Monoclonal antibodies isotyping

May be different for monoclonal antibody isotype (with associated activity)... 

Tissue cross-reactivity studies, although burdensome, provide a rational in vitro assay to determine the range and intensity of distribution of potential epitopes reactive with a monoclonal antibody test article prior to its administration to humans. In addition, cross-reactivity studies provide a useful tool to identify animal species for safety assessment. The cross-reactivity profiles of different species can be compared to the profiles obtained in human tissues. The predictive value of the assay lies in incorporating the characteristics of the monoclonal antibody (isotype, subtype, and other molecular modifications) with the biological activity of the molecule itself, and the potential in vivo distribution of it. 

Isotype andBinding Affinity/Capacity of ATI. The isotypes of secreted antibodies were determined through the use of a mouse monoclonal antibody isotyping kit (Cell Sciences, Norwood, MA). An anti-TPT IgGl antibody (ATI), secreted by hybridoma 8C2, was selected for further characterization. The 8C2 supernatant (with a constant ATI con-centration) was incubated with increasing concentrations of TPT (100,125,150,175,200,225, and 250ng/mL) for 2h to allow binding between TPT and ATI. Free TPT was then separated from antibody-bound TPT by ultrafiltration and assayed via HPLC. 

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. 

Fujiwara, K., Matsumoto, N., Yagisawa, S., Tanimori, H., Kitagawa, T., Hirota, M., Hiratani, K., Fukushima, K., Tomonaga, A., Hara, K., and Yamamoto, K. (1988) Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewisx using b-D-galactosidase coupled to a monoclonal antibody of IgM isotype./. Immunol. Meth. 112, 77-83. 

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). 

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... 

Figure 7.9. FcyRI expression in blood and synovial-fluid neutrophils. Neutrophils were isolated from the blood (trace ii) and synovial fluid (trace iii) of a patient with rheumatoid arthritis. Expression of CD64 (FcyRI) was then measured by FACS analysis. Trace (i) indicates the fluorescence distribution of a non-immune isotype control monoclonal antibody. Similar results were obtained in 6 out of 11 patients with rheumatoid arthritis.

Aroxmd the same time, Beckman Instruments (now Beckman Coulter) had begun an array-based product development program focused on the use of modified plastics. Silzel and coworkers (1998) and Matson et al. (2001) of Beckman Coulter were among the first to pursue printing of antibodies onto a plastic surface in a microarray format. Silzel et al. immobilized biotinylated monoclonal antibodies onto an avidin-coated polystyrene surface and performed micro-ELISA-based isotyping of IgG species. Matson et al. 

Monoclonal antibody (MAb) to PCNA. A suitable mouse MAb (cat. no NCL-PCNA IgG2a isotype) can be obtained from Euro-Path Ltd (Stratton, Bude, UK) (see Note 2 for alternatives). 

Because many potentially useful monoclonal antibodies do not possess the appropriate isotype and so are unable to activate human complement and/or trigger Fc-yR on human cells, treatment strategies are needed. One such strategy is the application of bispecific monoclonal antibodies that exploit the specificity of monoclonal antibody and ensure activation of cellular cytotoxic mechanisms (Fanger et al., 1993). 

Double immunofluorescence labeling in conjunction with microwave heating can be used to visualize two markers at the same cellular location in routine formalin-fixed and paraffin-embedded tissue sections (Mason et al 2000). The primary antibodies are either monoclonal antibodies of differing isotype/subclass or antibodies from different species. Labeling is visualized on a conventional fluorescence microscope equipped with a cooled analog monochrome CCD camera (Model C 5985, Hamamatsu Photonics, Billerica, MA) and recorded using off the shelf personal computer hardware and software. Contrary to general belief, paraffin-embedded tissue sections do not show excessive nonspecific fluorescence. 

Such a control antibody is known as an isotype control because it is of the same immunoglobulin isotype (subclass) as the staining antibody used in the experiment. It will allow you to determine how much background stain is due to irrelevant stickiness (dead cells, Fc receptors, and so forth). The only trouble with this scenario is that exactly correct isotype controls are not usually available. Various manufacturers of monoclonal antibodies will sell so-called isotype controls and will certainly recommend that they be used. These are, however, general purpose isotype controls that will be of an average... 

Step 4 Definition of the isotype of monoclonal antibodies obtained... 

Fab (50 kd) and scFv (27 kd) antibody fragments have shorter plasma half-lives (0.5 to 21 hours) because of more rapid glomerular filtration and clearance [7], Plasma clearance, particularly of the xenogenic (usually mouse) portions of humanized and/or chimeric monoclonal antibodies, might be accelerated by development of anti-mouse (or other species), anti-isotypic, anti-idiotypic, or anti-allotypic antibodies that foster immune complex formation or reticuloendothelial clearance via several different FcR forms or elicit allergic or anaphylactic responses [7,8]. 

Monoclonal antibodies can vary tremendously in terms of isotype, construction (animal derived, chimeric, humanized, bound to toxin), ability to activate complement, binding avidity, target specificity, and whether it binds and blocks or binds and activates the receptor. Monoclonal antibodies may be directed toward soluble or membrane bound receptors or receptor ligands, tumor antigens, growth factor or their receptors. Therefore toxicity and side effects are equally variable [68]. 

Baneijee, A., Roach, M. C., Trcka, P. and Luduena, R. F. (1992) Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain. Journal of Biological Chemistry, 267, 5625-5630. 

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