Purification and Characterization of Antibodies Directed against the α-Gal Epitope

Abstract

The α-Gal epitope is an immunogen trisaccharide structure consisting of N-acetylglucosamine (GlcNAc)β1,4-galactose (Gal)α1,3-Gal. It is presented as part of complex-type glycans on glycoproteins or glycolipids on cell surfaces of non-primate mammalians. About 1% of all antibodies in human sera are specific toward α1,3-Gal and are therefore named as anti-α-Gal antibodies. This work comprises the purification and characterization of anti-α-Gal antibodies from human immunoglobulin G (IgG). A synthetically manufactured α Gal epitope affinity resin was used to enrich anti-α-Gal antibodies. Selectivity experiments with purified antibodies were carried out using enzyme-linked immunosorbent assays (ELISA), Western blotting, and erythrocyte agglutination. Furthermore, binding affinities toward α-Gal were determined by surface plasmon resonance (SPR) and the IgG distribution of anti α Gal antibodies (83% IgG2, 14% IgG1, 2% IgG3, 1% IgG4) was calculated applying ELISA and immunodiffusion. A range of isoelectric points from pH 6 to pH 8 was observed in 2D gel electrophoresis. Glycan profiling of anti α Gal antibodies revealed complex biantennary structures with high fucosylation grades (86%). Additionally, low amounts of bisecting GlcNAc (15%) and sialic acids (13%) were detected. The purification of anti-α-Gal antibodies from human IgG was successful, and their use as detection antibodies for α Gal-containing structures was evaluated.

The Quantification of IgG Specific to α-Gal Could Be Used as a Risk Marker for Suffering Mammalian Meat Allergy

“…Galili et al (1984) established that the level of anti-α-Gal Abs in healthy donors was 1% [8]; Yu et al (1996) quantified by ELISA that the value was in the range of 0 to 15 µg/mL [27], and Tomlinson and Nussenzweig (1997) established this value between 0.25 and 0.5% of total Igs [48]; Obukhova et al (2007) indicated that the level of Igs against α-Gal in healthy donors was about 10-fold less than the established by Galili et al (1984) [49]. Recently, Zappe et al (2021) published that the level of IgG anti-α-Gal in a commercial concentrate of human IgG is about 10% (IgG1 isotype) [50].…”

“…There are several works on the Ab titers of the different Ig isotypes and IgG subclasses expressed in both AGS patients and healthy donors, including their relationship to the blood group of the individuals [46,47] and, the published data on the concentration of IgG to α-Gal in normal human serum are controversial depending on the methodology used for quantification. [37,[48][49][50] The objective of our study was to quantify, by means of an automated and standardized method, the levels of sIgG to α-Gal in four different groups of adult subjects: clinical diagnosed AGS-patients, atopic subjects, healthy donors, and in a group of subjects bitten by ticks but who had not developed AGS. The aim was to stablish a sIgG to α-Gal cut-off value.…”

α-Gal Nanoparticles Mediated Homing of Endogenous Stem Cells for Repair and Regeneration of External and Internal Injuries by Localized Complement Activation and Macrophage Recruitment

“…Anti-Gal is one of the most abundant natural antibodies in humans, constituting ~1% of immunoglobulins [ 26 , 27 , 28 , 29 , 30 ]. It is produced throughout life, starting few months after birth [ 26 , 31 ], in response to antigenic stimulation by gastrointestinal bacteria [ 32 , 33 , 34 , 35 , 36 , 37 ].…”