Conversion of an anti-single-stranded DNA active site to an anti-fluorescein active site through heavy chain complementarity determining region transplantation

• Published in: The Journal of biological chemistry Vol. 269; no. 11; pp. 7934 - 7940
• Main Authors: G A Gulliver, W D Bedzyk, R G Smith, S L Bode, S Y Tetin, E W Voss, Jr
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 18.03.1994
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - metabolism; Base Sequence; Binding Sites, Antibody; DNA Primers; DNA, Single-Stranded - immunology; Fluorescein; Fluoresceins; Immunoglobulin Heavy Chains - biosynthesis; Immunoglobulin Heavy Chains - metabolism; Mice; Mice, Inbred BALB C - immunology; Molecular Sequence Data; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Spectrometry, Fluorescence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)37141-7

Complementarity determining region (CDR) transplant studies were conducted between two monoclonal antibodies of distinctly different specificities (anti-fluorescein monoclonal antibody (mAb) 4-4-20 and anti-single-stranded DNA (ssDNA) mAb 04-01) which possessed nearly identical light chains but dissimilar heavy chains. The variations in binding specificities between the two immunoglobulins suggested that the active-site features of anti-fluorescein antibodies were dictated by characteristics intrinsic to the heavy chain (H-chain). To identify specific regions of the H-chain which influence the structure and function of an anti-fluorescein active site, CDR transplantation was systematically employed to convert the anti-ssDNA 04-01 antibody active site to an active site with anti-fluorescein activity. Each mAb 4-4-20 H-chain CDR (HCDR) was transplanted into the H-chain of a single-chain derivative of the 04-01 molecule. A fluorescence polarization ligand binding assay was utilized to determine the equilibrium dissociation constant, Kd, of hybrid transplant single-chain antibody HCDR1-HCDR2-HCDR3(4-4-20) for fluorescein (3.8 x 10(-7) M, indicating successful conversion of an anti-ssDNA active site to an anti-fluorescein binding site. A similar Kd (6.3 x 10(-7) M) was determined using a fluorescein fluorescence quenching assay. The transplantation results are discussed in terms of the relative contribution of each HCDR to a successful conversion in antibody specificity.