High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolith.
Reactive monoliths of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications.
D000327:Adsorption; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004852:Epoxy Compounds / Q000737:chemistry; D019544:Equipment Failure Analysis; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008420:Manufactured Materials; D008567:Membranes, Artificial; D008689:Methacrylates / Q000737:chemistry; D010949:Plasma; D011108:Polymers / Q000737:chemistry*; D016062:Porosity; D011786:Quality Control; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry*
D004852:Epoxy Compounds; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D008567:Membranes, Artificial; D008689:Methacrylates; D011108:Polymers; D013205:Staphylococcal Protein A; D006639:Histidine; C004919:ethylene dimethacrylate; C007870:glycidyl methacrylate