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The crystal structure of Fab R19.9, derived from an anti-p-azobenzenearsonate monoclonal antibody, has been determined and refined to 2.8- angstrom resolution by x-ray crystallographic techniques. Monoclonal antibody R19.9 (IgG2bκ ) shares some idiotopes with a major idiotype (CRIA) associated with A/J anti-p-azobenzenearsonate antibodies. The amino acid sequences of the variable (V) parts of the heavy (VH) and light (VL) polypeptide chains of monoclonal antibody R19.9 were determined through nucleotide sequencing of their mRNAs. The VL region is very similar to that of CRIA-positive anti-p-azobenzenearsonate antibodies as is VH, except for its third complementarity-determining region, which is three amino acids longer; it makes a loop, unique to R19.9, that protrudes into the solvent. A large number of tyrosine residues in the complementarity-determining region of VH and VL, with their side chains pointing towards the solvent, may have an important function in antigen binding.

The three-dimensional structure of FabR19.9 from a well-characterized anti-p-azobenzenearsonate monoclonal antibody has been determined by x-ray diffraction techniques in two crystalline forms (I and II) to a resolution of 2.8 and 2.7 0 Å, respectively. Essentially the same tertiary and quaternary structure of the Fab is observed in the two forms. The major difference resides in the intermolecular contacts, which are interpreted to favor an irreversible transition from the metastable form I to the more stable form II. The third complementarity-determining region of the heavy chain (H3) folds back over the combining site and requires rearrangement for hapten binding. This dynamic requirement on H3 is consistent with its mobility in the structure and can explain hapten binding to an otherwise inaccessible antibody combining site.

By using X-ray diffraction and immunochemical techniques, we have exploited the use of monoclonal antibodies raised against hen egg lysozyme (HEL) to study systematically those factors responsible for the high specificity of antigen-antibody interactions. HEL was chosen for our investigations because its three-dimensional structure and immunochemistry have been well characterized and because naturally occurring sequence variants from different avian species are readily available to test the fine specificity of the antibodies. The X-ray crystal structure of a complex formed between HEL and the Fab D1.3 shows a large complementary surface with close interatomic contacts between antigen and antibody. Thus single amino acid sequence changes in heterologous antigens give antigen-antibody association constants that are several orders of magnitude smaller than that of the homologous antigen. For example, a substitution of His for Glu at position 121 in the antigen is sufficient to diminish significantly the binding between D1.3 and the variant lysozyme. The conformation of HEL when complexed to D1.3 shows no significant difference from that seen in the free molecule, and immunobinding studies with other anti-HEL antibodies suggest that this observation may be generally true for the system of monoclonal antibodies that we have studied.

By using X -ray diffraction and immunochemical techniques, we have exploited the use of monoclonal antibodies raised against hen egg lysozyme (HEL) to study systematically those factors responsible for the high specificity of antigen -antibody interactions. HEL was chosen for our investigations because its three-dimensional structure and immunochemistry have been well characterized and because naturally occurring sequence variants from different avian species are readily available to test the fine specificity of the antibodies. The X-ray crystal structure of a complex formed between HEL and the Fab D1.3 shows a large complementary surface with close interatomic contacts between antigen and antibody. Thus single amino acid sequence changes in heterologous antigens give antigen-antibody association constants that are several orders of magnitude smaller than that of the homologous antigen. For example, a substitution of His for Glu at position 121 in the antigen is sufficient to diminish significantly the binding between D1.3 and the variant lysozyme. The conformation of HEL when complexed to D1.3 shows no significant difference from that seen in the free molecule, and immunobinding studies with other anti-HEL antibodies suggest that this observation may be generally true for the system of monoclonal antibodies that we have studied.