Separation of Immunoglobulin and Transferrin from Blood Serum and Plasma by Metal Chelate Interaction Chromatography

Abstract

Metal chelate interaction chromatography was used to separate Ig, transferrin, and albumin from blood serum and blood plasma. A column was packed with iminodiacetic acid: 1,4-butanediol diglycidyl Sepharose 6B or Sephacryl S-300 and loaded with copper, zinc, nickel, or cobalt ion. Radial immunodiffusion assay indicated that Ig-rich fractions of blood serum obtained from Zn-, Ni-, Co-, and Cu-loaded columns contained 23.2, 81.3, 79.4, and 98.1% active IgG, respectively. Transferrin was recovered from the second peak. When the same conditions of metal chelate interaction chromatography were used for blood plasma, hemoglobin tended to bind strongly to the Cu-loaded column and was eluted only with 50% ethanol. Modification of histidine residues in Ig and transferrin with diethyl pyrocarbonate almost completely destroyed their binding ability to the column. Immunoglobulin G separated showed antilipopolysaccharide antibody activity against Escherichia coli, Salmonella typhimurium, and Bordettella parapertussis.

Perspectives on Immunoglobulins in Colostrum and Milk

“…These include traditional methods of ammonium sulfate precipitation and column chromatography [3,5,145,184]. Affinity chromatographic methods used to isolate IgG include lectins [185]; protein A or G chromatography [186,187], and more recently, isolation with protein A/G immobilized electrospun polyethersulfone membranes [188]; metal chelate chromatography [189,190]; and adsorption with polyanhydride microparticles [191]. The range of detection and quantification methods for IgG, most often analyzed by radial-immunodiffusion [192] or enzyme-linked immunosorbant methods [193], are now expanding to include methods that detect multiple proteins, such as thermally addressed immunosorbant assays [194], and rapid methods that may be integrated into milking systems, such as surface plasmon resonance-based immunosensors [195].…”

Heterogeneity of Monoclonal Antibodies

“…224 Immunoglobulin has been reported to be separated from bovine serum using metal chelate chromatography. 225 Immobilized metal affinity chromatography has been used for the purification of humanized and murine IgG1 antibodies, 226 and the binding sites were localized to the C-terminal region of heavy chains. A copper binding IgG was found in a myeloma patient with hypercupremia, 227 where copper is bound to Fab region with the involvement of a light chain free Cys.…”

The In Vitro Anti-pathogenic Activity of Immunoglobulin Concentrates Extracted from Ovine Blood

“…Ovine immunoglobulin concentrate (OIC) was prepared according to procedures previously developed in our laboratory based on the standard precipitation technique [20]. Fresh ovine blood was mixed with sodium citrate (1%) and centrifuged (Sorvall Instruments, Du Pont, USA) at 11,000×g for 20 min at room temperature for separation of plasma.…”