Modifying specificity of antidigoxin antibodies using insertional mutagenesis.

Certain antibodies (Abs) elicited using the cardiac glycoside digoxin (digoxigenin tridigitoxoside) bind preferentially to analogs that differ from digoxin by substitutions on the cardenolide rings, the lactone, or by the presence or absence of attached sugars. Antibody 26-10 binds equally well to digoxin and digitoxin, which differ only by the presence in the former and the absence in the latter of an hydroxyl group at C12. Other antidigoxin Abs, however, can distinguish between these ligands by three orders of magnitude in binding.

Ouabain-binding protein(s) from human plasma.

Conservation of the binding site on mammalian Na+,K+-ATPase for cardiac glycosides and the importance of the Na+ pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form.

Complementary combining site contact residue mutations of the anti-digoxin Fab 26-10 permit high affinity wild-type binding.

Antibody 26-10, obtained in a secondary immune response, binds digoxin with high affinity (K(a) = 1.3 x 10(10) M(-1)) because of extensive shape complementarity. We demonstrated previously that mutations of the hapten contact residue HTrp-100 to Arg (where H refers to the heavy chain) resulted in increased specificity for digoxin analogs substituted at the cardenolide 16 position.

Selection of high affinity p-azophenyarsonate Fabs from heavy-chain CDR2 insertion libraries.

The length of the heavy chain complementarity-determining region two (HCDR2) of the unmutated anti-p-azophenylarsonate (Ars) monoclonal antibody (36-65 mAb) was extended by three residues in order to test whether this insertion can provide additional contacts between the Ab and the antigen. Two libraries were generated using 36-65 heavy and light chain genes which were cloned as Fab in the phage-display vector pComb3. In the first library, three randomized amino acids were inserted between residues Gly 54 and Asn 55, which are the most solvent exposed residues in the HCDR2 loop.

Structural analysis of mutants of high-affinity and low-affinity p-azophenylarsonate-specific antibodies generated by alanine scanning of heavy chain complementarity-determining region 2.

Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71. Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used.