Immunochemical Determination of Hemoglobin-A1c Utilizing a Glycated Peptide as Hemoglobin-A1c Analogon

We describe the development of a heterogeneous affinity-matrix based immunoassay for the determination of HbA1c which could in future be applicable to analytical devices. We developed an immunoenzymometric assay (IEMA) where the glycated pentapeptide Val-His-Leu-Thr-Pro (VHLTP) as HbA1c analogon is immobilized either to the surface of a microtiter plate by adsorption or to an amino-modified cellulose membrane by covalent linkage. The immobilized analogon competes together with the HbA1c in the sample for the antigen binding sites of the anti-HbA1c antibodies. Glucose oxidase-labeled antibodies have been used to indicate the antigen-antibody reaction indirectly and enzyme activity was detected optically. Calibration curves for HbA1c were obtained with a linear range of 1,5-10 µg ml-1 (23-155 nM). In a mixture of non-glycated and glycated hemoglobin with a total hemoglobin concentration of 30 µg ml-1 (465 nM) a linear range was obtained between 5-50 % HbA1c. Since the glycated peptide shows a high affinity for the anti-HbA1c antibody (Kd = 0,3 nM) only a low contact time (< 1 min) between the modified solid support and the preincubated mixture of HbA1c and anti-HbA1c antibody was required. Regeneration of the affinity-matrix was carried out with 10 mM HCl for 3 min without loss of antibody binding activity

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

<p>Abstract</p> <p>Background</p> <p>As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.</p> <p>Results</p> <p>The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit <it>in vitro </it>infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.</p> <p>Conclusion</p> <p>The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.</p

Tear proteome analysis in ocular surface diseases using label-free LC-MS/MS and multiplexedmicroarray biomarker validation

We analyzed the tear film proteome of patients with dry eye (DE), meibomian gland dysfunction (MGD), and normal volunteers (CT). Tear samples were collected from 70 individuals. Of these, 37 samples were analyzed using spectral-counting-based LC-MS/MS label-free quantitation, and 33 samples were evaluated in the validation of candidate biomarkers employing customized antibody microarray assays. Comparative analysis of tear protein profiles revealed differences in the expression levels of 26 proteins, including protein S100A6, annexin A1, cystatin-S, thioredoxin, phospholipase A2, antileukoproteinase, and lactoperoxidase. Antibody microarray validation of CST4, S100A6, and MMP9 confirmed the accuracy of previously reported ELISA assays, with an area under ROC curve (AUC) of 87.5%. Clinical endpoint analysis showed a good correlation between biomarker concentrations and clinical parameters. In conclusion, different sets of proteins differentiate between the groups. Apolipoprotein D, S100A6, S100A8, and ceruloplasmin discriminate best between the DE and CT groups. The differences between antileukoproteinase, phospholipase A2, and lactoperoxidase levels allow the distinction between MGD and DE, and the changes in the levels of annexin A1, clusterin, and alpha-1-acid glycoprotein 1, between MGD and CT groups. The functional network analysis revealed the main biological processes that should be examined to identify new candidate biomarkers and therapeutic targets

Development of a bifunctional sensor using haptenized acetylcholinesterase and application for the detection of cocaine and organophosphates

We developed a dual piezoelectric/amperometric sensor for the detection of two unrelated analytes in one experiment that uses propidium to anchor acetylcholinesterases (AChE) at the surface. This mass-sensitive sensor does not only allow the examination of the interaction between AChE and the modified surface but also the detection of in situ inhibition of the surface-bound AChE. Here we describe the application of the propidium-based sensor in combination with a modified AChE. For this reason the cocaine derivative benzoylecgonine (BZE) was coupled via a 10 Å long hydrophilic linker – 1,8-diamino-3,4-dioxaoctane – to carboxylic groups of the AChE after EDC/NHS activation. Thus the modified AChE (BZE–AChE) possesses an additional recognition element besides the inhibitor binding site. After the deposition of BZE–AChE on the sensor surface the binding of an anti-BZE-antibody to the BZE–AChE can be monitored. This makes it possible to determine two analytes – cocaine and organophosphate – in one experiment by measuring antibody binding and decrease in enzymatic activity, respectively. Furthermore it was also shown that other cocaine-binding enzymes, e.g., butyrylcholinesterase, can bind to the modified BZE–AChE. The competitive immunoassay allowed the detection of cocaine with a dynamic range from 10−9 to 10−7 M. The organophosphate chlorpyrifos-oxon could be detected in concentrations from 10−6 down to 10−8 M after 20 min of injection time (equals to 500 L sample volume

Anti-hemagglutinin antibody derived lead peptides for inhibitors of influenza virus binding

Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing