Active site structure and antigen binding properties of idiotypically cross-reactive anti-fluorescein monoclonal antibodies

• Published in: The Journal of biological chemistry Vol. 265; no. 1; pp. 133 - 138
• Main Authors: W D Bedzyk, J N Herron, A B Edmundson, E W Voss, Jr
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.01.1990
• Subjects: Amino Acid Sequence; Animals; Antibodies, immunoglobulins; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibody Affinity; Antigens - immunology; Base Sequence; Binding Sites; Biological and medical sciences; Cloning, Molecular; DNA - genetics; Fluorescein; Fluoresceins; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Hybridomas - immunology; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - immunology; Immunoglobulin Idiotypes - immunology; Immunoglobulin kappa-Chains - genetics; Immunoglobulin kappa-Chains - immunology; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Mice; Mice, Inbred BALB C; Molecular immunology; Molecular Sequence Data; Monoclonal antibodies; Sequence Homology, Nucleic Acid; Complementary DNA; Gene; Active site; Cross reaction; Hybridoma; Monoclonal antibody; Hapten; Aminoacid sequence; Idiotype
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)40205-6

This report includes complete VH and V kappa nucleotide and deduced amino acid sequences of idiotypically cross-reactive monoclonal anti-fluorescein antibodies that differed greater than 10(5)-fold in affinity. High affinity monoclonal antibody 4-4-20 and intermediate affinity antibodies 10-25, 5-14, 9-40, 12-40, and 3-24 utilized greater than or equal to 90% homologous VHIIIC germ-line genes. Extensive D segment length and sequence variability were observed; however, compensatory germ-line JH4 (4-4-20 and 3-24) or JH3 (10-25, 5-14, 9-40, and 12-40) sequence lengths resulted in H chain CDR3 + FR4 to be a constant 18 amino acids. In addition, each antibody and low affinity 3-13 rearranged greater than or equal to 96% homologous V kappa II genes to J kappa 1, except for 10-25 (J kappa 5) and 3-13 (J kappa 4). Resolved crystal structure of complexed fluorescein and 4-4-20 Fab fragments revealed residues HisL27d, TyrL32, ArgL34, SerL91, TrpL96, and TrpH33 acted as hapten contact residues. Antibodies 5-14, 9-40, 12-40, and 3-24 primary structures possessed identical contact residues as 4-4-20 except for the substitution of HisL34 for ArgL34. Thus, ArgL34 was implicated in the increased affinity of monoclonal antibody 4-4-20. Finally, it was difficult to correlate extensive H chain CDR3 residue heterogeneity directly with fluorescein binding and idiotypy.