Direct ELISA principles

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...

Direct ELISA was extensively described because it introduces the investigator to the ELISA. Many of the areas covered will need less explanation, so that protocols shown for ELISAs will have less detail. The major use for the direct ELISA is to be able to titrate antispecies conjugates and thus avoid using preparations that are too strong or two weak. Some of the major principles of ELISA were introduced Cplateau height, end point, nonspecific reactions, backgrounds, and titration curvesj and they will be constantly reviewed in all the assays described. 

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 ... 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

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. 

The most popular form of this technique is solid-phase heterogeneous ELISA, and the inherent use of passive adsorption facilitates flexibility in assay design. ELISA is generally classified as direct, indirect, sandwich (double-antibody) or competitive. Principles of each of these formats are briefly discussed below. 

Fig. 2. Principle of ELISA. Antibodies elicited against a hapten-KLH conjugate are tested against a hapten-BSA conjugate. Only hapten-specific antibodies are detected. A blocking agent is used to prevent direct binding of antibodies to the solid support...

The ETA for mycotoxins has been used since the late 1980s. It is based on the principle of the direct competitive ELISA. Anti-mycotoxin antibody is coated on the surface of a membrane. 

The direct, indirect, and capture ELIS As have now been examined. You should be able to optimize the conditions of the tests and be able to use them to measure antigen or antibody in a variety of formats. Competitive ELISAs involve the principles of all these types of assay. 

A typical principle of direct competitive ELISA is shown in Figure 11.3. After an aflatoxin is extracted from a ground sample with solvent, a portion of the sample... 

Figure 5. Principle of a direct competitive enzyme immunoassay (ELISA) with photometric detection...

On the plant, PPA are exposed to light and can undergo photolysis. An example is parathion (XXXVII). Nitrosoparathion is formed, among other compounds, and reacts with components of the cuticle. The resulting product is insoluble and is not detected by the conventional PPA analysis. It can be identified directly on the cuticle with the help of the ELISA technique (principle, cf. 2.6.3). 

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