IgG isotypes

Administration of Tyv-specific monoclonal antibodies to rat pups already infected with intestinal larvae causes larvae in the epithelium to be expelled (Carlisle et al, 1990). Only the LI stage is susceptible to expulsion once the larva has moulted to L2 it resists the effects of the antibodies (Carlisle et al, 1990). Expulsive immunity is transferred by three IgG isotypes, F(ab )2 fragments, as well as IgM (Carlisle et al., 1991a). These findings argue against a role for Fc-mediated effector functions and imply that antibodies against Tyv can disturb the larva s niche in a direct fashion. 

Bell, R.G., Appleton, J.A, Negrao-Correa, D.A. and Adams, L.S. (1992) Rapid expulsion of Trichinella spiralis in adult rats mediated by monoclonal antibodies of distinct IgG isotypes. Immunology 75, 520-527. 

Basic protocol for simultaneous immunoenzymatic double staining using primary antibodies of two different species or two different IgG isotypes... 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted primary antibodies raised in two different species (e.g., rabbit and mouse) or belonging to two different IgG isotypes (e.g., IgGl and IgG3). Wash sections in PBS or TBS for 3 x 3 min. 

Secondary antibodies incubate sections for 60 min at room temperature with a mixture of secondary antibodies conjugated with different enzymes (e.g., HRP and AP) and raised against the corresponding species IgG or IgG isotypes. Wash sections in a buffer recommended for developing the first enzyme label. The more stable HRP label should be developed before AP. 

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). 

When primary mouse monoclonal antibodies are of different IgG isotypes/ subclasses, they can be selectively detected with secondary antibodies directed against the corresponding IgG isotype (Fig. 8.2). 

Bound 1 st AB from different IgG isotypes are detected with 2nd AB directed against the corresponding IgG isptype... 

Fig. 8.2 Schematic presentation of double immunostaining using secondary antibodies (AB) raised against corresponding IgG isotypes...

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... 

Attachment via affinity interactions. These methods are based on the high affinity of Fc receptors, such as protein A (PrA), protein G (PrG) or recombinant protein A/G, for the amino groups in the Fc region of antibodies. In these procedures, the Fc receptor is immobilized on a solid support and is the key element for the oriented immobilization of the antibody (Fig. 5b). However, not all Fc receptors have the same affinity to all IgG isotypes. PrA has been successfully used to bind the Fc portion of antibodies from many mammalian species, except for goat, sheep, cow and horse. PrG has some advantages over protein A, as it reacts with more IgG isotypes and to a lower extent with other immunoglobulins, such as human IgM and IgA. How-... 

The antibody most frequently used for the preparation of ITs belongs to the IgG isotype of murine monoclonal antibodies (MAbs). However, Fab fragments as well as chimeric mouse-human IgG antibodies have also been used (16). 

Murine IgG does not bind to human FcRn, and this explains the shorter half life of murine antibodies in human patients, typically 12 to 48 h [91]. The half lives of endogenous human IgG isotypes have been well studied and they do differ - 3 weeks for IgGl, IgG2 and IgG4, while IgG3 has a half-Hfe of 1 week [88, 92]. 

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