Antigens antigen competition

The microplate ELISA testis conducted in standard 96-well microplates. A microplate consists of a 12 X 8 grid of wells for test solutions. The three most widely used ELISA formats are immobilized antigen competitive immunoassay, immobilized antibody competitive immunoassay and sandwich immunoassay. " ... 

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. 

FIGURE 16.9 Principle of reagentless amperometric immunosensor based on immobilized antigen, competitive immunological reaction, and direct electrochemistry of HRP label (adapted from [138]). 

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

Meulemans EV, Slobbe R, Wasterval P, Ramaekers FC, van Eys GJ, Selection of phage-displayed antibodies specific for a cytoskeletal antigen by competitive elution with a monoclonal antibody, J. Mol. Biol., 244 353-360, 1994. 

Ag reaction with catalase-labeled antigen. After competitive binding of free and catalase-labeled AFP, the sensor is examined for catalase activity by ampero-metric measurement after addition of H 2O2. AFP can be assayed in the rjuige 10-0.01 ng/mL. 

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. 

One of the earliest efforts of qualitative measurement of a protein (human serum albumin) in a microchip-based device was based on bead agglutination in a microchamber (approximately lOjaL). Subsequently, several quantitative immunoassays have been performed using microchip electrophoretic systems that permit separation and quantitation of free- and bound-labeled antigens in competitive assays (see Chapter 5). Most are carried out in channels micro-machined into fused silica substrates. Early work on quantitative assays achieved measurement of cortisol in serum.The assay used cortisol labeled with fluorescein and an argon laser detector at 488 nm and required only 80 pL of a 40x dilution of serum as the sample. Other capillary electrophoresis-based assays for a variety of antibodies have also been developed that include immunoglobulins (IgG, IgA, and IgM), antibovine serum albumin, and antiestradiol. ... 

In CE, analysis by immunoassays depends on the principles that the antigen and antibody migrate differently when they are bound compared to when they are free. One of these two compounds (mostly the antigen) is labeled with a fluorescent tag. The unknown sample is mixed with labeled antigen for competitive binding assay and the mixture is separated by CE. The label in the bound fraction (or... 

Figure 4.2 Quantitation of antigen by competitive (inhibition) assay. An antigen is fixed (attached) and binds a specific antibody from solution. However, additional antigen is provided in solution and antibody binding to the bound antigen is reduced in direct proportion to the concentration of free antigen. The bound antibody is then detected by binding a second, labeled (enzyme, isotope, etc.) antibody.

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