Antigen competitive assays

Chemiluminescent labels may also be used in labeled-antigen (competitive) assays. The antigen (analyte) competes with the labeled analyte for immobilized antibody, and, following a rinse step, reagents are added to generate chemiluminescence from the labels. 

Most modem RJAs utilize a competitive assay format (Fig. 2) in which radiolabled antigen, Ag, competes with unlabeled antigen, Ag, in a sample for binding to the antibody. Ah. The free antigens are then separated from the antigen—antibody complexes, and the amount of radioactivity in the... 

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

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). 

Figure 14.4. Diagram of three basic immunoassay formats, (a) Common competitive format (b) competitive assay with immobilized antigens bound to a carrier protein (immunometric assay) (c) two-site immunometric assays. Haptenic analytes are indicated as triangles, whereas larger-molecular-weight analytes are shown as teardrop shapes. Conjugated fluorescent probes are denoted by the letter "F. ...

The high molar absorptivities and quantum yields of the large protein fluorophore phycoerythrin (240,000 Da) have been exploited in energy transfer assays. Phyco-erythrin has been used as both donor and acceptor, with several bound antigen molecules per phycoerythrin molecule/86,94) The usefulness of BPE is indicated in competitive assays for human IgG that use fluorescein-labeled antibody as donor to... 

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.)... 

A competitive assay could also be used for quantitation. In a competitive assay, unlabeled antigen competes for labeled antigen. Examples include ELISAs for vaccine product antigens, such as recombinant proteins from viruses, or nonvaccine antigens such as growth factors or cytokines. 

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... 

In non-competitive or reagent excess immunoassays [26], an excess of immu-noreagent (antibody or antigen) is used so that all the analyte forms an immunocom-plex that is further quantified and related to the analyte concentration in the sample. These assays are also known as immunometric assays and their advantages over competitive assays include higher sensitivity, precision, and analyte working range. 

The conventional competitive or non-competitive assays do not allow continuous detection so that, for on-line measurements, the so-called displacement assays are usually applied. Typically, the antibody is immobilized onto a solid support and packed in a column, and the corresponding antigen is labeled. The antibody binding sites are saturated with labeled antigens and the sample, containing the free (unlabeled) analyte, is injected into the column, resulting in displacement of the bound labeled analyte, as the affinity of the antibody for the labeled analyte is usually much lower than its affinity for the unlabeled analyte. The displaced labeled analyte is eluted and detected at the outlet of the column and the measured signal is directly proportional to the analyte concentration in the sample. 

Fig. 6.4 A four-parameter logistic standard curve depicting a competitive assay. In this format the antibody (Ab) is present in a limited amount. The known amount of labeled drug competes with the drug analyte in the sample for the limited sites of the Ab. The response factor produced by the label antigen-antibody complex (Ag-Ab) is inversely proportional to the concentration of the drug analyte in the sample. In this example the % B/B0 (% label bound at concentration X divided by...

An immunoassay for TNT and its analogues was carried out on a glass chip. A competitive assay was adopted in which TNT or its analogues competed with fluorescein-labeled TNB (TNB-F1) for the anti-TNT antibody. The ratio of fluorescent intensity of the free TNB-F1 to that of the complex was plotted against the TNT concentration, and the LOD was determined to be 1 ng/mL [285], Dissociation kinetics of the antigen-antibody complex was also studied, thanks... 

Fig. 5 NRL Array Biosensor mixed format immunoassays (modified from [119]). Schematic of (a) the sandwich and (b) the competitive immunoassay fonnats used in the detection of the Campylobacter jejuni and aflatoxin Bi (AFBi), respectively, (a) Sandwich format antigen captured by the immobilized antibody then quantified by passing a second, fluorescently labeled, antibody over the surface, (b) Competitive format competition for binding sites on the fluorescently labeled antibody occurs between the unlabeled antigen in solution and the surface-bound antigen analog, (c) Final charge-coupled devices image taken with the NRL Array Biosensor of a waveguide exposed simultaneously to the C. jejuni (5 x 10" cfu/mL) sandwich assay (SAND) and the aflatoxin Bj (AFBi-1 ng/mL) competitive assay (COMP) in various combinations...

A brief description of techniques used in EIA follows. The various assays have been classified as either competitive or noncompetitive assays, depending on whether or not the technique involves a reaction step in which unlabeled and labeled antigen compete for a limited number of antibody sites (competitive assay) or whether the antigen (or antibody) to be measured is first allowed to react with antibody (antigen) on a solid phase followed by measurement of the binding of enzyme-labeled immune reactant (noncompetitive assay)... 

Determination of Antigen by Two-Step Competitive Assay Antigen can be quantitated by its capacity to inhibit the binding of antibody to the tmtigen adsorbed on the solid phase. The affinity of the antiserum is the most important factor among variables that determine the sensitivity of the assay. To obtain maximal sensitivity in the assay, the amount of antigen used for coating is decreased as far as practicable and the amount of antibody added should be limited. 

The competitive assay uses a limited amount of antibody, which is insufficient to bind all of the antigen. The antigen competes with a fixed amount of labeled antigen for the limited number of antibody binding sites. From the proportion of bound (or free) labeled... 

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