The influence of epitope availability on atomic-force microscope studies of antigen-antibody interactions.

The ability of the atomic-force microscope (AFM) to detect interaction forces between individual biological molecules has recently been demonstrated. In this study, force measurements have been obtained between AFM probes functionalized with the beta-subunit of human chorionic gonadotrophin (betahCG) and surfaces functionalized with anti-betahCG antibody. A comparison of the obtained results with previous anti-ferritin antibody-binding data identifies differences when the antigen molecule expresses only a single epitope (betahCG), rather than multiple epitopes (ferritin), for the monoclonal antibodies employed. Specifically, the probability of observing probe-sample adhesion is found to be higher when the antigen expresses multiple epitopes. However, the periodic force observed in the adhesive-force distribution, due to the rupture of single antigen-antibody interactions, is found to be larger and more clearly observed for the mono-epitopic system. Hence, these findings indicate the potential of the AFM to distinguish between multivalent and monovalent antibody-antigen interactions, and demonstrate the influence of the number of expressed epitopes upon such binding studies.

Detection of antigen-antibody binding events with the atomic force microscope.

An atomic force microscope (AFM) has been used to directly monitor specific interactions between antibodies and antigens employed in an immunoassay system. Results were achieved using AFM probes functionalized with ferritin, and monitoring the adhesive forces between the probe and anti-ferritin antibody-coated substrates. Analysis of the force distribution data suggests a quantization of the forces, with a period of 49 +/- 10 pN. This periodic force may be attributed to single unbinding events between individual antigen and antibody molecules. These results demonstrate that the AFM could be employed as an analytical tool to study the interactions between the molecules involved in biosensor systems. The potential of the technique to provide information relating to the manner in which the antibody molecule binds to its specific antigen is also discussed.

In situ observation of streptavidin-biotin binding on an immunoassay well surface using an atomic force microscope.

Polystyrene microtitre wells are commonly used as supports for the enzyme-linked immunosorbent assay (ELISA) method of biomolecular detection, which is employed in the routine diagnosis of a variety of medical conditions. We have used an atomic force microscope (AFM) to directly monitor specific molecular interactions between individual streptavidin and biotin molecules on such wells. This was achieved by functionalising an AFM probe with biotin and monitoring the adhesive forces between the probe and a streptavidin coated immunoassay well. The results demonstrate that the AFM may be employed as an analytical tool to study the interactions between biomolecules involved in immunoassay systems.

The discrimination of IgM and IgG type antibodies and Fab' and F(ab)2 antibody fragments on an industrial substrate using scanning force microscopy.

We have previously employed scanning force microscopy (SFM) to study antibody-antigen molecular interactions on microtiter wells used for enzyme linked immunosorbant assays (ELISA). Here we demonstrate the ability of SFM to image and discriminate different types of antibody and antibody fragments bound to an ELISA well surface. The samples studied include a type IgG antibody with a proportion of bound IgM and two-dimensional films of whole IgG antibody, and Fab' and F(ab)2 antibody fragments. Molecular resolution is achieved in each case despite the size of substrate features exceeding most of the molecular dimensions observed. Analysis of the data shows that the SFM overestimates molecular dimensions by an approximately constant amount, which is proposed to principally result from the effects of a finite probe size and not from deformation of the molecular species due to the imaging forces employed.

Scanning tunnelling microscopy and dynamic contact angle studies of the effects of partial denaturation on immunoassay solid phase antibody.

A range of partial denaturation antibody pre-treatments that affect immunoassay performance have been evaluated. Monoclonal anti-ferritin antibody was either partially denatured by heat, urea or pH pre-treatment or left untreated and then passively adsorbed to polystyrene microtiter wells. The adsorption characteristics and functionality of the different surfaces produced have been evaluated by dynamic contact angle (DCA) analysis and scanning tunnelling microscopy (STM) imaging respectively. The DCA data show that the effect of partial denaturation is to change the wetting characteristics of the antibody surfaces, while, in addition, STM imaging reveals marked effects seen in the aggregation properties of the denatured antibodies.

A scanning tunnelling microscopy comparison of passive antibody adsorption and biotinylated antibody linkage to streptavidin on microtiter wells.

An antiferritin antibody was either, (a) passively adsorbed to microwells or (b) biotinylated and immobilised to streptavidin coated microwells. Scanning tunnelling microscope (STM) imaging of these well surfaces coated with a platinum (95%) carbon (5%) coating (Pt/C) conductive layer showed a randomly oriented array of antibodies for passive adsorption whereas for biotin-streptavidin immobilisation there was a more uniform and even distribution of antibodies on the well surface. On further incubation with ferritin STM imaging showed that for passive adsorption approximately 5% of the surface was functional, while for the biotinylated antibody it was greater than 60%. The images presented in this paper show graphically the loss of functionality that occurs using passive adsorption and, conversely, the preservation of antibody functionality using the biotin-streptavidin linkage for antibody immobilisation. These results correlate well with the work of others in the field.