ELISA noncompetitive

Noncompetitive ELISA methods are based on sandwich assays in which an excess supply of immobilized primary antibody, the capture antibody, quantitatively binds the antigen of interest and an enzyme-labeled secondary antibody is then allowed to react with the bound antigen forming a sandwich. A color reaction product produced by the enzyme is then used to measure the enzyme activity that is bound to the surface of the microtiter plate. Sandwich ELISA (noncompetitive) methods yield calibration curves in which enzyme activity increases with increasing free antigen concentration. 

ELISA assays may be competitive or noncompetitive. As the name imphes, in a competitive ELISA, enzyme-labeled antigen competes with free antigen (the analyte of interest) for a fixed and limited quantity of immobihzed antibody binding sites. After incubation, the microtiter plate (sohd support) is rinsed to remove all unbound species and the enzyme substrate is added in saturating concentration. The conversion of substrate to produce can be measured continuously (kinetic assay) or, more commonly. 

Very recently, a new noncompetitive assay principle called an open sandwich ELISA has been reported (S7) (Fig. 15). The assay mechanism could be regarded... 

Noncompetitive ELISA. The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtitre plates a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Figure 8.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed activity is directly related to the concentration of antigen. 

This method is further divided into two major types. One type is competitive ELISA, which can be used for the analysis of both hapten and macromolecule the other is noncompetitive sandwich-type ELISA, which is only used for divalent and multivalent antigens. Two major types, i.e., direct competitive ELISA (dC-ELISA) and indirect competitive ELISA (inC-ELISA), are used most commonly in food analysis. 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

Both competitive and noncompetitive ELISA methods employ microtiter technology for automation. Robotics are used for reagent addition and timing, while... 

More recently, noncompetitive immunoassays (IRMA, ELISA, and ICMA) have been reported for calcitonin. Com-... 

More recently, noncompetitive methods have been developed for measuring human OC. These immunoassays use monoclonal antibodies and affinity-purified polyclonal antisera against synthetic peptides of human OC or intact human or bovine OC. Many antibody pairs have been reported including capture [and signal] antibodies, respectively, against amino acid sequences 12-33 [and 34-49], 43-49 [and 5-13], 25-37 [and 5-13], 20-43 [and 7-19], 1-19 [and 20-49], and 1-12 [and 15-30 or 38-49], These noncompetitive methods have used signal antibodies labeled with radioactivity (IRMA), enzymes (EIA/ELISA), or chemiluminescent compounds (ICMA). 

Noncompetitive sandwich ELISA. The bound AbE increases in proportion to the amount of Ag. 

Among noncompetitive assays, sandwich ELISA is one of the most commonly used (Figure 12.2). Capture antibodies bound to the plate bind the target in the first step of... 

FIGURE 12.2 Noncompetitive immunoassay (A) direct ELISA (B) enhanced assay using a biotin-streptavidin system (C) indirect ELISA. 

ELISA Technologies, Inc., ELISA-TEK, noncompetitive. Casein, whey 25 mg/kg NA... 

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. 

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