Antibodies antigen binding

Four possible mechanisms for solid-state extraction (a) adsorption onto a solid substrate (b) absorption into a thin polymer or chemical film coated on a solid substrate (c) metal-ligand complexation in which the ligand is covalently bound to the solid substrate and (d) antibody-antigen binding in which the receptor is covalently bound to the solid substrate. 

Figure 20.9 Polysaccharide groups on antibody molecules may be oxidized with periodate to create aldehydes. Modification with biotin-hydrazide results in hydrazone linkages. The sites of modification using this technique often are away from the antibody-antigen binding regions, thus preserving antibody activity.

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable marker . The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay RIA) although immunoassay systems using enzymes (enzyme immunoassays EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags. 

Antibodies raised against the antigen of interest (i.e. the therapeutic protein) are first adsorbed onto the internal walls of microtitre plate wells. The sample to be assayed is then incubated in the wells. Antigen present will bind to the immobilized antibodies. After an appropriate time, which allows antibody-antigen binding to reach equilibrium, the wells are washed. 

Antibodies, 20 831 functional, 12 475 liquid separation adsorption, 1 678 oligonucleotides attached to, 17 635 yeast-derived, 26 486 Antibody-analyte complex, 14 138 Antibody-antigen binding event, 14 137-138... 

Other immunoassays are based on the same antibody-antigen binding reaction but use a different labeling system for detection. Instead of an enzyme label, there are radioactive isotopes, and fluorescent and luminescent labels. Some important immunoassays are defined below ... 

Sapsford etal. (2001) examined microarray-based antibody-antigen binding kinetics in real time to determine the effect of spot size. Capture antibodies were immobilized in an array pattern onto silver-clad microscope slides. Antimouse IgG was directly attached to the surface or attached via neutravidin capture of the biotinylated antibody. Cy5-labeled mouse IgG capture was monitored based upon the signal generated from the excitation of an evanescent wave guide (slide) with a 635-nm laser source detection was achieved by a charge-coupled device (CCD) camera system. Both static and flow-through conditions were employed. 

If weak antibody-antigen binding is suspected, the binding may be stabilized by fixation in 1% glutaraldehyde in PBS for 10 min... 

A resistive pulse method of particle sizing was used to detect antibody-antigen binding events at a pore fabricated on a PDMS chip. The pore was typically 7-9 pm long and 1 pm in diameter. Mouse monoclonal anti-streptavidin antibody (0.75-10 pg/mL) was supposed to bind to the surface of latex colloidal particles coated with streptavidin (the antigen). This binding, which caused an increase of 1-9 nm of the particle diameter, was measured by the resistive pulse method [1031],... 

Saleh, O.A., Sohn, L.L., Direct detection of antibody-antigen binding using an on-chip artificial pore. Proc. Natl. Acad. Sci. USA 2003, 100, 820-824. 

High concentrations of sodium chloride and azides are used frequently as preservatives in commercial preparations, but these components can reduce antibody reactivity. Excessive ionic strength can decrease specific staining by interfering with antibody-antigen binding. 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

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