ELISA competitive assays

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. 

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

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. 

Competitive binding immunoassay (e.g., RIA) Enzyme-linked immunosorbent assay (ELISA) Immunoradiometric assay (IRMA) — dual monoclonal antibody assay Receptor binding assay Cell binding assay... 

The competition assay was designed which followed the standard indirect ELISA format (17-18 . The methoprene conjugate was bound to a solid support in the form of a microtiter plate. Free methoprene in methanol (5 fib) was added to the pre-coated wells followed by methoprene-specific antiserum. The antibodies were allowed to compete for both immunogen-bound and free methoprene. Enzyme-conjugated, goat-antirabbit antibody was added, followed by substrate, and the color was allowed to develop. The absorbance of substrate over a range of methoprene concentrations can be drawn as a standard curve, which is presented as percent inhibition of the assay (Figure 6). The 50% inhibition (I5g) of methoprene was at a concentration of approximately 50 ng/mL. 

Many types of assay are available to be used in HTS protocols to identify inhibitors of PPIs, but a competition assay, in which inhibition of complex formation is measured, is most common. Fluorescence polarization (FPA), fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assays (ELISA), and other assay formats have been used. The interacting proteins can be used in their full-length forms though, more frequently only the interacting domains are employed, and if possible the excised interacting peptide is usually preferred. 

Table 3. Specificity of anti-CCK antisera raised with CCK-10 and CCK-13 iso-1-cytochrome c conjugates in guinea pigs as determined by competition assays using ELISA techniques. Crossreactivities of CCK- and gastrin peptides are expressed by the respective IC50 values using CCK-12 as coated antigen.

Figure 2 Decoding of active peptide sequence by activity screening of pools of sublibraries in competition assays. The ability of sublibraries to interfere with a receptor/ligand interaction is measured in a competition assay using ELISA microplates coated with the target receptor. Identification of the sublibrary with the highest inhibitory activity will lead to the characterization of the first residue responsible for activity. Resynthesis of sublibraries with the known first residue and subsequent screening of their activity will lead to the identification of the second residue responsible for activity. The process is repeated until the full sequence is decoded. w...

This stabilization effect appears to be mediated by the C-region scaffolds on which the V-regions sit and, critically, by the disulphide bonds that covalently join the C-regions. It has recently been shown that the Fab antibody format is likely to be the most reliable and sensitive format for use in small molecnle competition assays. The strict monovalency of the Fab format can lead to 2-12 fold improvements in assay sensitivity in both ELISA and competition SPR analyses [121],... 

Competitive enzyme-linked immunosorbent assays (ELISA) Two types of ELISA have been used for the analysis of mycotoxins and both types are heterogenous competitive assays. One type, i.e. direct ELISA, involves the use of a mycotoxin-enzyme conjugate and the other system, i.e. indirect ELISA, involves the use of a protein-mycotoxin conjugate and a secondary antibody to which an enzyme has been conjugated. Although horseradish peroxidase (HRP) is most commonly used as the enzyme for conjugation, other enzymes such as alkaline phosphatase and beta-galactosidase, also have been used (5, 9, 13). 

Among the very few examples for chemical signal amplification after analyte binding to the MIP site, a secondary reaction in a competitive ELISA-analogous assay had been established with a glued MIP particle film [4], Furthermore, with an about 100 nm thin protein-imprinted functional polyaniline film, the enzyme activity of bound peroxidase was used to retrieve quantitative data about MIP site capacities [62]. 

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