Wipe test for the detection of indoor allergens.

BACKGROUND: The determination of house dust mite and cat allergens is a prerequisite for qualified assessment of exposure risk indoors. We describe the validation of integrated wipe tests comprising a porous sampling pin for the collection of settled house dust and an immunochemical test set-up for specific analysis of dust associated allergens. METHODS: Wipe tests comprised an immunochromatographic test system and a porous sampling pin for collecting dust by manually wiping a surface. Two different wipe tests were developed with specificity to cat allergen (Fel d 1) and to dust mite allergen (Der p 2/f 2). One-hundred and fifty on-site determinations for each of the allergens in 50 different households completed the field study to validate the analytical system. Reference analysis was based on collection of dust with a vacuum cleaner and analysis of dust associated allergens by ELISA. RESULTS: The wipe tests were found to produce semiquantitative results (class 0-3) on-site within 10 min of analysis time. For dust mite (cat) allergen the median of class 0-3 was 119 (263), 812 (844), 2708 (5670) and 8000 (56 800) ng/g dust, respectively. With respect to cut-off levels for dust mite (cat) allergens the clinical sensitivity was 62% (79%) and the clinical specificity was 94% (92%). CONCLUSIONS: The wipe tests proved to be a reliable tool for indoor allergen exposure screening, suitable for use even by untrained persons. This may have an impact on compliance with allergen avoidance measures.

Real-time observation of affinity reactions using grating couplers: determination of the detection limit and calculation of kinetic rate constants.

The use of integrated optical grating couplers for the analysis of bioaffinity reactions in order to calculate kinetic rate constants was investigated. The specificity of the sensor surface was determined by adsorptive or covalent attachment of the specific ligands. As an evanescent field sensor, the specific interaction of the corresponding ligand could be observed in real time and without labels. The detection limit in terms of the molecular weight of the analyte was studied by the specific binding of biotinylated proteins of different molecular weights to avidin-loaded sensors. It was shown that grating coupler sensors allowed detection of compounds of at least 2000 daltons using high-affinity receptors, while the direct sensing of low molecular analytes, such as biotin, could not be significantly achieved. Association rate constants were calculated for the interaction of the different biotinylated proteins to avidin-covered sensors from single binding curves. Due to the strong binding between avidin and biotin, the dissociation of the formed complex could not be observed. Kinetic rate constants and equilibrium constants were determined by studying the interaction of human immunoglobulin with the immobilized receptor, protein G. For the four human immunoglobulin subclasses a high affinity to protein G was determined with affinity constants ranging from 3.3 to 8.4 x 10(8) M-1.

Optimization of biosensing using grating couplers: immobilization on tantalum oxide waveguides.

This paper presents a comparative study of immobilization strategies for integrated optical grating couplers using tantalum oxide waveguides. As a model system the affinity reaction between protein G and human IgG was investigated. The receptors were coupled to the waveguide by adsorption, covalent attachment and avidin-biotin bridges after modifying the sensor surface by silanization introducing epoxy and amino groups. In addition, the results obtained by a monolayer coverage of the sensors with the receptors were compared with those using a carboxymethyl-dextran matrix allowing the immobilization of an increased amount of protein. The different coupling procedures were assessed by the system response due to the specific binding of human IgG to the sensing layer and are discussed with respect to their effectiveness, stability and the absence of non-specific binding.

On-line monitoring of monoclonal antibodies in animal cell culture using a grating coupler.

A grating coupler was used for the on-line determination of monoclonal antibodies produced in perfused animal cell bioreactor. The device was connected with the culture vessel via a flow-injection analysis (FIA) system, which was controlled automatically. Specific antimouse lgG antibodies were immobilized on the surface of the sensor-chip. After injection of the sample, the binding of mouse lgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the on-line detection of produced monoclonal antibodies in the range of 10 to 150 microg/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off-line) and a competitive immunochemical assay using the grating coupler (off-line).