Single step, direct fluorescence immunoassays based on metal enhanced fluorescence (MEF-FIA) applicable as micro plate-, array-, multiplexing- or point of care-format.

Although Enzyme Linked Immuno Sorbent Assay (ELISA) technology is approaching it's 45th year of existence since first described in 1971, it is still the main diagnostic tool in clinical research and routine diagnostics. However, despite its broad usage it suffers from some drawbacks, limiting its use especially in more advanced assay formats like multiplexing platforms, point of care devices or protein arrays. Those limitations result from the need for an enzyme label, a soluble enzyme substrate, washing steps (multiplexing, point care, arrays) and in some cases also insufficient sensitivity, because the majority of circulating proteins and thus potential biomarkers may be found in lower sub-picomolar concentrations. We hereby present a new assay platform based on metal enhanced fluorescence (MEF), that remedies these problems since it eliminates the need for washing steps, for using enzyme labels and allows detection of analytes down to sub-picomolar concentrations. In addition this technology is fully compatible to standard fluorescence reader equipment as it is found in many laboratories nowadays. Since our present work is focused on single biomarker evaluation, we chose a 96 well plate format for convenience, but any other formate like antibody arrays, strip-like point of care devices etc. is feasible too.

Serum level of the phosphaturic factor FGF23 is associated with abdominal aortic calcification in men: the STRAMBO study.

CONTEXT: Calcification inhibitor deficiencies, mineral imbalance, and phenotypic transformation of vascular cells to osteogenic cells initiate and sustain vascular calcification. Fibroblast growth factor-23 (FGF23) is a key molecule regulating mineral homeostasis. OBJECTIVE: Our objective was to assess the association of serum FGF23 levels with mineral metabolism parameters and abdominal aortic calcification (AAC) in men. DESIGN: This was a cross-sectional analysis in the STRAMBO cohort. SETTING: Men holding a private health insurance cover with Mutuelle de Travailleurs de la Région Lyonnaise were included in the study. PARTICIPANTS: Participants included male volunteers aged 20-87 (n = 1130). INTERVENTIONS: Nonfasting blood collection was done. AAC was semiquantitatively assessed from vertebral fracture assessment scans obtained using dual-energy x-ray absorptiometry. MAIN OUTCOME MEASURES: We evaluated the association between FGF23 concentration and AAC severity in men. RESULTS: In 350 men aged 60 yr or younger, FGF23 levels decreased with age (r = -0.21; P < 0.001) but were not associated with any other parameter. In 780 men aged over 60 yr, serum FGF23 correlated with age (r = 0.37; P < 0.001) and, after adjustment for confounders, with glomerular filtration rate (r = -0.31; P < 0.001) and PTH levels (r = 0.25; P < 0.001). After adjustment for confounders, self-reported ischemic heart disease, diabetes mellitus as well as higher concentrations of C-reactive protein and osteoprotegerin were all associated with higher FGF23 levels. After adjustment for confounders, subjects in the highest FGF23 quartile had higher prevalence of severe AAC compared with the three lower quartiles combined (odds ratio = 1.88; 95% confidence interval = 1.22-2.85; P < 0.005). CONCLUSIONS: In healthy older men, circulating FGF23 is associated with parameters of mineral metabolism, including bone metabolism-regulating cytokines, and with severe AAC independent of traditional risk factors.

Cortical bone status is associated with serum osteoprotegerin concentration in men: the STRAMBO study.

