Thiol-disulfide redox buffers maintain a structure of immunoglobulin A that is essential for optimal in vitro binding to secretory component.

We have shown that human secretory component (SC) binds in vitro to different samples of human and murine dimeric immunoglobulin A (IgA). The binding ratio in the IgA/SC complex is 1:1. IgA which is stably bound to SC is separated from unreacted IgA by anion exchange chromatography. A part of IgA/SC complexes formed in vitro is unstable to this elution; the proportion varies between different samples of IgA; it increases following prolonged incubation of IgA at 37 degrees C. Incubation of IgA with glutathione/glutathione disulfide (GSH/GSSG) redox buffers increases the proportion able to form a stable complex with SC to approximately 90%. The presence of bound SC is not essential for this process but does allow it to occur at a lower GSH/GSSG concentration. The stable IgA/SC complex consists of a structure with a disulfide bond between IgA and SC apparently in equilibrium with a structure in which this bond is absent. The proportion bound covalently is similar for different samples of IgA and is insensitive to incubation with GSH/GSSG. It is significantly greater for secretory IgA (sIgA) and for IgA and SC incubated together with a starting mixture of cysteine/cystine. Monoclonal, antigen-specific IgA, all of which is optimally bound to SC in essentially the same way as in native sIgA, can be isolated in high yield. Our results support a mechanism for optimal binding of IgA to SC, that can occur both in vitro and in vivo, in which a thiol disulfide interchange occurs between a free IgA thiol and a sensitive SC disulfide following the initial non-covalent interaction.

The isolation of digoxin-specific antibody and its use in reversing the effects of digoxin.

Specific antibodies to digoxin were isolated from antisera of sheep immunized with a digoxin-human serum albumin conjugate. The antibody was purified by adsorption to an immunoadsorbent, synthesized by coupling a ouabain-ribonuclease conjugate to bromoacetyl-cellulose, followed by elution with 25 mM ouabain. Ouabain was dissociated from antibody by denaturation in 6 M guanidine. The renatured antibody bound 1.6 mol of digoxin per mol and had an association constant of 1.6 x 10(8) M(-1). At near-stoichiometric concentrations, either purified antibody to digoxin, or its papain-digested product (Fab-Fc), reversed digoxin-induced: (a) inhibition of (86)Rb transport in human erythrocytes, (b) increase in developed tension in isolated guinea-pig atrial strips, and (c) ventricular tachycardia in intact dogs, and also corrected digoxin-induced automaticity in isolated guineapig atrial strips.

Isolation and characterization of structurally homogeneous antibodies from antipneumococcal sera.

Antibodies of sufficient homogeneity for sequence studies were readily obtained in high concentrations from rabbits immunized with pneumococcal vaccines. By taking advantage of slightly differing immunologic specificity for Type III and Type VIII capsular polysaccharides, an antibody with unique electrophoretic mobility could be isolated from serum containing several distinct antibody components by using appropriate cross-reacting immunoadsorbents. A unique sequence for the N-terminal 11 amino acid residues of the light chain of the antibody was found, in contrast to several sequences in the antibody mixture from which this component was isolated. The sequence of a nonimmune light chain pool demonstrates even greater heterogeneity. Chymotryptic peptide maps of the antibody light chain show two unique cysteine-containing variable region peptides not seen in maps of nonimmune light chain pool of the same allotypic specificity as that of the antibody light chain. The experimental approach described here may provide further insight into the structure-function relationship of several homogeneous antibodies of closely related specificity for the same polysaccharide antigen.