PURIFICATION OF DUCK IMMUNOGLOBULINS - AN EVALUATION OF PROTEIN-A ANDPROTEIN-G AFFINITY-CHROMATOGRAPHY

Duck serum proteins binding to protein A Sepharose CL-4B and protein G Sepharose 4 Fast Flow and eluted at pH 2.8 or 11.5 were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, radial /immunodiffusion against defined anti-immunoglobulin (Ig) reagents, an d by the reactivity in immunoelectrophoresis of antisera raised in rab bits inoculated with the eluates. The results indicated that IgY (prev ious nomenclature 7.8S IgG) and IgY(Delta Fc) (previously 5.7S IgG) bo und to protein A efficiently and to protein G weakly, while IgM bound to protein A and protein G weakly. Some binding of non-Ig proteins als o occurred. Attempts to separate the non-Ig proteins from the Igs by e lution at different pHs (5.0, 4.0, 3.0 and 2.5) were unsuccessful, but it was found that precipitation of Igs in day-old duck serum with Na2 SO4, followed by chromatography on protein A Sepharose, yielded relati vely pure IgY. The efficient binding of the duck IgYs to protein A res embles high affinity binding of mammalian Igs but cannot be attributed to the Fc, as it is in mammals, since the IgY(Delta Fc) does not have an Fc region. Instead, binding probably occurs through unique histidi ne residues occurring predominantly in the C(H)1 domain.