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Biotechnology and Applied Biochemistry (1998) 28, (189–199) (Printed in Great Britain)

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Capture of anti-(Gala1-3Gal) antibodies by immobilized Gala1-3Gal oligomers derived from carrageenan

Elias Klein*¹, Marco Euler* and John Vercellotti†

*Kidney Disease Program, University of Louisville, Louisville, KY 40202, U.S.A., and †V-LABS, Covington, LA 70433, U.S.A.

¹ To whom correspondence should be addressed.

An immobilized Gala1-3Gal-bearing affinity ligand was prepared as a possible prophylactic binding site of human anti-(Gala1-3Gal) antibodies for the prevention of hyperacute rejection of pig xenografts. l-Carrageenan, a natural polysaccharide known to possess alternately a1-3 and b1-4-linked D-galactopyranose sulphate residues, was selectively degraded by acetolysis and subsequently deacetylated to obtain l-carrageenan oligosaccharides. The resulting syrup was ultrafiltered to remove higher polymers and chromatographed on Sephadex G-15 to produce a series of sized oligosaccharide fractions. An immuno-dot-blot method was established on the basis of the competition between the l-carrageenan oligosaccharides in solution and bovine thyroglobulin (BTG), a glycoprotein known to express the Gala1-3Gal marker, as the solid-phase antigen. Increasing amounts of l-carrageenan fractions added to normal human plasma resulted in a progressive decrease in Gala1-3Gal-specific human IgM and IgG binding to BTG. Complete inhibition of Gala1-3Gal-specific human immunoglobulin was attained at concentrations less than 0.6 mg of carbohydrate/ml of human plasma with the more active fractions. These bioactive l-carrageenan oligosaccharides were immobilized on hydrazide-modified microporous nylon membranes, yielding capacities between 2.18 and 2.86 mg/ml of membrane. A subsequent decrease in the human anti-(Gala1-3Gal) antibody level in normal human plasma was demonstrated and proved with the established immuno-dot-blot procedure.

Received 20 March 1998/14 May 1998; accepted 14 May 1998

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