Primary 1 Antibodies

Competitive inhibition. A constant amount of anti-analyte antibody (primary antibody) and a series of solutions containing increasing amounts of analyte are added to the prepared microtiter plate wells. During incubation, the free analyte and bound... 

Visualizing more than one epitope on one section can be accomplished by different fluorescence labeling or different sizes of colloidal gold coupled to primary or secondary antibodies. Primary antibodies from different species and adequate secondary antibodies labeled differently can be used. In case of primary antibodies from the same species, the hapten technique can be applied. A hapten is a small molecule that can be bound to antibodies dinitrophenol and arsinilate are typically used as haptens. Again, adequate secondary antibodies labeled differently can be used (14,17,32). A collection of protocols for multiple immu-nolabeling has been described by Beesley (37). 

Indirect methods for immunofluorescent detection of multiple tissue antigens in their simplest form make use of primary antibodies that are raised in different species. Bound primary antibodies can accordingly be visualized with differently labeled species-specific secondary antibodies. Primary antibodies to a pair of different antigens (e.g., rabbit anti-antigen A and mouse anti-antigen B) can be mixed and applied as a cocktail. The same is valid for a mix of secondary antibodies (e.g., goat anti-rabbit-FITC and goat anti-mouse-Cy3) as shown in Fig. 8.1. 

Primary and secondary antibodies Primary antibodies are raised against the antigen under study, whereas secondary antibodies are raised against the corresponding IgG species or isotype of the primary antibody. 

Biotinylated Secondary Antibody Primary Antibody Tissue Antigen... 

Fig. 12. Noncompetitive hapten immunoassay procedures (A and B) using a combination of the a-type and j6-type anti-idiotype antibodies, each recognizing the framework and paratope of the anti-hapten antibody. Anti-hap, anti-hapten antibody (primary antibody) a-Id, a-type anti-idiotype antibody /J-Id, /i-type anti-idiotype antibody S, signal-generating group B, biotin SA, streptavidin.

Incubate with primary antibody Primary antibodies. 

At this point, the anti-/3-galactosidase antibody (primary antibody) diluted in blocking buffer is added to the well. During the incubation, it will bind to the /3-galactosidase adsorbed to the bottom of the well. Several washes are then performed to remove primary antibody that may have bound nonspecifically to proteins on the bottom of the well other than /3-galactosidase. As in the Western blot experiment, the wash solution contains a small... 

Why has the two-antibody (primary and secondary) technique become so popular for use in immunochemistry experiments such as the Western blot Enzyme-conjugated antibodies that recognize... 

Anti-Peroxidase Antibody Peroxidase Enzyme Anti-Peroxidase Antibody Secondary Linking Antibody Primary Antibody... 

Finally we want to stress that, whenever it is possible, all antibodies, primary as well as secondary, should be affinity purified to obtain the best results. 

The sandwich immunoassay is the most commonly used analytical technique for the detection and quantification of specific proteins of interest (Jiang et al., 2008 Qiu et al., 2009). In this type, the antigen is sandwiched between two antibodies (primary and labeled secondary antibody) as shown in Figure 4.10. A constant amount of primary antibody (capture antibody) specific for the antigens is first coated on the microplate/ sensor surface. After coating, a series of dilutions of the antigens (proteins) standards... 

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