levels. FGF-23 concentrations are elevated in chronic kidney disease (CKD). oncogenic osteomalcia and a number of rare hereditary disorders. Studies systematically evaluating the pre-analytical stability of intact FGF-23 are lacking.Methods: The stability of FGF-23 was assessed in timed experiments using blood taken into K-2-EDTA plasma specimen tubes from a group of healthy participants and from a group with mild-to-moderate CKD. We evaluated the use of aprotinin, a serine protease inhibitor, and a commercially available protease inhibitor cocktail to preserve intact FGF-23 after blood collection. FGF-23 measurements were made using both intact and C-terminal assays.Results: Both whole blood and separated sample studies demonstrated a rapid loss of intact FGF-23 within 2 h, while concentrations increased using the C-terminal assay. The addition of protease inhibitor cocktail stabilised FGF-23 concentrations for 4 h after blood collection. Intact and C-terminal assay FGF-23 measurements showed poor correlation in both healthy and CKD cohorts.Conclusion: K-2-EDTA plasma samples, even when promptly separated, are unsuitable for measurement of FGF-23 unless stabilised with a protease inhibitor cocktail. (C) 2011 Elsevier B.V. All rights reserved.

Background: Fetuin-A is a circulating inhibitor of ectopic calcification. Low plasma levels have been associated in some studies with increased vascular calcification, aortic stiffness and mortality in patients with Chronic Kidney Disease (CKD). However, there are other studies examining the association of fetuin-A with vascular parameters and mortality, which do not show these associations. These conflicting data may be explained by methodological differences.Methods: We compared plasma fetuin-A measurements made with two widely-used commercial fetuin-A ELISA kits (Biovendor, Modrice, Czech Republic: Epitope Diagnostics Inc., San Diego, US) in samples from patients with and without CKD. We evaluated the effect of differences in fetuin-A glycosylation status on assay specificity.Results: Deming regression analysis showed poor agreement between methods (for CKD cohort: y = -0.05 + 2.52x, S(y|x) = 0.099 g/L, R(2) = 0.694). The Epitope Diagnostics kit demonstrated significant positive bias and greater specificity for deglycosylated fetuin-A relative to the Biovendor assay.Conclusion: The apparently contradictory nature of reports of the association of fetuin-A with biological variables may reflect differences in the specificity of different ELISA methods for glycosylated plasma fetuin-A. (C) 2010 Elsevier B.V. All rights reserved.

There is a disproportionate burden of vascular disease in patients with chronic kidney disease (CKD). Both aortic stiffness, as measured by carotid-femoral pulse wave velocity (C-F PWV), and deficiency in inhibitors of vascular calcification, such as Fetuin-A, have been implicated in the higher rates of cardiovascular mortality observed in this population. We sought to determine whether Fetuin-A concentration was inversely associated with progressive aortic stiffness. 54 stable outpatients enrolled in a prospective cohort study of cardiovascular risk in CKD stages 3 and 4 underwent measurement of C-F PWV using Complior under standardized conditions at baseline and12months. Baseline plasma Fetuin-A concentration was determined using the Biovendor ELISA kit. The population was aged 68.0±10.4years, 80

A method for the simultaneous analysis of asymmetric dimethylarginine, symmetric dimethylarginine, monomethylarginine and arginine in human plasma and urine, with short analysis time and isotopic internal standardisation for each analyte is described. The method requires neither sample derivatisation nor the need for chromatographic separation of analytes. The method described shows good precision and accuracy and is suited for both research purposes and implementation in the busy, routine clinical laboratory. In addition the synthesis and utilisation of isotopically labelled symmetric dimethylarginine and monomethylarginine is described for the first time, avoiding the use of surrogates such as homoarginine for internal standardisation.

