Regeneration of antidigoxin binding activity in an antibody by changes surrounding the original binding defect.
A panel of antibodies which differ in their L chain structures and which bind to structurally defined haptens, would be useful in investigating L chain structure and function. In a previous study, chain recombinant antibody CR24 (26-10 H, 45-20 lambda) was produced by hybridoma-hybridoma fusion. Although both parental antibodies bound digoxin with high affinity, CR24 lacked detectable digoxin-binding activity. Hybridoma CR24 was subsequently fused with H chain-loss hybridomas in order to produce a panel of antibodies composed of 26-10 H chains and 26-10 "like" L chains. Two antibodies produced were CR260 which demonstrated digoxin-binding activity and CR256 which did not. CR260 and CR256 expressed only one amino acid difference (Pro to Leu at L-96). This difference resulted in the CR256 binding defect. In this report, two new antidigoxin antibodies are described. One, SR2E7, contained the Pro to Leu (L-96) defect, but still bound digoxin. Binding affinities and binding specificity patterns, as well as complete VL DNA sequence and corresponding protein sequence of the new digoxin binding antibody L chains (SR2E7 and SR1C7) are presented. Both kappa L chains are highly homologous to the 26-10 kappa L chain as well as the BALB/c germline gene K5.1. These results suggest that antibodies which are initially defective in binding activity can be cured by changing specific amino acids involved in determining the binding-site structure. Molecular modelling studies of the binding-site region were completed to address L chain structural changes induced by specific amino acid substitutions.
Hudson
N W
NW
Northwest Center for Medical Education, Indiana University School of Medicine, Gary 46408.
Albrecht
M
M
Randhawa
Z I
ZI
Steinrauf
L K
LK
Hamilton
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003198:Computer Simulation; D004077:Digoxin / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000276:immunology*; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology
D000906:Antibodies; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004077:Digoxin