Antibody analyte interaction, immunoassay

There is a continuing demand for fast and simple analytical methods for the determination of many clinical, biochemical and environmental analytes. In this respect, immunoassays and immunosensors that rely on antibody-antigen interactions provide a promising means of analysis owing to their specificity and sensitivity. High specificity... 

Since immunoassays are primarily analytical techniques, in addition to studies for a better understanding of the nature of antibody-antigen interaction, there are continuous efforts to improve immunoassay performance (e.g., sensitivity, selectivity, precision and accuracy) in terms of robustness and reliability when analysing complex samples. The present chapter attempts to summarize the most commonly used immunoassay concepts, as well as the main approaches employed for the improvement of immunoassay sensitivity, selectivity and precision. The discussion is focussed aroimd the main thermodynamic and kinetic principles governing the antibody-antigen interaction, and the effect of diverse factors, such as assay design, concentration of reactants, incubation time, temperature and sample matrix, is reviewed in relation to these principles. Finally, particular aspects on inummoassay standardization are discussed as well as the main benefits and limitations on screening vs. quantification of analytes in real samples. 

Immunoassays, along with all other methods based on biorecognition, are a great achievement for the field of analjdical chemistry. The first area to benefit from the advantages of this technique is probably chnical analysis, where selective and sensitive determination of macromolecules is often necessary, as it is very difficult, or sometimes impossible to identify and quantify macromolecules by any alternative method. Recently, the immunoassay application field was extended, more or less successfiilly, to the determination of small molecules (below 1000 Da) as well. The acceptance of immunoassays is, however, relatively hmited in some fields, probably due to the differences between the classical analytical methods and immunoassays, the latter requiring special conditions of operation, characterization and data interpretation, due to the extraordinary nature of the antibody-antigen interaction, as well as that of many possible interfering reactions. 

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... 

In fluorescent immunoassay (FIA), a fluorescent molecule is covalently bound to a molecule of analytical interest. The labeled analyte interacts reversibly with an antibody (usually derived from a rabbit or goat) which is specific for the analyte and will bind with about equal affinity to the labeled or unlabeled analyte. The labeled analyte usually shows different fluorescent properties according to whether it is bound by the antibody or free to diffuse in solution. The serial additional of different concentrations of the analyte to the labeled analyte-protein... 

Immunological methods make use of antibodies as analytical tool for detecting a plethora of clinical, environmental, and food-relevant analytes. The special features that had made immunoassay widely increased in the last decades are the highly sensitivity and specificity of the antibody-antigen interaction. Although enzyme-linked immunosorbent assay (ELISA), currently performed in microtiter plates is the most common technique, a variety of assay types can be performed depending of the analytes or samples. 

The analytical response generated by an immunoassay is caused by the interaction of the analyte with the antibody. Although immunoassays have greater specificity than many other analytical procedures, they are also subject to significant interference problems. Interference is defined as any alteration in the assay signal different from the signal produced by the assay under standard conditions. Specific (cross-reactivity) and nonspecific (matrix) interferences may be major sources of immunoassay error and should be controlled to the greatest extent possible. Because of their different impacts on analyses, different approaches to minimize matrix effects and antibody cross-reactivity will be discussed separately. 

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