The non-human, immunogenic epitope Gal-a-(1→3)-Gal can be found on glycoprotein therapeutics produced in murine derived cell lines (SP2/0 and NS0) or Chinese hamster ovary cells (CHO). This modification has been demonstrated to produce a hyperacute immune response reaction in a number of patients, leading to fatality in severe cases. Thus it is of importance to be able to detect and measure the presence of this epitope in glycoproteins. Within our research group, a panel of single chain antibody fragments (scFv) against this glycan epitope have been generated. Recognition profiling of these scFvs, utilising neoglycoconjugate (NGC) microarrays, demonstrated that they were highly specific and selective for the Gal-a-(1→3)-Gal target.
The scFvs were immobilised on a functionalised glass slide surface in microarray format and were tested for their specificity using NGCs and inhibitory sugars, including the monosaccharide galactose, the Gal-a-(1→3)-Gal disaccharide, the trisaccharide Gal-a-(1→3)-Gal-b-(1→4)-Gal and the Gal-a-(1→3)-Gal-b-(1→4)-Gal-a-(1→3)-Gal tetrasaccharide. The scFvs were found to maintain their unique specificity in this format. Binding constants from these data were also ascertained and compared to the same assay in an ELISA format. In addition, inhibition was carried out with Gal-a-(1→3)-Gal NGC analogues, which differed in their linker lengths, and the scFvs recognised the epitopes in both analogues. The stability of these scFvs on a solid surface was also evaluated to demonstrate the suitability of these recognition molecules for deployed industrial and laboratory use.
