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 β2-glycoprotein I and cardiolipin.     

Recent advances in immunodiagnostics based on biosensor technologies—from central laboratory to the point of care

Immunological methods are widely applied in medical diagnostics for the detection and quantification of a plethora of analytes. Associated analytical challenges usually require these assays to be performed in a central laboratory. During the last several years, however, the clinical demand for rapid immunodiagnostics to be performed in the immediate proximity of the patient has been constantly increasing. Biosensors constitute one of the key technologies enabling the necessary, yet challenging transition of immunodiagnostic tests from the central laboratory to the point of care. This review is intended to provide insights into the current state of this transition process with a focus on the role of biosensor-based systems. To begin with, an overview on standard immunodiagnostic tests presently employed in the central laboratory and at the point of care is given. The review then moves on to demonstrate how biosensor technologies are reshaping this landscape. Single analyte as well as multiplexed immunosensors applicable to point of care scenarios are presented. A section on the areas of clinical application then creates the bridge to day-to-day diagnostic practice. Finally, the depicted developments are critically weighed and future perspectives discussed in order to give the reader a firm idea on the forthcoming trends to be expected in this diagnostic field.

     

Solid-phase synthesis, isolation and analysis of a mouse protein, the macrophage migration inhibitory factor.

Murine macrophage migration inhibitory factor (mMIF) is an Mr 12,500 protein composed of all natural amino acid residues. Using the fluorenylmethoxycarbonyl chemistry for solid-phase peptide synthesis (SPPS) under special conditions, a stepwise approach was very successful leading to a crude product in unexpected high purity. After RP-HPLC isolation, a little mass difference of -12 u was determined for the received protein by matrix-assisted laser desorption ionization MS and ion spray MS and the failure sequence [Ser15]-mMIF identified by Edman degradation. The total solid-phase synthesis was repeated in the stepwise manner under the same conditions leading to the expected mMIF protein in high purity, which was confirmed by different analytical methods. Our results shows the reproducibility of our SPPS approaches to proteins and point out the importance of high-resolution mass spectrometry for a rapid and accurate analysis of such biopolymers.     

Application of ion pair high performance liquid chromatography to the analysis of porphyrins in clinical samples

Reversed phase ion pair chromatography is a highly selective separation technique for the determination of free porphyrin carboxylic acids from human materials. Isocratic and gradient elution methods can be used to analyse porphyrin isomers and to establish porphyrin profiles for the biochemical diagnosis of porphyrias. Ion pair high performance liquid chromatography led to the discovery of the atypical isomers II and IV of uroporphyrin and coproporphyrin in human urine. Advantages and limitations of the ion pair technique are discussed.     

Application of ion-pair high-performance liquid chromatography to detection of the atypical coproporphyrin isomers II and IV in human faeces

A highly selective and sensitive method has been developed for the detection of small amounts of the atypical isomers II and IV of coproporphyrin in human faeces. This method combines liquid-liquid extraction and solid-phase sampling techniques using talc and C18-modified silica gel as the sorbents. Simultaneous separation of the four coproporphyrin isomers I-IV was achieved by isocratic ion-pair high-performance liquid chromatography. Stool samples of healthy subjects (n = 12) contained 1.1 +/- 0.4% (mean +/- S.D.) isomer II and 2.2 +/- 0.9% isomer IV of total coproporphyrins. A somewhat higher content of isomer II (2.7%) and isomer IV (5.4%) was found in faeces of a patient suffering from porphyria variegata.