Diagnostic performance study of an antigen microarray for the detection of antiphospholipid antibodies in human serum.

BACKGROUND: The parallelization of clinically relevant antigens in a microarray format is of growing importance due to the ability to measure multiple antigen-antibody interactions. With the development of a microarray for the detection of antiphospholipid antibodies we focussed on one important autoimmune disease that is still diagnostically challenging. Reasons are the heterogeneity of the autoantibodies and the unspecific clinical symptoms. METHODS: For the covalent immobilization of antigenic structures, glass transducers were coated with 11-aminoundecyltrimethoxysilane (11-AUTMS). In total 35 antiphospholipid syndrome (APS) patients, six patients with lupus erythematosus and 24 healthy controls were investigated on a microarray format using polarized imaging reflectometric interference spectroscopy. RESULTS: The novel surface modification based on the short derivative 11-AUTMS resulted in a selective biosensor allowing a clear differentiation of patient and control samples. It combined proteinogenic as well as phospholipid-derived antigens, namely ß2-glycoprotein I (ß2-GPI), prothrombin, cardiolipin (CL) and a ß2-GPI/CL complex. With optimized regeneration conditions, up to 20 consecutive measurements could be performed on one chip. Sensitivity was determined to be 0.800-0.929, specificity was between 0.733 and 0.969, depending on the respective antigen. CONCLUSIONS: Multiplexed determination of serological parameters has a great potential. We have shown that our biosensor is capable of detecting four different APS relevant antibodies in parallel exhibiting a sensitivity and specificity comparable to existing ELISA methods.

Size does matter! Label-free detection of small molecule-protein interaction.

This review is focused on methods for detecting small molecules and, in particular, the characterisation of their interaction with natural proteins (e.g. receptors, ion channels). Because there are intrinsic advantages to using label-free methods over labelled methods (e.g. fluorescence, radioactivity), this review only covers label-free techniques. We briefly discuss available techniques and their advantages and disadvantages, especially as related to investigating the interaction between small molecules and proteins. The reviewed techniques include well-known and widely used standard analytical methods (e.g. HPLC-MS, NMR, calorimetry, and X-ray diffraction), newer and more specialised analytical methods (e.g. biosensors), biological systems (e.g. cell lines and animal models), and in-silico approaches.

Development of a new parallelized, optical biosensor platform for label-free detection of autoimmunity-related antibodies.

Autoimmune diseases are characterized by the presence of autoantibodies in serum of affected patients. The heterogeneity of autoimmune relevant antigens creates a variety of different antibodies, which requires a simultaneous detection mode. For this reason, we developed a tool for parallelized, label-free, optical detection that accomplishes the characterization of multiple antigen-antibody interactions within a single measurement on a timescale of minutes. Using 11-aminoundecyltrimethoxysilane, we were able to immobilize proteinogenic antigens as well as an amino-functionalized cardiolipin on a glass surface. Assay conditions were optimized for serum measurements with a single spot antigen chip on a single spot 1-λ detection system. Minimized background signal allows a differentiation between patients and healthy controls with a good sensitivity and specificity. Applying polarized imaging reflectometric interference spectroscopy, we evaluated samples from three APS patients and three control subjects for this proof-of-principle and already obtained good results for ß2-glycoprotein I and cardiolipin.

A biomimetic sensor surface to detect anti-ß2-glycoprotein-I antibodies as a marker for antiphospholipid syndrome.

A biomimetic sensor has been developed, that allows for quantification of autoantibodies related to the antiphospholipid syndrome (APS). Autoantibodies directed against the ß(2)-glycoprotein-I (ß(2)GP-I) are known as the best markers for diagnosis of APS, however, detection of such antibodies is still a challenge. The epitopes of ß(2)GP-I are exposed upon binding to negatively charged membranes. The surface of the sensor chips was therefore modified with such type of membranes, on which ß(2)GP-I molecules were subsequently immobilized as recognition elements. Using the label-free method, reflectometric interference spectroscopy, it was possible to quantify anti-ß(2)GP-I antibodies and to calibrate the sensor chip in buffer. A mild regeneration procedure allows for many consecutive measurements without stripping off the membrane in between.

Label-free quantification of cystatin C as an improved marker for renal failure.

A label-free biosensor has been developed, allowing quantification of cystatin C in human serum. This was achieved by using reflectometric interference spectroscopy as detection method. Cystatin C is a small serum protein that allows detection of renal failure more reliably than established parameters as creatinine. The protein was immobilized on the surface of a glass transducer, forming the sensitive layer of the sensor chip. Based on a binding-inhibition assay, two different types of monoclonal cystatin C antibodies were compared, by their behavior and their obtained working range in buffer and serum as matrix. Both antibodies allowed quantification of the protein in serum as matrix within the required clinical ranges of 0.53-1.02 mg/L. Detected recovery rates are in a range between 84.8% and 116.1%. The developed sensor shows high inner chip reproducibility and low cross-sensitivity.