Competitive assays enzyme-labeled antibody

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

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. 

In this mode, a fixed amount of unlabelled antigen (Ag) is bound to microtitre plates. A food sample containing antigen is added, followed by a fixed amount of enzyme-labelled antibody (Ab-E) (Figure 8.2a). There is competition between the fixed and free antigen for the limited amount of Ab-E. After an appropriate reaction time, unbound Ag (and other materials) are washed from the plate and the amount of bound enzyme activity assayed. As above, the amount of enzyme activity is inversely proportional to the concentration of antigen in the food sample. 

A disadvantage of both competitive assays is that the enzyme conjugate solution has to be mixed with the sample solution. A sample solution, however, may contain inhibitory substances such as proteases that can alter the activity of the antibody and/or the enzyme-label used. Where enzyme-labeled antibodies are employed, such problems may be circumvented by using an unlabeled antibody in the competition phase, followed by incubation with an enzyme-labeled second antibody that is directed against the primary antibodies (109). 

The analytical detection limits of competitive and noncompetitive immunoassays are determined principally by the affinity of the antibody and the detection limit of the label used, respectively. Calculations have indicated that a lower limit of detection of lOfmol/L (Le., 600,000 molecules of analyte in a typical sample volume of 100 jiL) is possible in a competitive assay using an antibody with an affinity of iO L/mol. Table 9-2 illustrates the detection limits for isotopic and nonisotopic labels. A radioactive label, such as l, has low specific activity (7.5 million labels necessary for detection of 1 disintegration/s) compared with enzyme labels and chemiluminescent and fluorescent labels. Enzyme labels provide an amplification (each enzyme label produces many detectable product molecules), and the detection limit for an enzyme can be improved by replacing the conventional photometric detection reaction by a chemiluminescent or bioluminescent reaction. The combination of amplification and an ultrasensitive detection reaction makes noncompetitive chemiluminescent EIAs among the most sensitive types of immunoassay. Fluorescent labels also have... 

ELISA ELISA is based on the principle of competition between enzyme-labeled and vmlabeled antigens for a specific antibody present in a limited concentration in the assay mixture. The labeled antigen and antibody form a reversible complex. The complex is then detectable by assay of the Hnked enzyme. Since a pure sample of antigen is required for preparation of standards, the method has not been applied extensively to the determination of enzyme concentrations. 

Heterogeneous EEIA takes place on the surface of a solid matrix where the molecules of the antibody used are bound. The binding of antibodies to a solid support enables easy separation of free and bound enzyme labels. Two main alternatives to heterogeneous EEIA are known competitive and uncompetitive (sandwich) assay (Fig. 17). In the competitive assay a labelled analyte competes with the unlabelled... 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

The competitive assay is another format used to quantitate an analyte. An unlabeled analyte competes with a labeled analyte (enzyme-conjugated molecule) for binding to a specific capture antibody (Figure 11.1c). 

Direct competition. The solid phase (a microtiter plate) is coated with an antibody specific for the antigen being assayed. The sample and enzyme-labelled antigen (antibiotic) are added. There is a competition for the antibody between the labelled and unlabelled antigen (antibiotic). Substrate is added and the color produced by the enzymatic hydrolyse is inversely proportioned to the concentration of antigen in the sample... 

On the other hand, the main types of immunoassays that can be performed by using labelled antibodies or antigens are direct sandwich, competitive and indirect assays. The labels can be enzymes (alkaline phosphatase, peroxidise or glucose oxidase) metal NPs (gold) fluorescent or electrochemiluminescent probes. 

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