CONTEXT: Osteoprotegerin (OPG) is an inhibitor of bone resorption, but its relationship to bone microarchitecture remains unclear. OBJECTIVE: Our objective was to study the relationship between OPG concentration and bone microarchitecture in men. DESIGN, SETTING, AND PARTICIPANTS: We conducted a cross-sectional study of a population-based cohort of 1149 men aged 20-87 yr. INTERVENTIONS: We assessed bone microarchitecture at the distal radius and tibia by high-resolution peripheral quantitative computed tomography (XtremeCT Scanco) and measured serum OPG concentration and bone turnover markers: osteocalcin, bone-specific alkaline phosphatase, N-terminal extension type I collagen propeptide, C-terminal type 1 collagen telopeptide, and urinary deoxypyridinoline. MAIN OUTCOME MEASURES: Differences were assessed in bone microarchitectural parameters across the OPG quartiles in the models adjusted for age, weight, height, physical activity, ischemic heart disease, diabetes mellitus, calcium intake, serum levels of free testosterone, bioavailable 17ß-estradiol, PTH, 25-hydroxycholecalciferol, and creatinine. RESULTS: After adjustment for the confounders, men in the highest (fourth) quartile of OPG levels (>4.55 pmol/liter) had higher total cross-sectional area and trabecular area at the distal radius and distal tibia (3.3-6.0%, P < 0.05). At both skeletal sites, the highest OPG quartile was associated with lower cortical thickness (8.2%, P < 0.001, and 3.7%, P < 0.05) and volumetric bone mineral density (vBMD, 2.7%, P < 0.001, and 1.6%, P < 0.005) compared with the three lower quartiles combined. Associations of OPG level with trabecular vBMD, number, thickness, and distribution were not significant. Men in the fourth OPG quartile had higher levels of bone resorption markers (11.8-13.1%, P < 0.01-0.001). CONCLUSIONS: Men with higher serum OPG concentration had lower cortical thickness and vBMD, probably due to accelerated endo- and intracortical bone turnover, but higher cross-sectional area possibly due to periosteal apposition.

Association of plasma N-terminal pro-B-type natriuretic peptide concentration with mitral regurgitation severity and outcome in dogs with asymptomatic degenerative mitral valve disease.

BACKGROUND: The clinical outcome of dogs affected by degenerative mitral valve disease (MVD) without overt clinical signs is still poorly defined, and criteria for identification of animals that are at a higher risk of early decompensation have not yet been determined. HYPOTHESIS: N-terminal pro-B-type natriuretic peptide plasma concentration (NT-proBNP) is correlated with mitral regurgitation (MR) severity and can predict disease progression in dogs with asymptomatic MVD. ANIMALS: Seventy-two dogs with asymptomatic MVD, with or without heart enlargement (International Small Animal Cardiac Health Council: ISACHC classes 1a and 1b), and a control group of 22 dogs were prospectively recruited. METHODS: Severity of MR was quantitatively assessed from the regurgitation fraction (RF) by the proximal isovelocity surface area method. Consequences of MR were evaluated from measurements of the left atrium/aorta ratio (LA/Ao), fractional shortening (FS), end-diastolic and end-systolic left ventricular volumes indexed to body surface area (EDVI and ESVI). The relevance of these echo-Doppler indices and NT-proBNP for prediction of outcome at 12 months was studied. RESULTS: A significant correlation was found between NT-proBNP and RF, LA/Ao, FS, and EDVI (P < .05). NT-proBNP was higher in dogs with MVD (ISACHC classes 1a and 1b) compared with the control group (P= .025 and < .001, respectively). The difference was not significant when only dogs from ISACHC class 1a with RF < 30% were considered. Lastly, NT-proBNP was higher in dogs that underwent MVD decompensation at 12 months (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE: NT-proBNP is correlated with MVD severity and prognosis in dogs with asymptomatic MVD.

Serum levels of cathepsin K decrease with age in both women and men.

Bone turnover increases with age. In a previous study, we reported on bone metabolism in young and elderly women and men. The aim of the present investigation was to evaluate potential age- and gender-related changes in cathepsin K, a cysteine protease that plays an important role in the degradation of the organic matrix of bone. Twenty-five healthy premenopausal women, 24 young healthy men, 26 elderly women, and 25 elderly men participated in the study. Elderly women and men had significantly lower cathepsin K levels than younger ones. In both men and women, serum levels of cathepsin K were negatively correlated with age. In men there was a statistically significant negative correlation between serum levels of cathepsin K and osteoprotegerin, which inhibits osteoclast differentiation and activation. No association was found between serum levels of cathepsin K and bone-specific alkaline phosphatase, osteocalcin, or 25-hydroxy vitamin D. Thus, the age-related increase in OPG, which markedly inhibits the expression of cathepsin K, may also reduce serum levels of cathepsin K. Despite the age-related increase in bone resorption, this study shows lower cathepsin K values in elderly women and men than in younger subjects. It might be speculated that a different enzyme could compensate for the decline in cathepsin K during old age.

