Production of Native Bispecific Antibodies in Rabbits

BACKGROUND: A natural bispecific antibody, which can be produced by exchanging Fab arms of two IgG4 molecules, was first described in allergic patients receiving therapeutic injections with two distinct allergens. However, no information has been published on the production of natural bispecific antibody in animals. Even more important, establishment of an animal model is a useful approach to investigate and characterize the naturally occurring antibody. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that a natural bispecific antibody can also be generated in New Zealand white rabbits by immunization with synthesized conjugates. These antibodies showed bispecificity to the components that were simultaneously used to immunize the animals. We observed a trend in our test animals that female rabbits exhibited stronger bispecific antibody responses than males. The bispecific antibody was monomeric and primarily belonged to immunoglobulin (Ig) G. Moreover, bispecific antibodies were demonstrated by mixing 2 purified monospecific antibodies in vivo and in vitro. CONCLUSIONS/SIGNIFICANCE: Our results extend the context of natural bispecific antibodies on the basis of bispecific IgG4, and may provide insights into the exploration of native bispecific antibodies in immunological diseases

Regional and segmental flexibility of antibodies in interaction with antigens of different size.

The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range

Evaluation of IgG subclass responses against Dermatophagoides farinae allergens in healthy and atopic dogs

The definitive version is available at www3.interscience.wiley.comA semiquantitative chemiluminescent Western blot analysis system was developed and validated to evaluate antigen-specific IgG subclass responses to electrophoretically separated proteins of Dermatophagoides farinae in healthy and atopic dogs. Both groups mounted similar D. farinae-specific IgG1 and IgG4 responses to multiple antigens, but IgG2 and IgG3 responses were difficult to detect. The most commonly recognized bands in both groups were 18 and 98 kDa antigens for IgG1 and 18, 45, 66, 98, 130 and 180 kDa for IgG4. The number of bands recognized per dog did not differ significantly, but significantly more atopic dogs had an IgG1 response to a 180 kDa protein. The overall D. farinae-specific IgG1 and IgG4 responses were slightly higher, but not significantly different, in the healthy group. The results suggest that some antigens produced by D. farinae can induce different subclass responses. However, as most of these responses are seen in both healthy and atopic dogs, they are likely to merely represent recognition of foreign proteins presented to the immune system, rather than involvement in the pathogenesis of atopic dermatitis. The role of the 180 kDa antigen warrants further study.Chia-Chun Hou, Michael J. Day, Timothy J. Nuttall and Peter B. Hil