Development of a Convenient Competitive ELISA for the Detection of the Free and Protein-Bound Nonhuman Galactosyl-α-(1,3)-Galactose Epitope Based on Highly Specific Chicken Single-Chain Antibody Variable-Region Fragments

Abstract

The presence of the nonhuman galactosyl-α-(1,3)-galactose (Gal-α-(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-α-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-region fragment (scFv) libraries targeting the Gal-α-(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-α-(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-α-(1,3)-Gal on therapeutic glycoproteins.

Distinctive Surface Glycosylation Patterns Associated With Mouse and Human CD4+ Regulatory T Cells and Their Suppressive Function

“…For example, higher binding to Treg compared to Tconv was also observed for the lectins GSL-I and GSLB4 (Figure 4 A). These lectin-binding characteristics were shown to reflect surface levels of the terminal glycan motif composed of a Gal residue in α-(1,3)-linkage to another Gal residue (Gal-α-(1,3)-Gal) using a single-chain fragment with specificity for this motif (scFv-A4) ( 24 ). As for PHA-L, Treg with higher scFv-A4 binding exhibited higher surface expression of CD73, ICOS, GITR, and PD-1 (Figures 4 B,C).…”

“…For the evaluation of surface glycosylation, cells were incubated with biotinylated lectins (Table S2 in Supplementary Material; Vector labs, Burlingame, CA, USA; EY labs, San Mateo, CA, USA) and HA-tagged, engineered chicken single chain, scFv-A4. The scFv-A4 has been demonstrated to be highly specific for the Gal-α-(1,3)-Gal terminal motif ( 24 ). The cells were washed with FACS buffer and labeled with fluorochrome-conjugated streptavidin (SA).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…MALDI has also helped in the development of an ELISA assay for the Gal‐ α ‐ (1 → 3)‐Gal epitope that is responsible for rejection in tissue transplants from pigs (Cunningham et al, ).…”

Alpha-Gal on the Protein Surface Hampers Transcytosis through the Caco-2 Monolayer

“…The membranes were probed with monoclonal anti-α-Gal antibody M86 (Enzo Life Sciences, Farmingdale, NY, USA) diluted 1:5 in 0.1% HSA in tris-buffered saline-Tween 20 (TBS-T) for 3 h followed by alkaline phosphatase (AP)-labeled goat anti-mouse IgM antibody (Southern Biotech, Birmingham, AL, USA) diluted 1:3000 in TBS-T for 1 h at room temperature (RT). Alternatively, the membranes were incubated for 2 h at RT with the chicken ScFV anti-α-Gal [25] (ScFV, a kind gift from the Glycoscience group at the National University of Ireland, Galway, Ireland) diluted 1:7500 in 0.2% HSA in phosphate buffered saline-Tween 20 (PBS-T). Subsequently, the membrane was incubated with 1 µg/mL monoclonal mouse-anti-HA antibody (H3663, Sigma-Aldrich) in 0.2% HSA in PBS-T for 1 h at RT followed by goat-anti-mouse IgG conjugated to AP (Jackson Immunoresearch, West Grove, PA, USA) diluted 1:1000 in 0.2% HSA in PBS-T for detection.…”