One of the latest attempts to improve monoclonal antibodies in their use as radiolabeled diagnostic markers has involved the cross-linking of novel Fab’ fragments. It is expected that a relationship exists between the spacing of the antigen binding sites at the extremes of the Fab’ frag-ments in solution and their immunological performance in vitro and in vivo.
In order to properly understand the function of these new antibody fragments in vivo, it is critical to have an appreciation of their size, self-association behavior (or preferably lack of) and solution conformation. Appropriate application of the ProteomeLab XL-A analytical ultracentrifuge can provide this data. No other single technique provides this breadth of information.
The flexibility conferred upon the cross-linked F(ab’)2 protein x-ray crystallography. However, the sedimentation coefficients of both the F(ab’)2 combined with radius of gyration data obtained from small angle x-ray scattering experiments provide a useful gauge of solution conformation.
In this study the monodispersity and absence of selfassociation phenomena of Fab’ and F(ab’)2 solutions are strongly indicated by sedimentation velocity and sedimentation equilibrium experiments. The weight average molecular weights measured are shown to be in complete agreement with the molecular weights as calculated from amino acid sequences. Finally, sedimentation coefficients have been measured, and we indicate how these can be used to access conformation when combined with other solution measurements.
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