Analyte-antibody interactions

Assay Diluents As the noncovalent interactions between antibody and analyte are influenced by pH, ionic strength, and temperature, typical assay buffers are isotonic solutions at or near neutral pH. Assay buffer and diluent formulations should not only promote analyte antibody interactions but also minimize the nonspecific interactions between the critical reagents and the variety of biomolecules in the sample matrix. Components such as BSA, HSA, and nonionic detergents are often included in assay buffers. Phosphate buffered saline (PBS) or 10 mM Tris-HCl solutions (both near pH 7.4) containing 1% BSA and 0.05% Tween-20 are common buffers that can also be used for dilution of test samples and detection reagents, as well as wash buffers. When wash buffers are used in large quantities and stored at room temperature, preservatives such as sodium azide or thimerasol are often added to increase their shelf life. It should be noted, however, that some components of the wash buffer may have adverse effects... 

With the view of selectively extracting target compounds from environmental waters, there has been a certain interest in developing and employing selective adsorbents based on analyte-antibody interactions achieved by immunosorbents. In the immunosorbent, the antibody... 

Fig. 9. Immunosensor approaches where A is the analyte, is the labeled analyte, and Y is the antibody, (a) Direct immunosensors where the actual antigen—antibody interaction is measured (b) indirect immunosensors 1 and 2 which utilize formats similar to competitive and displacement...

The combination of the specificity of the antigen-antibody interaction with the exquisite sensitivity of fluorescence detection and quantitation yields one of the most widely applicable analytical tools in cell biology (1). Within the last decade, flow cytometry (FCM) has become an integral part of basic immunological research. Elaboration of this technology has been intensively stimulated by a rapidly growing sophistication in monoclonal antibody technology and vice versa (2). 

Combination of electrophoresis in different formats with affinity reactions started in the early 1950s, when Tiselius cells were used for the first time to characterise antigen-antibody interactions. A real milestone report in the field was the determination of the equilibrium constants for the binding of Ca2+ and Zn2+ to serum albumin by gel electrophoresis in 1960 [1]. Finally, the term "affinity electrophoresis" was proposed [2,3]. Its modification or, better said, its particular case, named immunoelectrophoresis, has been used for many years in reports relying on agarose gel separation in conjunction with immunoprecipita-tion. Appearance of precipitated zones on a gel is indicative for the antigen-antibody reaction and can be used for the identification of an analyte and also for its quantification. 

Protein arrays Identification/separation techniques based on protein-protein interactions including antigen-antibody interactions. Analytical... 

The potential usefulness of any analytical procedure should be assessed by carrying out control experiments in samples comparable to those collected for the field studies. Analytes in environmental samples are often determined at the ppm-ppb levels. While an immunoassay may appear to be specific, sensitive and reliable under the pristine conditions of the laboratory (e.g. when the test substance is in buffer), it may give false results with environmental samples that vary sufficiently (e.g. in pH, ionic strength, viscosity, solubility, humic content, etc.) to alter the antigen-antibody interaction or interfere with the monitoring systems. Samples collected at the point source where the analyte is most concentrated (e.g. at the... 

In affinity chromatography the unique and specific biological interaction of the analyte and ligand is used for the separation (Figure 6-4). The specificity resulting from enzyme-substrate, hormone-receptor, or antigen-antibody interactions has been used in this type of chromatography. 

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