Amino acid sequence of the light chain variable region from a mouse anti-digoxin hybridoma antibody

Abstract

A hybridoma cell line (26-10) derived from the A/J strain of mice secretes IgG2a-.kappa. which binds digoxin [a cardiac glycoside] with an association constant of 1.2 nM. Such high-affinity antibodies have been utilized in clinical radioimmunoassays and in the reversal of toxicity due to excess digoxin. The amino acid sequence of the L chain variable region of this antibody was derived by automated sequencing of the following: the intact chain; a fragment beginning C terminal to the tryptophan residue 40, obtained by cleavage with iodosobenzoic acid; a fragment beginning C terminal to arginine residue 82, obtained by trypsin cleavage on the completely reduced, alkylated and succinylated chain. Difficulties which had previously prevented the automated Edman sequencing of this chain (and, presumably, similar ones of the same subgroup) were overcome by increasing the duration of the cleavage step at proline residues 8 and 12. The sequences of the first 2 hypervariable and framework regions of this chain are virtually identical with those of the dinitrophenol- and menadione-binding myeloma L chain MOPC 460 (95% homology). This anti-digoxin hybridoma from the A/J strain makes use of a V.kappa. gene which is similar to that utilized by some BALB/c 2,4-dinitrophenol-binding myelomas. Anti-digoxin antibodies are excellent models for the study of antigen-antibody interactions at the molecular level.