Antigen presentation plate

Antibodies raised against the antigen of interest (i.e. the therapeutic protein) are first adsorbed onto the internal walls of microtitre plate wells. The sample to be assayed is then incubated in the wells. Antigen present will bind to the immobilized antibodies. After an appropriate time, which allows antibody-antigen binding to reach equilibrium, the wells are washed. 

ELISAs can be used in two modes, qualitatively to determine the presence or absence, or quantitatively to determine the amount of antigen present. ELISA kits often depend on the adsorption of either the antibody or antigen to a solid phase, e.g., wells of a microtiter plate, surface of plastic beads, or plastic stick. The choice of antibody (or antibodies) used determines the specificity of the ELISA assay, which can range from genus-specific to strain-specific. The principle on which ELISA methods are based usually prevents them from being used for the determination of total microbial counts. However, they can be used to detect pathogens such as Salmonella spp.. Listeria spp. 

Antibody present in liquid binds to the antigen.The plate is washed. 

Fig. 8. Schematic presentation of a enzyme linked immunosorbent assay (ELISA). An antigen ( ) is immobilized on the surface of a microtiter plate and incubated with its antibody (abl). A second antibody (ab2) with a covalently linked enzyme ( , e.g., horseradish peroxidase) binds to the primary one and catalyzes a color reaction with its enzyme. All incubations are separated by washing steps...

In the indirect format (see Fig. 2b), the coating antigen is coated on the plate, but in this case the amount of analyte present in the sample is indirectly measured by measuring the bound Ab with a second Ab that is conveniently labeled (AntilgG-enzyme). Although this format has a step more, it has often proved to be more robust. 

Fig. 20c. 1. ELISA assay, (a) Antibodies to the drug of interest are secured to a solid substratum such as a test tube or micro-well plate. The sample containing the analyte antigen is added to the reaction surface, (b) After the analyte has bound to the antibody, the vessel is rinsed to remove unbound antibody. A second antibody to the analyte is added. This antibody has a bound enzyme which has been chosen because its reaction produces a colored product which can be detected spectrophotometrically. (c) After this second antibody has bound to the first antibody-antigen complex, the surface is again rinsed to remove unbound-antibody enzyme. The enzyme substrate is added in sufficient excess such that the rate of product formed is proportional to the amount of enzyme present. The enzyme-linked assays are very sensitive, since each enzyme can rapidly catalyze thousands of substrate to product reactions.

The sandwich assay is the format used most often to quantitate a target antigen or analyte. In the sandwich assay, two antibodies are used that bind to different parts of the antigen. One of the antibodies is bound to, or coated on, the solid surface (mictotiter plate wells), whereas the other has a label attached to it (Figure 11.1a). Alternatively, a secondary conjugated antibody can be used to detect the bound primary antibody (Figure 11.1b). If the antigen is present in the sample solution, it links the two antibodies. Therefore, the label is retained on the plate where it can be detected by use of a colorimetric substrate. 

Antibody inhibition. Antibody is preincubated with the sample being assayed. If any antigen is present in the sample it will bind with antibody. When the assay mixture is added to a microtiter plate coated with antigen, there is a decrease in the intensity of the color produced. 

To perform a microplate ELISA for the measurement of antigen, plates sensitized with the specific antigen are Incubated with a mixture of reference antibody and the test sample. If antigen is present in the test solution, it combines with the reference antibody which cannot then react with the sensitized plate. The amount of antibody attached to the solid phase is then indicated by... 

Fucose was tested as an inhibitor for both types of antibodies by the micro-inhibition test. These results are presented in Fig. 16. The center well of plate A contains native anti-fucose antibodies, with the native antibodies diffusing against decreasing concentrations (20 to 1 jug) of Fuc-BSA in the outer wells (1 to 6). The anti-fucose antibody yielded precipitin bands with 4 concentrations of antigen. Antibodies incubated with L-fucose (plate B) yielded a precipitin complex only at the highest concentration of antigen. A calculation from the concentrations shows that the L-fucose... 

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