Immunochemical binding

The evolution of modem bioanalysis has, to a large extent, been coupled to the development of immobilization technology. Most immunochemical binding assays as well as all biosensors are dependent on the use of immobilized reagents. Some major challenges for the future include the fabrication of biosensors based on microchips and the validation of biosensor technology in routine applications. 

The use of protein-coated acoustic wave devices for detection of gas-phase species has also been reported with claims of good sensitivity and selectivity. Guilbault et al. reported TSM sensors for the reversible gas-phase detection of formaldehyde [227], and organophosphorous pesticides [228,229]. More recent studies have cast some doubt as to whether the gas-phase sensitivity was the result of selective immunochemical binding, or simply due to nonspecific adsorption. In work reported by Thompson et al. [230], sensors coated with parathion antibody exhibited sensitivities to the pesticides parathion, malathion, and disul-foton that were remarkably similar to sensors coated with nonspecific proteins (valproic acid antiserum, human immunoglobulin G, and bovine serum albumin). The fact that the previous study [228] reported significantly larger sensitivity at... 

The use of the avidin-biotin system enables a separation of the primary and secondary steps. Previous employment of an antibody-marker for direct detection was problematic since such conjugates are very large and subject to a variety of irrelevant interactions that interfere with the desired immunochemical binding. In contrast, the biotinylated antibody can first be incubated with the cell or tissue sample following this initial interaction, the sample can be fixed and incubated subsequendy with... 

Flow injection analysis offers many attractive features to biosensor analysis. The reproducibility and the speed are two dominant characteristics when combining FIA with proper sampling and sample handling. It can be used for both enzyme-based assays and immunochemical binding assays. 

Iron is, as part of several proteins, such as hemoglobin, essential for vertebrates. The element is not available as ion but mostly as the protein ligands transferrin (transport), lactoferrin (milk), and ferritin (storage), and cytochromes (electron transport) (Alexander 1994). Toxicity due to excessive iron absorption caused by genetic abnormalities exists. For the determination of serum Fe a spectrophoto-metric reference procedure exists. Urine Fe can be determined by graphite furnace (GF)-AAS, and tissue iron by GF-AAS and SS-AAS (Alexander 1994 Herber 1994a). Total Iron Binding Capacity is determined by fuUy saturated transferrin with Fe(III), but is nowadays mostly replaced by immunochemical determination of transferrin and ferritin. 

The concept of immunoassay was first described in 1945 when Landsteiner suggested that antibodies could bind selectively to small molecules (haptens) when they were conjugated to a larger carrier molecule. This hapten-specific concept was explored by Yalow and Berson in the late 1950s, and resulted in an immunoassay that was applied to insulin monitoring in humans. This pioneering work set the stage for the rapid advancement of immunochemical methods for clinical use. 

An important factor in all these experiments is the choice of bead used to immobilize the probe. Biochemists have considered cross-linked agarose beads to be exceptionally hydrophilic with a low tendency to bind proteins nonspecifically, and these beads have the further attraction of being commercially available in activated forms (succinimidyl esters, epoxides, and maleimides, for example). However, early trials of bead-based chemical proteomics have shown that many proteins in mammalian cell lysates bind tenaciously to agarose beads. This was unimportant in many studies in which protein-protein interactions were detected by coimmunoprecipitation with immunochemical... 

A most important technique which has been developed as an extension of the isotope dilution principle is that of radioimmunoassay (RIA). Analyses by this method employ substoichiometric amounts of specific binding immuno-chemical reagents for the determination of a wide range of materials (immunogens) which can be made to produce immunological responses in animals such as sheep or rabbits. It is possible to combine the specificity of an immunochemical reaction with the extreme sensitivity of radiotracer detection. Analytical methods based upon these principles have achieved wide applicability in the determination of organic compounds at trace levels. 

Immunochemical methods are valuable because of their sensitivity and specificity. The sensitivity depends on the method used to determine an end point. One of the reaction components may be tagged with radioactivity, or tagged by covalent binding of an enzyme capable of being detected, or by covalent binding of a totally unrelated species (i.e., fluorescein). 

Immunochemical techniques are based on the immunological reaction derived from the binding of the antibody to the corresponding antigen. This reaction is reversible and is stabilized by electrostatic forces, hydrogen bonds, and Van der Waals interactions. The formed complex has an affinity constant (k j that can achieve values around the order of 1010 M. This great affinity and specificity between the specific antibody and the antigen (or the analyte) have turned these techniques into powerful analytical tools to detect and quantify... 

Cribb AE, Nuss CE, Alberts DW, et al. Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by immunochemical detection. Chem Res Toxicol 1996 9(2) 500-507. 

The kinetics of disulfide formation, the demonstration of specific binding, and the immunochemical results all support the conclusion that native-like structure results from the oxidative folding of reduced peptide 13-105. These three independent lines of evidence support the conclusion that lysozyme has a continuous chain independent assembly region somewhere in the sequence 13-105. 

Good overall agreement was obtained in the conclusions reached on folding-by-parts in albumin by immunochemical (Teale and Benjamin, 1976, 1977) and small ligand binding measurements (Johanson et al., 1977, 1981). This serves to increase our confidence in the usefulness of immunochemical approaches to this kind of problem. 

There was neither report of yield nor investigation of the kinetics of return of native characteristics. Attempts to prepare intact fragment 16-125 have not yet been successful. The immunochemical and substrate binding evidence support the claim that refolding to native-like structure did indeed occur. It clearly would be desirable to have more evidence on this system. 

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