The objectives of this proposal are (1) to extend current information about immunoglobulin (Ig) structure, derived from X-ray crystallographic studies of IgG1 antibodies, to the full range of Ig isotypes, by image analysis of IgCa, IgG2b, IgG3, IgA, IgM, and IgE antibodies; and (2) to investigate the functional correlates of Ig structure by image analysis of antibody-complement complexes and Ig variants. The proposed research will be carried out on a collaborative basis by members of three laboratories, and will employ hybridoma, two-dimensional (2D) crystallization, and monolayer X-ray diffraction techniques. Isotype switch variants will be produced from existing hybridomas. Novel antibody variants will be generated by genetic engineering techniques and transformation with expression vectors. The variants will include Ig's with modified, deleted, and rearranged domains. Variants will be crystallized in 2D monolayers of lipid haptens and imaged at 15 resolution by electron microscopy and computer processing. The results should be informative about the shape, dimensions, domain structure, and hinge angles of the antibody molecules. Crystals of IgG's will be complexed with components of complement and imaged to reveal the structural basis of antibody-complement interaction. The monolayer X-ray diffraction technique will be developed and applied to obtain information missing from the electron microscope image analysis.
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