Osteoprotegerin and the receptor activator of NF-kappa B ligand in the serum and synovial fluid. A comparison of patients with longstanding rheumatoid arthritis and osteoarthritis.

We examined OPG and soluble RANKL in the serum (sOPG, sRANKL) and synovial fluid (synOPG, synRANKL) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). OPG and RANKL were measured in 85 patients (44 with RA, 41 patients with OA) in serum and synovial fluid as well. For measuring of OPG and RANKL ELISA tests were used. The results of OPG and RANKL were compared with clinical and radiological scores. We found a negative correlation for OPG and RANKL in synovial fluids: not only for the whole group of patients (P < 0.003, r = -0.32), but also for the subgroups (RA: P < 0.04, r = -0.28, OA: P < 0.002, r = -0.54). SRANKL and synRANKL were positively correlated in the whole group (P < 0.01, r = 0.25) and in the OA group (P < 0.02, r = 0.35); the RA group was showing a trend (P < 0.063, r = 0.24), however. Serum OPG was lower in RA, synOPG higher in OA. The difference between the two patient groups was only significant for synOPG (P < 0.03, r = 0.056), but not for sOPG (P < 0.09, r = 0.19), sRANKL (P < 0.43, r = 0.85) or synRANKL (P < 0.11, r = 0.22). The synOPG:synRANKL ratio was significantly correlated with the Larsen score (P < 0.004, r = 0.38). Synovial OPG is significantly decreased in rheumatoid joints, whereby synovial RANKL is increased. Lower synOPG could reflect a lower protective effect on bone, thus leading to an earlier and more pronounced bone destruction in RA. However, the effect of different mediators for joint destruction in RA and OA seems not to be important to the pathophysiological changes in the joints. The upregulation of serum OPG might be the result of the inflammation; in contrast, an upregulation of RANKL could not be found in the serum of patients with RA and OA.

Direct monitoring of molecular recognition processes using fluorescence enhancement at colloid-coated microplates.

Direct monitoring of recognition processes at the molecular level is a valuable tool for studying reaction kinetics to assess affinity constants (e.g. drugs to receptors) and for designing rapid single step immunoassays. Methods currently used to gain information about binding processes predominantly depend on surface plasmon resonance. These systems use excitation with coherent light in attenuated total reflection geometry to obtain discrimination between surface-bound and free molecules in solution. Therefore labeling of the compounds is not necessary, but due to the complexity of the measuring setup the method is rather costly. In this contribution we present a simple method for performing kinetic single step biorecognition assays with fluorophore labeled compounds using the fluorescence enhancement properties of surface bound silver colloids. Silver colloids are bound to standard microplates via silanization of the plastic surface. Fluorophores close to this colloid coated surface show a significant gain in fluorescence compared to fluorophores farther away in the bulk solution. Therefore discrimination between surface bound and free fluorophores is possible and the binding of, for example, fluorophore labeled antibodies to antigens immobilized on the colloid surface results in increasing fluorescence intensity. Utilization of standard microplates makes this method fully compatible with conventional microplate processing and reading devices. Neither excitation with coherent laser light nor ATR geometry is required, the measurement is performed in a standard fluorescence microplate reader in front face geometry with a xenon flash lamp as excitation source. Methods for the preparation of colloid-coated microplates and fluorescence-enhanced biorecognition assays are presented. Additionally the dependence of the system performance on the structure and properties of the metal colloid coated surface is described. A two-component biorecognition model system shows a detection limit in the subnanomolar range. The ease of colloid-surface preparation and the high sensitivity makes fluorescence enhancement at colloid-coated microplates a valuable tool for studying reaction kinetics and performing rapid single-step immunoassays.

Sandwich ELISA for proANP 1-98 facilitates investigation of left ventricular dysfunction.

