1 research outputs found
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
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