Background: A method utilizing liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) has been developed and evaluated for the determination of total homocysteine, cysteine and methionine in plasma and urine. The simultaneous measurement of homocysteine and methionine concentrations may help explain the underlying mechanism responsible for hyperhomo-cysteinaemia. Methods: Samples were prepared by simple protein precipitation after reduction of disulphides by dithiothreitol. Reduced analyte signal caused by ionization suppression effects, seen with plasma samples, was compensated for with matrix-matched standards, and the use of isotopically labelled internal standards. Recovery for each analyte was better than 94%. Results: Concentrations of plasma homocysteine determined by LC-MS/MS were compared with those obtained by two automated commercially available FDA-approved procedures: (i) high-performance liquid chromatography (HPLC) with pre-column derivatization and fluorescence detection and (ii) by fluorescence polarization immunoassay (FPIA). Agreement with the LC-MS/MS method is given by the Deming regression equations LC-MS/MS = 1.062 HPLC-0.01 and LC-MS/MS = 1.104 FPIA-0.43. Conclusion: Low reagent costs together with the relative simplicity of sample preparation make the LC-MS/MS method well suited, not only for research work but also in those laboratories with a tandem mass spectrometer, for the measurement of routine clinical samples.

In this flow-Injection system for direct determination of lithium in serum by atomic absorption spectroscopy, the 10-rL sample is manually injected into a continuously flowing non-segmented stream of de-ionized water, which is pumped, via a dispersion tube, to the spectrometer’s nebulizer. Controlled dispersion of the sample zone, before It is introduced into the nebulizer, produces the required sample dilution. Effects of varying the length of the dispersion tube, the flow rate, and the sample size were studied. Analytical readout is obtained, in the form of transient peaks, 5 s after sample injection. ft is necessary to include physiological concentrations of sodium and potassium in the standards because each of these cations enhances the lithium absorbance signal. Analytical recovery (98.5 to 101 %) andCV (about 2%) are good, and resufts compare well with those obtained by aspiration of prediluted samples (n = 121, r = 0.99).

A miniaturized multi ion-sensor (IS) array based on coated silver-stripe (CSS) technique is described. The K+-CSS-IS, Na +-CSS-IS, and Ca2+CSS-IS studied so far show very good coated-wire electrode properties comparable to conventional ion-selective electrodes (ISE); the main characteristics are the near-Nernstian response over clinical range, a better selectivity and short response times. A very thin (300 nm), wide (1 mm), and long (70 mm) with circular tip area of 7 mm2 silver metallic layer deposited on a glass substrate using chemical vapor deposition. It is shown that this approach could be used for the development of a cost-effective miniaturized multi-sensor array systems for clinical application for the determination of the ion activity or concentration by zero-current potentiometry method

The use of immunogold-silver enhancement has its roots in immunohistocytochemistry, in which it has gained widespread use as a specific and sensitive staining tool. More recently this technology has been applied to such other areas as immunoassays. A novel immunoassay- silver enhanced gold-labeled immunosorbent assay (SEGLISA) - is described for the detection of specific analytes in human blood. This non-enzymic, non-isotopic immunoassay has been developed from established microtitration technology incorporating the knowledge and use of immunogold-silver staining. This technique is being developed in several formats, ranging from indirect assays for antibody detection to competitive assays for detection of lipoproteins.We describe a sandwich-type SEGLISA for the measurement of human growth hormone. It has been compared with an equivalent commercially available enzyme-linked immunosorbent assay (ELISA). This novel use of immunogold-silver staining allows an immunoassay that is quick and easy to carry out, is relatively inexpensive, and doesn't suffer from many of the drawbacks associated with immunoassays incorporating enzymic or radioactive labels. The result is permanent and can be read either visually, or quantitatively on a conventional microplate reader.

A competitive silver-enhanced gold-labelled immunoassay has been developed for the direct measurement of low density lipoprotein (LDL) in whole blood. Immobilized LDL and sample LDL compete for added antibody. Quantitation of the bound antibody/antigen complex is achieved by the addition of gold-labelled antiimmunoglobulin G followed by enhancement of absorbance by addition of silver ions. Whole-blood samples from fasting patients were assayed directly for LDL by the procedure and the corresponding plasma samples were assayed for total cholesterol, high density lipoprotein and triglycerides followed by the indirect calculation of LDL cholesterol. The correlation between the two methods was good (r = 0·82) and the SEGLISA exhibited good precision.

