Though first reported over 30 years ago, there remains considerable
interest in the Gal-α-(1,3)-Gal motif, commonly found as a terminal motif on
glycoproteins and glycolipids in a range of species. This glycan structure
still remains relevant to xenotransplantation efforts, due to hyperacute
rejection (HAR). Recent reports of the occurrence of this immunogenic motif on
glycoprotein biopharmaceuticals, with resulting adverse reactions in patients
(HAR), has particularly highlighted the need for specific binding agents and a
convenient analytical method for Gal-α-(1,3)-Gal.
We have recently reported the successful generation of single chain
antibody fragments (scFv) targeting the Gal-α-(1,3)-Gal motif1 and have
used one of these antibodies to develop a competitive ELISA for both free and
protein-bound Gal-α-(1,3)-Gal. The ELISA was shown to be specific for the
target, to allow detection of the disaccharide at low ng/ml levels
(approximately 3.9 ng/ml [~10 nM]) and to give an estimate of target loading on
glycoproteins.
Here, we extend these studies and report the detection of the Gal-α-(1,3)-Gal
motif on both natural proteins and recombinant glycoprotein therapeutics using
the competitive ELISA. The results obtained were validated by conventional HPLC
analysis following enzymatic release of the terminal Gal. This study has
provided evidence of the suitability of the scFv-ELISA for the detection of Gal-a-(1→3)-Gal in a
range of different presentation modes, indicating its potential for commercial
application.
1Cunningham S, Starr E, Shaw I, Glavin J,
Kane M, Joshi L. (2013) Anal Chem. 2013 Jan 15;85(2):949-55.
