Antibodies, fluorescence label covalent attachment

The detection of flu viruses via a fluorescent sandwich immunoassay was reported by Bucher.(10) However, the method sensitivity was too low for direct detection of the virus. A novel sandwich immunoassay was described by Ogcr((lff7 for the detection of Botulinum Toxin A. Antibodies specific for Clostridium botulinum were covalently attached to the surface of a tapered fiber. After the capture of the antigen, a sandwich was formed with a rhodamine-labeled anti-toxin IgG, and the evanescent wave was measured. The assay was highly specific with detection limits near 5 ppb. 

Here, the antibody-antigen reactions are observed indirectly by use of labels which are attached to either the antibody or antigen. The labels can be conjugated covalently and include radioisotopes, enzymes, labeled second antibodies, fluorescent tags, luminescent molecules and phages (12, 41, 45, 48). The use of labels helps in increasing the sensitivity of the assays considerably compared to the precipitation or agglutination assays. 

Specific labelling can be achieved by coupling the chromophore covalently to a vehicle, ustially an immunoglobulin or a hormone, that binds specifically to cellular receptors. This is the technique widely used to identify specific sub-cellular compartments/locations. Antibody specificity is coupled to the sensitivity of fluorescence to permit the highlighting of particular objects for which specific, often monoclonal, antibodies are available. Polyclonal antibodies specific for determinants on the monoclonal antibody are available with covalently attached chromophores, whose spectral properties match the excitation and emission criteria available in most fluorescence microscopes. 

Imine. A molecule containing a nitrogen atom attached to a carbon atom by a double bond. The nitrogen is also covalently linked to a hydrogen. Immunofluorescence. A cytological technique in which a specific fluorescent antibody is used to label an antigen. Frequently used to determine the location of an antigen in a tissue or a cell. 

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