Noncompetitive heterogeneous

Figure 11.15 Noncompetitive heterogeneous electrochemical immunoassay based on gold NP label and using ASV detection. Adapted from Ref. 50 with permission.

An electrochemical immunoassay has been developed by Chu et al. [75], based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by ASV at a glassy carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an IgG... 

CAE employing antibodies or antibody-related substances is currently referred to as immunoaf-hnity capillary electrophoresis (lACE), and is emerging as a powerful tool for the identification and characterization of biomolecules found in low abundance in complex matrices that can be used as biomarkers, which are essential for pharmaceutical and clinical research [166]. Besides the heterogeneous mode utilizing immobilized antibodies as described above, lACE can be performed in homogeneous format where both the analyte and the antibody are in a liquid phase. Two different approaches are available competitive and noncompetitive immunoassay. The noncompetitive immunoassay is performed by incubating the sample with a known excess of a labeled antibody prior to the separation by CE. The labeled antibodies that are bound to the analyte (the immuno-complex) are then separated from the nonbound labeled antibody on the basis of their different electrophoretic mobility. The quantification of the analyte is then performed on the basis of the peak area of the nonbonded antibody. 

Electrophoretic heterogeneity of whole antibody or even of active Fab proteolytic fragments is still an issue in the development of noncompetitive assay. To get a uniform peak in CE, Hafner et al. [29] proposed using singlechain antibody variable-region fragments (scFv), which can be produced by recombinant technology. 

Assays inherently require the ability to detect unknown levels of antibody or antigen in a biological sample and they may be competitive or noncompetitive, and can also be heterogeneous or homogeneous. 

The three most common t3q>es of reversible inhibition occurring in enzymatic reactions are competitive, uncompetitive, and noncompetitive. (See Problem P7-12b) The enzyme molecule is analogous to the heterogeneous catalytic surface in that it contains active sites. When competitive Inhibition occurs, the substrate and inhibitor are usually similar molecules that compete for the same site on the enzyme. Uncompetitive inhibition occurs when the inhibitor deactivates the enzyme-substrate complex, usually by attaching itself to both tlie substrate and enzyme molecules of the complex. Noncompetitive inhibition occurs with enzymes containing at least two different types of sites. The inhibitor attaches to only one type of site and the substrate only to the other. Derivation of the rate laws for these three types of inhibition is shown on the CD-ROM. 

NNRTls are a chemically heterogeneous group of agents that bind noncompetitively to reverse transcriptase close to the catalytic... 

IAs are classified by several groupings. If the antigen-antibody complex is separated from the unbound reactants before quantitation, it is referred to as a heterogeneous assay. For homogeneous assays, the detection of the bound complexes can be differentiated from the unbound reactants, and no separation process is needed. Another classification is competitive and noncompetitive IA. The measure of occupied or unoccupied Ab determines whether an IA is competitive or noncompetitive, not whether the label is on the Ag or Ab. A broader classification is limited-reagent assay and excess-reagent assay (7,8). They will be discussed in more detail. 

The three most common types of reversible inhibition occurring in enzymatic reaction.s are cotnpeiitive, uncompetitive, and noncompetitive. The enzyme molecule is analogous to a heterogeneous catalytic surface in that it contains active sites. When competitive inhibition occurs, the substrate and... 

This heterogeneous ELISA for DES is, in its present form, noncompetitive ( 5). The single sandwich design of the ELISA (see Materials and Methods) is a necessity due to the sole epitope present on the small DES molecule. DES by itself will not elicit an immune response, thus, it must be conjugated to BSA or another substance (e.g. keyhole limpet hemocyanin) for the production of an antibody. The anti-DES antibody used in this study was produced against a carboxymethylether derivative of DES that was conjugated to KLH. 

Table 1 The plethora of formats of immunoassay. Ab = antibody, Ag = antigen. Noncompetitive and competitive heterogenous binding assays where the probe is labelled with an enzyme are known as enzyme-linked immunosorbent assay (ELISA)...

Figure 4 Schematic representation of (a) heterogenous noncompetitive and (b) heterogenous competitive immunoassays. The noncompetitive format involves the formation of antibody-antigen-antibody sandwich . Signal is proportional to the sites occupied by analyte. The competitive format involves the analyte competing with probe (here, labelled antigen) for the available sites. Signal is proportional to sites not occupied by analyte.

Currently, one of the most sensitive EIA methods is the immune complex transfer immunoassay, which is a heterogeneous and noncompetitive EIA. This system can measure zeptomole per assay levels of protein antigens and antibodies as well as attomole per assay levels of several haptens. In this method, the primary immune reaction occurs on a solid support, after which the immune complexes are specifically... 

In some aqueous polymer solutions, hydration is noncompetitive with association. For instance, in solutions of telechelic polymers, main chain hydration only indirectly affects the end-chain association. There is interference only in the region very close to the chain end. Dehydration and chain collapse start near the core of the flower micelles in the form of heterogeneous nucleation. The solutions with such coexisting hydration and association turn into gels on cooling (low-temperature gelation), while they phase separate at high temperatures. 

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