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 molecules as a heterogeneous population of monomeric (IgA(m)), dimeric (IgA(d)), and polymeric (IgA(p)) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatography, and size exclusion chromatography for the simultaneous and qualitative separation of the three molecular forms. Milligram quantities of purified IgA molecules were recovered allowing for direct comparison of the biological properties of the three forms, LPS binding specificity was tested after purification; IgA(d) and IgA(p) mere found to bind strongly to LPS whereas IgA(m) did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgA(d) or IgA(p), but not IgA(m). Surface plasmon resonance-based binding experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (K-A 1.0 x 10(8) 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. K-A falls to 6.6 x 10(5) M(-1) when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.