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 (IgAm), dimeric (IgAd), and polymeric (IgAp) 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; 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 experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (KA 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. KA 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.
Lüllau
E
E
Institut de Génie Chimique et Institut de Chimie Physique, Ecole Polytechnique Fédérale, CH-1015 Lausanne, Switzerland.
Heyse
S
S
Vogel
H
H
Marison
I
I
von Stockar
U
U
Kraehenbuhl
J P
JP
Corthésy
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D002151:Calorimetry; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas; D015151:Immunoblotting; D007070:Immunoglobulin A / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D007071:Immunoglobulin A, Secretory / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D007700:Kinetics; D008070:Lipopolysaccharides / Q000276:immunology*; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D014462:Ultrafiltration; D014734:Vibrio cholerae / Q000276:immunology
D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007071:Immunoglobulin A, Secretory; D008070:Lipopolysaccharides; D046911:Macromolecular Substances