Competitive immunoassay steps

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. 

I.A. Darwish and D.A. Blake, One-step competitive immunoassay for cadmium ions development and validation for environmental water samples. Anal. Chem. 73, 1889-1895 (2001). 

In contrast with sensors, sensing systems are ideal for exploitation of immunochemical selectivity. This accounts for various highly sensitive and successful competitive immunoassays. Incorporation of the manipulative step(s) opens the door for regeneration of the antibody or even for operation under virtually reversible conditions. 

In an indirect competitive immunoassay, the competitor is commonly immobilized on a solid surface while the antibody and the analyte are added in the adjacent solution. After the competition, the fractions that are unbound to the solid surface are removed and the bound primary antibody is usually measured by the addition of a labelled secondary antibody (see Fig. 9.7). Even though this assay format is not as frequently used as the direct competitive immunoassay, it provides an advantage when analysing complex samples, i.e., the label (usually an enz5rme) does not come into direct contact with the sample matrix, so that interferences with the detection step are minimized. 

S Separation of fractions in heterogeneous immunoassay Separation of free from antibody-bound analyte/competitor fractions in heterogeneous immunoassays is very seldom complete and the efficiency of fraction separation has a direct effect on the quality of the assay. For example, in competitive immunoassays that employ a labelled analjde derivative as tracer and measurement of the antibody-bound fraction, interferences with the separation step can be due to ... 

Immunoassay is well acknowledged since the introduction of ELISA (enzyme-linked immunosorbent assay) which was developed over 20 years ago. ELISA can be classified into three formats direct, sandwich, and competitive [2]. Direct ELISA is a simple process that the excessive antibody is reacted with antigen. After incubating, a washing step is conducted to eliminate the unbound antibody. The sensitivity is proportional to the amount of the present antibody in the solution. In a sandwich immunoassay, the antigen (e.g., sample) is between the primary antibody and the second antibody (detection antibody). Generally, the primary antibody is immobilized on the solid surface and the second antibody is labeled with certain enzyme or fluorophore. The amount of sample is proportional to the amount of the labeled second antibody which is measured by the different detection strategies. In a competitive immunoassay, the... 

For a competitive immunoassay, a fixed quantity of tracer is incubated in the presence of a fixed (lesser) quantity of antibody and variable quantities of the analyte. After incubation, the free and bound fractions of the tracer are separated. Quantification of one or other of the fractions allows calculation of the amount of analyte present. This technique is partictdarly suited to hapten assay. This assay method, requiring a separation step, is known as a heterogeneous assay. In a few particular cases, the free and bound fractions of the tracer give different signals, eliminating the need for the separation step. This is known as a homogeneous-phase assay. 

Once specific antibodies of the proposed analyte and a tracer have been obtained, an analytical method must be chosen to quantify the tracer. The next step in developing the immunoassay is to establish a dilution curve for the antibodies, obtained by incubation of a fixed quantity of tracer in the presence of variable dilutions of antibodies. From this it is possible to determine the titer of the antibodies (the inverse of the dilution of antibodies binding 50% of the tracer), which gives an indication of the quantity of specific antibodies of the analyte contained in the antiserum. Finally a calibration curve must be established to allow quantification of the analyte. In the case of a competitive immunoassay, this is obtained by incubation of a fixed quantity of antibody (equal to the titer value) in the presence of a fixed quantity of tracer (able to bind -50% of the antibody) and variable quantities of the analyte to be assayed. Note most of the organometallic complexes reported here are insoluble in water and in the buffers used for immunological analyses, but their solubility in these media can easily be attained by addition of a small percentage of alcohol (-5%). 

Avidin (an egg yolk protein) exhibits an extremely strong afEnity (ca. lO L/mol) for biotin (vitamin H). This irreversible interaction can be utilized in the design of heterogeneous immunoassays with biotinylated. antigen-specific antibodies [68], [69]. Biotinylation can be performed without interfering with antibody function. The antibodies are applied in the usual manner in a competitive immunoassay (Fig. 8). In the last step, enzyme-labeled avidin is added in excess. Since there are four possibilities for the binding of avidin... 

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). 

Vilja, P. (1991) One- and two-step non-competitive avidin-biotin immunoassays for monomeric and het-erodimeric antigen./. Immunol. Meth. 136, 77. 

Figure 3 Layout of competitive solid-phase antibody immunoassay format in an incubation step antigen (Ag) and labeled antigen (Ag-L) compete for the solid-phase antibodybinding sites (Ab) after the solid-phase antibodies are washed, the activity of the bound labeled antigen is measured, and a calibration curve constructed. The measured signal is inversely proportional to the antigen concentration.

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors require multiple steps for analyte determination, and either sandwich assays or competitive binding assays may be used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. 

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