Background: For the laboratory diagnosis of the antiphospholipid syndrome (APS) we developed a biosensor with the ability to distinguish between diseaserelevant anti-2-glycoprotein I (2GPI) autoantibodies (anti-2GPI) and pathogen-specific 2GPI cross-reactive antibodies that occur transiently during infections. Methods: We used a surface plasmon resonance (SPR) biosensor device. For the detection of anti-2GPI in serum samples, affinity-purified human 2GPI was covalently attached to a functionalized n-alkanethiol self-assembling monolayer on the biosensor chip. After verifying the specificity of the biosensor system with a panel of monoclonal antibodies to 2GPI, we analyzed sera from healthy donors and patients suffering from APS, systemic lupus erythematosus (SLE), syphilis, or parvovirus B19 infections. The SPR results were compared with 2GPI-specific ELISA. Results: Using the SPR biosensor, we recorded antigen binding curves with response levels in the range of 50 -500, resonance units (RU) for anti-2GPI ELISApositive APS patient sera. The amplitudes of the antiphospholipid antibody (APL) responses in the biosensor correlated with the overall IgG and IgM anti-2GPI ELISA titers with a correlation coefficient of 0.87. Moreover, we observed immunoglobulin isotype-specific association and dissociation profiles for APL binding of
6421 28 26652 Methods for rapid and reliable detection of clinically relevant bacteria are of highest interest. For such purposes, many analytical methods, e.g. ELISA, have been developed. Also optical biosensors like reflectometric interference spectroscopy (RIfS) are suitable analytical platforms. Here, results are described to capture and detect Legionella pneumophila serogroup 1 on differently functionalized surfaces of RIfS glass chips. As a major problem, availability of suitable antibodies was found. Immobilization of capturing antibodies on the chip surface followed by trapping of L. pneumophila gave only insufficient results. For this approach, the chip surface was partially blocked with bovine serum albumin (BSA) in different ratios between BSA and the Legionella specific antibody in order to obtain a site-directed immobilization. Further on, antibodies were immobilized by hydrophobic interaction followed by BSA treatment and exposition to L. pneumophila. However, also this approach did not allow sufficient capturing of bacteria cells. It can be assumed that binding forces between the antibody and bacteria are too low. Alternatively, L. pneumophila bacteria were directed immobilized on a hydrophobic chip surface. A sufficient sensor signal for quantification was obtained in a range between 8.25 Â 10 6 and 8.25 Â 10 8 bacteria cells/mL. This approach might open up the possibility for safe identification of L. pneumophila after subsequent addition of specific antibodies.
Autoimmune disorders are rare human diseases characterized by the presence of circulating autoantibodies that bind the body's own structural compounds as target antigens. The detection of autoantibodies is important for the diagnostic process. Immunofluorescence and immunoassay methods do not allow a reliable characterization of binding characteristics. Therefore, novel analytical techniques should be considered. This review describes the application of surface plasmon resonance biosensor systems for the diagnosis of autoimmune disorders. The covalent attachment of native antigens to the sensor chip is a suitable method for obtaining highly reproducible analyses of autoantibodies, allowing the evaluation of kinetic rate and affinity constants, and it may enable the identification of disease-relevant autoantibodies linked to disease progression. The autoantibody microarray is another future-oriented technique. Patterns of differential antigen recognition should allow early diagnosis. This is due to the fact that a broad range of autoreactive B cell responses in autoimmune disorders can only be mirrored by including a sufficient number of antigens in a microarray format.
Immobilization of biomolecules on solid surfaces is often combined with a partial loss of functionality. Therefore, smooth immobilization procedures are urgently required. Most recently, a Concanavalin A-Streptavidin (Con A-SAv) fusion protein was obtained, which allows the design of functionalized interfaces via self-assembling. The protein was successfully produced in Escherichia coli and the functionality was tested by surface plasmon resonance (SPR) measurements as well as by the mean of reflectometric interference spectroscopy. A re-generation of the mannan-coated surfaces, by washing with buffer containing 10% methyl a-D-mannopyranoside, could be demonstrated. This procedure should allow multiple measurements without replacing the chip. Investigation of the functionalized surfaces by atomic force microscopy showed a rather uniform coating with mannan and the fusion protein. In conclusion, the designed Con A-SAv fusion protein can be used as a universal linker between mannan-coated surfaces and biotinylated biomolecules, e.g. biotinylated antibodies.
Lateral flow assays (LFAs, e.g. the well established pregnancy test) are frequently used, fast and easy‐to‐handle immunoassays with a broad application range. Nevertheless, the restriction to small sample volumes is one of the major drawbacks and can be the reason for lack of sensitivity. In order to detect even small amounts of analyte in big sample volumes without the need for time consuming sample preparation or sophisticated labelling or detection technologies, the aim of the here presented research was the development of an ‘enrichment module’, which can be integrated into the workflow of a LFA. The core element of this enrichment module was a cleavable biological interface structure. A fusion protein, which was expressed recombinant and consisted of a monomeric concanavalin A (Con A) domain and a monomeric streptavidin domain (SAv), was used as interface structure. For the construction of the entire module, the polysaccharide mannan was firstly covalently attached to a porous polyethylene (PE) sintered body in a four‐stage procedure. This immobilisation procedure was monitored by a modified Bradford assay. In the next step, the Con A–SAv fusion protein was added. In order to assess the functionality of the enrichment module, a model assay was developed. The SAv domain of the attached fusion protein was specifically recognised by a primary and a secondary, horseradish peroxidase (HRP)‐labelled, antibody. The HRP‐reaction was used for photometric detection in order to characterise binding properties of the enrichment module and to prove binding characteristics of the fusion protein.
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