Conversion of an anti-single-stranded DNA active site to an anti-fluorescein active site through heavy chain complementarity determining region transplantation.
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.
Gulliver
G A
GA
Department of Microbiology, University of Illinois at Urbana-Champaign, 61801.
Bedzyk
W D
WD
Smith
R G
RG
Bode
S L
SL
Tetin
S Y
SY
Voss
E W
EW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000378:metabolism*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000276:immunology*; D019793:Fluorescein; D005452:Fluoresceins; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004277:DNA, Single-Stranded; D005452:Fluoresceins; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D019793:Fluorescein