CONVERSION OF AN ANTI-SINGLE-STRANDED DNA ACTIVE-SITE TO AN ANTIFLUORESCEIN ACTIVE-SITE THROUGH HEAVY-CHAIN COMPLEMENTARITY-DETERMINING REGION TRANSPLANTATION

Complementarity determining region (CDR) transplant studies were condu cted between two monoclonal antibodies of distinctly different specifi cities (anti-fluorescein monoclonal antibody (mAb) 4-4-20 and anti-sin gle-stranded DNA (ssDNA) mAb 04-01) which possessed nearly identical l ight chains but dissimilar heavy chains. The variations in binding spe cificities between the two immunoglobulins suggested that the active-s ite features of anti fluorescein antibodies were dictated by character istics intrinsic to the heavy chain (H-chain). To identify specific re gions of the H-chain which influence the structure and function of an anti-fluorescein active site, CDR transplantation was systematically e mployed to convert the anti-ssDNA 04-01 antibody active site to an act ive 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 o f the 04-01 molecule. A fluorescence polarization ligand binding assay was utilized to determine the equilibrium dissociation constant, K-d, 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 simil ar K-d (6.3 x 10(-7) M) was determined using a fluorescein fluorescenc e quenching assay. The transplantation results are discussed in terms of the relative contribution of each HCDR to a successful conversion i n antibody specificity.