Proatrial natriuretic peptides are proposed to be sensitive and specific markers for various stages of heart deficiency. Here we present a rapid and specific sandwich immunoassay for the measurement of proANP 1-98 in biological fluids like plasma, serum urine. No sample preparation prior to the assay is necessary. Polyclonal antibodies specific for distinct epitopes of proANP 1-98, predicted to be highly immunogenic, were raised in sheep and purified by immunoaffinity chromatography prior to use in the assay. Antigen binding sites of the antibodies were determined by epitope mapping with a set of peptide fragments. The capture antibody, specific for proANP 16-23, is coated directly onto microtiter plates. Recombinant proANP 1-98 is used as a standard. The biotinylated detection antibody, specific for proANP 80-88, is incubated simultaneously with 20microl of sample for 150 min. Signal is generated with a streptavidin-HRPO-conjugate and TMB as substrate. The detection limit of the assay is 50 pmol/l; intraassay CVs are below 5%, interassay CVs are lower than 10%. Dilution curves show good linearity, recovery of synthetic peptide ranged between 85% and 91%. Reference values from healthy volunteers range from 0 to 2000 pmol/l in human plasma. We conclude this assay is a easy to handle, quick and reliable method for routine measurement of proANP 1-98 and the presented manuscript describes the procedure and performance of this ELISA in detail.

Enzyme immunoassays for fragments (epitopes) of human proatrial natriuretic peptides.

Several peptides derived from the N-terminal sequence of pro-atrial natriuretic peptide (proANP) have been tested successfully as markers of heart disease. We have developed specific and sensitive competitive enzyme immunoassays for fragments [1-30] and [31-67] of proANP. Antisera were raised in sheep against synthetic peptides predicted to be highly immunogenic. Binding specificity was determined by epitope mapping. Microtiter plates were coated with antibody specific for the Fc region of sheep IgG to capture the affinity-purified specific anti-proANP antibodies in an oriented and reproducible form. Synthetic proANP calibrators or diluted samples were incubated simultaneously with biotinylated peptide and binding was quantitated using streptavidin-peroxidase and TMB. Immunoreactive proANP could be measured in diluted plasma, serum and urine. The detection limits of the proANP[1-30] and proANP[31-67] assays were 2.5 and 10 pmol/l respectively. The linearity of samples diluted beyond the recommended assay conditions was good. Recoveries of added standard peptides ranged from 102 to 112%. Circulating concentrations of immunoreactive proANP in 115 healthy subjects ranged from 0.11 to 0.47 nmol/l proANP[1-30] and 0.18 to 0.79 nmol/l proANP[31-67]. In patients with cardiac disease, proANP levels were increased significantly. The reference interval of proANP[31-67] in urine was 0.09 to 1.7 nmol/l, several-fold higher than proANP[1-30] (

Novel method for coupling of poly(ethyleneglycol) to carboxylic acid moieties of proteins.

Lack of toxicity, excellent solubility and superb biocompatibility make polyethylene glycol (PEG) one of the most popular modifiers of biologicals. The most common method for attachment of PEG is based on modification of amino groups of proteins with methoxy- or succinimide-derivatives of PEG. In the case of proteins with amino groups important for biological activity, this modification can lead to inactivation of proteins. A new strategy for covalent attachment of PEG to carboxylic groups of proteins using O,O-bis-(2-aminopropyl)polyethylene glycol and carbodiimide/N-hydroxysuccinimide-mediated reaction was developed. The reaction is carried out under mild aqueous conditions. The attached PEG serves as a hydrophilic spacer for further bioconjungation with biomolecules and haptens. Lipase from Candida rugosa was used as a model protein. Characteristic data of the modified protein such as activity, isoelectric points and stability were compared with that of the unmodified protein.

Photostructurized electrochemical biosensors for bioreactor control and measurement in body fluids.

This article details a new type of biosensor for the simultaneous analysis of glucose, glutamate and glutamine in complex biological fluids like fermentation broths and blood. Simultaneous analysis was made possible by the application of different enzyme layers onto different electrodes of one photostructurized sensor. Photostructuring was done by means of a new developed photopolymer. Preparation of the photopolymer and the enzyme layers as well as the characterization of the sensors thus constructed with respect to linearity, response time and sensitivity are described.