ABSTRACT
The aim of this work is to detect immune complexes without any kind of labelling of each of the immunological species, with a view to create a very sensitive biosensor. This is achieved by using the atomic force microscopy. We have proceeded by imaging the antibody (anti-rabbit IgG) or anti-rabbit IgG moieties adsorbed onto mica surface, before and after incubation of two kinds of antigens: a specific (rabbit IgG) and a non-specific one (sheep IgG). The analysis using the height histograms reveals many interesting features. We propose a general framework for interpreting these analysis, which enables the discrimination between specific and non-specific complexes.
Subject(s)
Aluminum Silicates/immunology , Antigen-Antibody Complex/analysis , Antigen-Antibody Reactions , Immunoassay/methods , Immunoglobulin G/analysis , Microscopy, Atomic Force/methods , Adsorption , Animals , Antibodies/analysis , Antibodies/immunology , Antibodies/ultrastructure , Antigen-Antibody Complex/immunology , Antigen-Antibody Complex/ultrastructure , Antigens/analysis , Antigens/immunology , Antigens/ultrastructure , Binding Sites, Antibody , Goats , Immunoglobulin G/immunology , Immunoglobulin G/ultrastructure , SheepABSTRACT
In the present study the atomic force microscope (AFM) was used to image the surface morphology of red blood cells (RBC) for the first time. The AFM yielded very reproducible images without appreciable modifications of the sample surfaces. In addition to this topographical imaging, we have developed an experimental approach to measure the binding strength between antibody (anti-A), and the RBC antigen A, when reversible bonds between specific molecules such as antigen and antibody mediate the adhesion. The experimental results suggest that the procedure established here may be used for specific antibody detection. This study has also enhanced our understanding under physiological conditions of molecular interaction in particular antigen-antibody.
Subject(s)
Antigen-Antibody Complex/ultrastructure , Antigen-Antibody Reactions , Binding Sites, Antibody , Erythrocytes/immunology , Erythrocytes/ultrastructure , Microscopy, Atomic Force/methods , ABO Blood-Group System/immunology , ABO Blood-Group System/ultrastructure , Humans , Immunoassay/instrumentation , Immunoassay/methods , Microscopy, Atomic Force/instrumentationABSTRACT
The potentialities of an electrodeposited biotinylated polypyrrole film as an immobilisation matrix for the fabrication of impedimetric immunosensors are described. Biotinylated antibody (anti-human IgG), used as a model system, was attached to free biotin groups on the electrogenerated polypyrrole film using avidin as a coupling reagent. This immobilization method allows to obtain a highly reproducible and stable device. The resulting immunosensor has a linear dynamic range of 10-80 ng ml(-1) of antigen and a detection limit of 10 pg ml(-1). Furthermore, this immunosensor exhibited minor loss in response after two regeneration steps.
