Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies

The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA mols., as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatog., and size exclusion chromatog. for the simultaneous and qual. sepn. of the three mol. forms. Milligram quantities of purified IgA mols. were recovered allowing for direct comparison of the biol. properties of the three forms. LPS binding specificity was tested after purifn.; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm. Surface plasmon resonance-based binding expts. using LPS monolayers indicated that purified reconstituted sIgA and IgA mols. recognize LPS with identical affinity (KA 1.0 * 108 M-1). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. KA falls to 6.6 * 105 M-1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is crit. for recognition by the antibody. [on SciFinder (R)]