Antibody analyte interaction, immunoassay development

The use of antibodies has allowed the development of a technique known as immunodiagnostics, especially attractive for the direct analysis of intrinsically complex samples such as blood, serum, urine, food, etc. An immunoassay is a test that uses antibody-antigen complexes as a means of generating a measurable result. The specific interaction between epitopes and paratopes lies at the heart of every immunoassay. The role of antibodies in immunoassays is based on the observation that, in a system containing the determinant and a specific antibody, the distribution of the former between its antibody-bound and antibody-free forms is quantitatively related to the total analyte concentration. 

Miniaturized immunosensors, which combine the analytical power of nuCTofluidic devices with the high specificity of antibody-antigen interactions, have been intensively developed [9-11, 47-51]. These platforms have proven to be highly suitable vehicles for conducting various immunoassay protocols. Our research groups have described a new approach to the performance of miniaturized electrochemical flow immunoassay system (on-chip typed flow immunoassay system) by using ferrocene-conjugated... 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

Like FRET, today BRET is predominantly used in biological sciences, especially in the monitoring of protein-protein interactions such as hormone-receptor interaction [223, 224] and protein-DNA interaction in living systems. However, BL resonance energy transfer can also be applied in immunoassays by using for instance a peptide-tagged luciferase and a fluorescein-labeled antipeptide antibody [225]. The development of more BRET assays for small-molecule analytes is thus awaited. 

When the concentration of the analyte in the biological solution is too lowfor detection using spectrophotometry, more sensitive methods such as immunoassays are used for the measurement. Immunoassays utilize antibodies developed against the analyte of interest. Since the antigen and the antibody have a very specific interaction and has very high affinity toward each other, the resulting detection system also has a very high sensitivity. A specific example, the enzyme linked immunosorbent assay (ELISA), will be described here. 

Antibodies form complexes with their respective antigens (here analytes). These complexes can show a particular strength, which can be quantified by the affinity constant (or equilibrium constant). This affinity constant is about 10 -10 L mol for most analytically useful antibodies. The higher this number, the more stable is the complex. The highest known affinity in the biochemical field is the interaction between avidin (an egg protein) and biotin (vitamin H), for which a value of around 10 L mol has been determined. The affinity constant plays an important role in immunoassays and other immunological techniques. The development of new methods is greatly facilitated if this constant is known. 

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