Rapid isolation of human plasma anti-alpha-galactoside antibody using sugar-specific binding to guar galactomannan or agarose.
A method of purifying the naturally occurring antibody to alpha-galactoside moiety (anti-alpha-Gal) in human plasma by a single-step affinity chromatography on cross-linked guar galactomannan (CLGG) or agarose (Sepharose 4B) is described. IgG nature of the two preparations, as revealed by agar gel diffusion, as well as their preference for alpha-anomer of galactose, as revealed in inhibition of their agglutination of trypsinized rabbit erythrocytes by sugars, identified them with anti-alpha-Gal. The antibody binding capacity of Sepharose 4B was only a third of that of CLGG. Both gels showed similar dependence on ionic strength for binding. The pH optimum for binding of anti-alpha-Gal to CLGG was 8.0. Significantly anti-alpha-Gal binding to Sepharose was unaffected by CNBr activation and ligand coupling to the gel, thus warning that contaminating plasma could introduce artifacts in agarose-based chromatography of human tissue biomolecules.
Jaison
P L
PL
Neurochemistry Division, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram.
Appukuttan
P S
PS
D000906:Antibodies / Q000097:blood / Q000302:isolation & purification*; D002241:Carbohydrates; D002846:Chromatography, Affinity / Q000379:methods; D005690:Galactose / Q000031:analogs & derivatives; D005697:Galactosides / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008351:Mannans; D009994:Osmolar Concentration; D012685:Sepharose
D000906:Antibodies; D002241:Carbohydrates; D005697:Galactosides; D008351:Mannans; C012990:galactomannan; D012685:Sepharose; D005690:Galactose