Immunoassay competitive assays

Fig. 13. General protocol for heterogeneous enzyme immunoassay preparation of reagent cuvettes by coating Ab, competitive assay format, and sandwich assay format. (Reprinted with permission from W. R. Heineman and H. B. Halsall, Anal. Chem, 1985, 57, 1321A. Copyright 1985, American Chemical Society)...

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],... 

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 Abbott IMx , a dedicated commercial immunoassay analyzer that employs FPIAs for small molecules, can also determine larger analytes by a fluorescence-based microparticle capture enzyme immunoassay (MEIA).(44) In this system, antibody-coated0.47- mlatexparticles are used for both sandwich and competitive assays, and alkaline phosphatase conjugates that bind to the particles cleave 4-methylumbelliferyl phosphate to generate the fluorophore. 

Schmerr and Jenny established a CE-based immunoassay for the detection of prion protein (24). In this competitive assay, peptides derived from the prion protein and labeled with fluorescein were used. This allowed them to distinguish scrapie-infected brain preparations from noninfected. For identification, the ratio between the peaks resulting from the free and the com-plexed peptide with a specific antibody was used. The results were in agreement with other data on the brain preparations achieved by Western blot analysis. The CE-based assay provides the advantage of direct detection of the scrapie protein in blood and tissue preparations with high sensitivity. Furthermore, due to the small sample amount needed for analysis, the CE-based assay is applicable to the putative diagnostics of prion protein in body fluids. 

Fig. 8 On-chip immunoassay for the quantification of cortisol in serum samples. Fluorescein-labeled cortisol (Ag ) was used in a competitive assay for the detection of unlabeled analyte cortisol. Three replicate injections for each of three samples with cortisol levels as indicated in the figure. Fluorescein was used as an internal standard (I.S.). (Reprinted with permission from Ref. 32. Copyright 1996 American Chemical Society.)...

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 competitive immunoassays, as the name indicates, are based on a competition reaction between two reagents for a third one. A competitive assay can be carried out in two different ways ... 

Figure 12 Examples of immunoassay systems and DNA assays performed with magnetic bead technology (a) sandwich assay (b) competitive assay (c) bridge assay to determine proteins (d) DNA and RNA assays.

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... 

Alternatively, Briggs et al. (B4) reported a fluorescent immunoassay based on the correlation of fluctuations in particle number measures of the amount of tagged species bound to micrometer-sized beads. A homogeneous competitive assay based on this principle can detect 1 ng of gentamicin per ml from a total sample volume of 10 p.1. 

Cyclooxygenase catalyzes the first step in the biosynthesis of arachidonic acid to PGH2. PGF2a produced from PGH2 by reduction with stannous chloride, is measured by enzyme immunoassay. This assay is based on the competition between PGs and... 

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...

Fig. 8.1. Immunoassays using antibodies. A. Competitive assay. B. Non-competitive sandwich" assay.

In principle, immunoassays with labelled compounds can be carried out in the following ways, (a) In competitive assays, the labelled analyte competes with the unlabelled analyte for the epitope-binding site of the antibody. The key feature of a competitive assay is that maximal assay sensitivity is attained... 

Bound fraction Antigen/competitor present as a complex, attached to the corresponding antibody the fi-action of the reaction mixture which contains the antigen-antibody complex Carrier protein Large non-immunogenic protein that, when coupled to a hapten, induces production of anti-hapten antibodies in an animal Competitive assay Immunoassay based on the principle of competition between the analyte in the sample ( unknown ) and competitor Competitor Antigen derivative that is modified (i.e., labelled, coupled to a large protein) and serves for indirect detection of the analsde in competitive immunoassays... 

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