Separation of immunoglobulin G from human serum by pseudobioaffinity chromatography using immobilized L-histidine in hollow fibre membranes.
L-Histidine, intended as a pseudobiospecific ligand, was immobilized on poly(ethylenevinyl alcohol) hollow fibre membranes after their activation with epichlorohydrin or butanediol diglycidyl ether. The affinity membranes obtained allowed the one-step separation of immunoglobulin G (IgG) from untreated human serum. Elution was possible under mild conditions with discontinuous pH or salt gradients. IgM was also adsorbed to a certain extent and partially separated from IgG by pH gradient elution. The bound IgG fractions showed pI values between 8 and 9.5 and contained IgG1 and IgG3. The dissociation constants for IgG on the bisoxirane- and epichlorohydrin-activated membranes coupled with histidine were determined by equilibrium binding analysis to be 2.5 x 10(-5) and 2.0 x 10(-5) M, respectively. The maximum binding capacity of the affinity hollow fibre membranes was 80 and 70 mg of IgG per gram of support, respectively. With a cartridge of surface area 1 m2 (about 19 g of fibres), during a 60-min run, theoretically up to 1.5 g of IgG can be removed from human serum. The histidine affinity membranes are very stable owing to the simple nature of the ligand and the coupling via an ether linkage. Reproducible results were obtained over more than 1 year even with untreated human serum being used regularly.
Bueno
S M
SM
Laboratoire d'Interactions Moléculaires et de Technologie des Séparations, Université de Technologie de Compiègne, France.
Haupt
K
K
Vijayalakshmi
M A
MA
D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008024:Ligands; D008567:Membranes, Artificial
D007074:Immunoglobulin G; D008024:Ligands; D008567:Membranes, Artificial; D006639:Histidine