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Volume 271, Number 27, Issue of July 5, 1996 pp. 16300-16309
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Antigen Binding Properties of Purified Immunoglobulin A and Reconstituted Secretory Immunoglobulin A Antibodies

(Received for publication, January 18, 1996, and in revised form, March 13, 1996)

Elke Lüllau , Stephan Heyse ^§ , Horst Vogel ^§ , Ian Marison , Urs von Stockar , Jean-Pierre Kraehenbuhl ^¶ and Blaise Corthésy ^¶

From the  Institut de Génie Chimique et ^§ Institut de Chimie Physique, Ecole Polytechnique Fédérale, CH-1015 Lausanne, Switzerland, the ^¶ Institut Suisse de Recherches Expérimentales sur le Cancer et Institut de Biochimie, Université de Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland, and the  Institut de Biologie Animale, Université de Lausanne, CH-1015 Lausanne, Switzerland

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 were 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 × 10⁸ M¹). 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 × 10⁵ M¹ when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

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