Using Haptenylated Primary Antibodies

The protocol for double/multiple immunolabeling using haptenylated primary antibodies is essentially the same as with primary antibodies of different IgG isotypes. These protocols can be easily customized depending on the availability of primary antibodies for your research requirements. For instance, you may have at your disposal a pair of monoclonal antibodies of the same IgG isotype, and only one of them is haptenylated. In this case, you have to carry out the immunostaining in two steps in the first step you visualize the unlabeled first primary antibody with a secondary species-specific antibody, and in the second step you can detect the second primary haptenylated antibody via another secondary antibody directed against the corresponding hapten. Should the hapten be a fluorophore, it can be visualized directly in a fluorescent microscope and you do not need the second step... 

However, if primary antibodies of the same species belong to different IgG isotypes, they can be selectively detected with secondary antibodies directed against the corresponding isotype (see Sect. 8.2). If the primary antibodies are of the same species and of the same IgG isotype, they can be modified via their haptenylation. Haptenylated primary antibodies are subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (see Sect. 8.3). 

Another approach to this persistent problem relies on haptenylation of primary antibodies. Hapten (e.g., biotin, digoxigenin or any fluorophore) can be covalently bound to the antibody via A-hydroxysuccinimide esters (NHS-ES) (see Sect. 2.1), or conjugated employing monovalent IgG Fc-specific Fab fragments (see Sect. 2.2). Haptenylated primary antibodies can be subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (Fig. 8.5). Fluorophore-labeled primary antibodies can be directly visualized in a fluorescent microscope. 

Haptenylation of Primary Antibodies with the Following Use of Secondary Antibodies Recognizing the Corresponding Hapten... 

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