We describe a new immunoassay for the detection of antibodies to the human immunodeficiency virus. The method is based on a silver enhanced gold-labelled immunosorbent assay (SEGLISA). Test sera are incubated in microtitre wells on which antigens have been coated. If present in the test sera, antibodies to the human immunodeficiency virus bind to the solid-phase antigens. Bound antibodies are quantitated with anti-human immunoglobulin labelled with gold. Positive specimens produce a faint pink deposit which is better visualised by silver enhancement which gives an intense black colour. The intensity of the colour is proportional to the bound antibody concentration. All the reagents are stable and the silver enhancement takes place under ambient light conditions. The assay has many of the advantages of micro enzyme-linked immunosorbent assays but does not suffer from the drawbacks associated with the use of an enzyme label. It requires fewer manipulations and is quicker to carry out than an equivalent enzyme-linked test. As the silver layer is permanent dried wells may be stored for future reading or checking.

A silver-enhanced gold-labelled immunosorbent assay (SEGLISA) for the detection of antibodies to the immunodeficiency virus (HIV) in whole-blood samples is described. This new non-isotopic, non-enzymic immunoassay incorporates use of solid phase viral antigens which bind any HIV antibodies present in the test sample. The antigen/antibody complex is then detected by gold-labelled anti human immunoglobulin G (IgG) followed by silver amplification. We found that whole blood samples give false positives when using a horseradish peroxidase label, whereas the SEGLISA correctly identified 50 HIV antibody positive samples and 50 HIV antibody negative samples when using whole blood. The use of whole blood collected on filter paper is also described. The SEGLISA has good precision (CV = 7.5%) and sensitivity.

Attempts to increase the sensitivity of fluorescein-based fluorescence immunoassays by using multiple labelling have generally been unsuccessful because of concentration quenching. We have labelled antibodies to human immunoglobulin G with multiple fluorescein fluorophores attached by means of a disulphide linkage: this linkage can be rapidly and easily broken by treatment with dithiothreitol, allowing fluorescein to be released from the antibody and measured in free solution. Application of this technique to a fluorescence labelled immunosorbent assay for antibodies to the human immunodeficiency virus gave an approximately 20-fold increase in signal compared with an equivalent assay using fluorescein isothiocyanate.

Human immunoglobulin G, human serum albumin and testosterone were labelled with the 4-aminosalicylic acid derivative of diethylenetriaminepentaacetic acid complexed with terbium ions. An exceptionally large amount of label, of the order of a few hundred moles of complex per mole of analyte, could be conjugated to the compounds tested by the use of poly-L-lysine. Self-quenching appears to be minimal, even with this high local concentration of fluorophores. The tracers were stable at 4 degrees C, and gave competitive calibration graphs at physiological concentrations.

M. Valcárcel, M.D. L. de Castro, M. Harriott, D. T. Burns, J. N. Miller, J. Tyson and B. F. Rocks, Anal. Proc., 1989, 26, 313 DOI: 10.1039/AP9892600313

A series of aminoaromatic derivatives of diethylenetriaminepentaacetic acid (DTPA) has been prepared, in a search for terbium chelates suitable for use in fluorescence immunoassay. Most of the derivatives contained heterocyclic rings with at least one nitrogen atom. The fluorescence properties of the terbium chelate of each compound were examined. Although none of the products proved suitable for use in immunoassays, the terbium chelate formed from the product of the reaction between DTPA anhydride and cytosine (4-amino-2-hydroxypyrimidine) was particularly fluorescent and had a long fluorescence lifetime. It was unstable, however, in aqueous solution below pH 9. The fluorescence properties of some europium complexes were also examined.