Purification of duck immunoglobulins: an evaluation of protein A and protein 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, and by the reactivity in immunoelectrophoresis of antisera raised in rabbits inoculated with the eluates. The results indicated that IgY (previous nomenclature 7.8S IgG) and IgY (delta Fc) (previously 5.7S IgG) bound 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 also occurred. Attempts to separate the non-Ig proteins from the Igs by elution 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 Na2SO4, followed by chromatography on protein A Sepharose, yielded relatively pure IgY. The efficient binding of the duck IgYs to protein A resembles 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 histidine residues occurring predominantly in the CH1 domain.
Higgins
D A
DA
Department of Pathology, University of Hong Kong, Queen Mary Hospital.
Cromie
R L
RL
Liu
S S
SS
Magor
K E
KE
Warr
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity / Q000662:veterinary*; D004372:Ducks / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D005260:Female; D005779:Immunodiffusion / Q000662:veterinary; D007136:Immunoglobulins / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D011817:Rabbits; D013205:Staphylococcal Protein A
C029152:G-substrate; D007136:Immunoglobulins; D009419:Nerve Tissue Proteins; D013205:Staphylococcal Protein A