Home » Key Medical Diagnostics Articles » Development of a convenient competitive ELISA for the detection of the free and protein-bound nonhumangalactosyl-{Alpha}-(1,3)-galactose epitope based on highly specific chicken single-chain antibody variable-regionfragments.

Development of a convenient competitive ELISA for the detection of the free and protein-bound nonhumangalactosyl-{Alpha}-(1,3)-galactose epitope based on highly specific chicken single-chain antibody variable-regionfragments.

Cunningham S, Starr E, Shaw I, Glavin J, Kane M, Joshi L.

Anal Chem. 2013 Jan 15;85(2):949-55.

Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.

Abstract 

The presence of the nonhuman galactosyl-{Alpha}-(1,3)-galactose (Gal-{Alpha}-(1,3)-Gal) carbohydrate epitope on a number of recombinant therapeutic proteins has recently been reported, renewing interest in this immunogenic carbohydrate epitope. It is well-known that this motif is the primary contributing factor in hyperacute rejection of porcine organ xenograft, due to the existence of natural antibodies against this epitope in human serum. Though the number of epitopes on recombinant glycoproteins may be low when compared directly to whole tissue, circulating anti-Gal-{Alpha}-R immunoglobulins can still induce anaphylaxis. Therefore, there is a need for rapid and convenient methods for detection and monitoring of this epitope in biopharmaceuticals produced in recombinant mammalian systems. To this end, we have generated immune-challenged chicken single-chain antibody variable-regionfragment (scFv) libraries targeting the Gal-{Alpha}-(1,3)-Gal motif and have selected a panel of scFv’s that bind the target. We have used one of these antibodies to develop a competitive ELISA for both free and protein-bound Gal-{Alpha}-(1,3)-Gal and have demonstrated that the ELISA is specific for the target and can be used to determine the loading of the target on glycoproteins. This competitive ELISA will provide a convenient method of detecting and quantifying Gal-{Alpha}-(1,3)-Gal on therapeutic glycoproteins.

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Additional Information

All cells are covered with complex sugars and most circulating proteins in the body are glycosylated. These sugars play critical roles in inter-cellular and inter-molecular interactions in most biological processes such as cell growth, migration, adhesion, fertilization, cell death, host-microbial interactions, immune response and half-life of circulatory glycoproteins. In addition to acting as signaling and recognition molecules, some of the glycan epitopes are prominent immunogens because nature has devised glycosylation as an evolutionary phenomenon for self-non self recognition. One such epitope is Galactose alpha-1-3 Galactose, also known as the alpha-Gal epitope, which is commonly found at the non-reducing end of glycolipids and glycoproteins in most non-primate animal species and some parasites and pathogens, but not humans.

 

Alpha-Gal is one of the major challenges in xenotransplantation where non-primate (especially pig) organs are used that are covered with the alpha-Gal epitope. This leads to acute rejection of transplants unless strong immune-suppressors are used. This epitope is increasingly becoming a challenge also in the field of recombinant protein therapeutics. Many highly valuable protein therapeutics are glycosylated and control of their glycosylation is still a challenge in bioprocessing, affecting their in-vivo pharmacokinetic and pharmacodynamic properties and efficacy. Achieving human-like glycosylation is a major focus during bioprocessing, with efforts to increase the sialic acid content of the protein receiving much attention. Attention has shifted, however, to the alpha-Gal epitope with recent reports highlighting the presence of this epitope on the Fab domain of chimeric monoclonal antibody (mAb) drugs resulting in anaphylaxis and an acute hypersensitive response in patients.

 

One of the reasons this epitope ‘slipped’ through bioprocess analytics is because of its low abundance and the lack of convenient detection methods. Glycoanalytics in general has come a long way but still lacks rapid and high-throughput methods for the detection of glycan epitopes of interest. Our group is developing a library of glycan-specific binders that can be integrated into various detection platforms for fast and sensitive detection of desirable or undesirable glycan epitopes for quality control during biomanufacturing. In this paper, we describe the development of chicken single-chain antibody fragments (scFv) that are more specific for the alpha-Gal epitope than existing lectins (Figure 1) and have utility in ELISA format to detect the epitope in free or protein-bound form. This will enable the development of rapid, sensitive and cost-effective detection systems for this epitope for use in bioprocess monitoring.

 

Figure legend:

 

Figure 1.  Specificity profiles of the three anti-Galactose alpha-1-3 Galactose scFvs and the lectin, GS-1-B4, showing the differences in their recognition of neoglycoconjugates and glycoproteins printed on a microarray. The structures of the three sugars reacting most strongly with the binders are given in the insets.

 

Development of a Convenient copy