Direct ELISA labeled antibody

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. 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

Radioimmunoassay (RIA), like ELISA, is based on the radioactive labelling of the antibody molecules. The labelled antibody reacts with the antigen present in the tube the amount of radioactivity present in the bound complex is directly proportional to the amount of antigen added to the tube . ... 

As shown in Fig. 21, in a direct ELISA the unlabeled antigen (a range of standard antigen concentrations or unknown samples) is attached to the solid phase. Enzyme-conjugated (labeled) primary antibody is then added. After incubation and washing of the plate... 

Fig. 21. Principles of ELISA. A In a direct ELISA the unlabeled antigen is attached to the solid phase. Enzyme-conjugated antibody is then added, followed by the enzyme substrate solution and color is allowed to develop. B ELISA with unlabeled antibody attached to the solid support. A variable amount of antigen is then added. A secondary antibody labeled with enzyme, followed by substrate solution, is added to all wells. The amount of color produced is proportional to the amount of antigen present. C Sandwich ELISA assay with the antigen sandwiched between an immobilized, antigen-specific primary antibody and an antigen- or species-specific secondary antibody. An enzyme-labeled tertiary antibody increases the assay specificity and sensitivity...

For ELISA, an enzyme is linked chemically to the antibody. The labeled antibody is allowed to bind to the unlabeled antigen, under conditions where nonspecific adsorption is blocked, and any unbound antibody and other proteins are washed away. Binding is detected by a reaction that converts a colorless substrate into a colored reaction product. The color change can be read directly in the reaction tray, making data collection very easy, and ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for most direct-binding assays (Plested et al. 2003). 

Noncompetitive ELISA. The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtitre plates a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Figure 8.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed activity is directly related to the concentration of antigen. 

Fig. 3. Comparison of different enzyme-linked immuno sorbent assay (ELISA) methods adapted for immuno-polymerase chain reaction (IPCR). Dependent on the purification grade of the sample to be analyzed and the availability of specific and functionalized antibodies, several typical ELISA protocols were adapted to IPCR. In the direct approach (A), the pure antigen is immobilized to the microplate surface and subsequently detected by a labeled specific antibody. If no labeled antibody is available (e.g., because of unpurified ascites fluid containing the antibody or loss in activity following labeling), a standardized labeled secondary species-specific antibody is used for detection of the primary antigen-specific antibody (B). For the detection of the antigen from matrices such as serum, plasma, tissue homogenate, and so on, a capture antibody immobilized to the microplate surface was used either in a direct (C) or indirect (D) sandwich approach, with the latter one additionally including a secondary species-specific detection antibody. For different methods of coupling antibody and DNA, abbreviated by in this figure, compare Fig. 2. Note that protein A chimeras (Fig. 2A) are not compatible with capture antibodies (Fig. 3C, D).

There are two forms of direct ELISA, namely labelled-antibody and labelled-antigen. Both follow a similar protocol, but as the former is arguably the most widespread it is described below (Eigure 10.8). 

Since a single enzyme-conjugated antibody is used, the system is limited to the specificities and properties inherent in that particular antibody set. This limits the versatility of the testj e.g., each antibody preparation used must be labeled (for different antigens) in the same way as the direct ELISA was limited to single antibody preparations. 

Again there is a requirement to titrate the direct ELISA system, which is then challenged by the addition of test antibodies. The competitive aspect here is between any antibodies in the test sample and the labeled specific antibodies for antigenic sites on the solid-phase bound antigen. The test sample and... 

Direct I-ELISA for antigen testing is not an available alternative since test antigen has to be mixed with pretitrated labeled antibody Thus, competitive conditions apply. One variation is that test antigen can be premixed with the labeled antibody and incubated for a period before the mixture is applied to the antigen-coated plates. In practice, this makes no difference to the assays in which antigen is added to the coated plates initially. 

Direct C-ELISA for antibody. The degree of inhibition by the binding of antibodies in a serum for a pretitrated enzyme-labeled antiserum reaction is determined. 

Direct sandwich competition ELISA for antibody. This system exploits the competition of antibodies in a sample for the binding of a pretitrated quantity of labeled antibody specific for the antigen captured by the coating antibodies on the wells. The extent of competition depends on the relative concentrations of the test and labeled antibodies. 

The direct sandwich I-ELISA for antibody is as described for the previous competitive system except that the sample under test is added to the captured antigen for a time preceding the addition of the labeled antibodies. Following this incubation step, there are two alternatives. The first is to add the pretitrated labeled antibodies directly to the reaction mixture followed by incubation. The second is to wash the wells, thereby washing away any excess test antibodies before the addition of labeled antibodies. For each alternative, there is an incubation step for the labeled antibodies followed by washing and then addition of... 

Sandwich ELISA Direct (3 components) Antibody + Antigen + Labeled Antibody... 

The indirect aspect therefore refers to the fact that the specific antiserum against the antigen is not labeled with an enzyme, but a second antibody specific for the particular species in which the first antibody was produced is labeled. Such assays offer flexibility and form the bases of other ELISAs. In principle, the optimization of reagents is similar to the direct ELISA. However, three factors have to be considered ... 

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