Enhanced Detection of αGal Using a Novel Monoclonal IgG1 Antibody: Comparative Evaluation with IgM Antibody [clone M86]

Introduction. Over the past two decades, the αGal (Galα1–3Galβ1–4GlcNAc–R) epitope, a carbohydrate found in many non-primate mammals, has gained significant relevance in medicine due to its association with an increasing number of allergic reactions to animal-derived foods, drugs, and medical devices. Due to the mutated gene coding for α1,3-galactosyltransferase (α1–3GT), humans lack αGal and, therefore, naturally produce anti-α-Gal antibodies (IgM, IgA, and IgG), especially in the context of a xenotransplantation, which can lead to extreme immunological reactivity, including hyperacute rejection of the transplant. Recently, these uncontrollable immune reactions have driven demand for more accurate procedures to better detect αGal in animal-derived foods or bioprosthetics. The currently most widely used α-Gal-specific monoclonal antibody is an IgM antibody (clone M86), developed in Ggta1 KO mice and isolated from hybridoma tissue culture. As the IgM isotype has limited purification properties, specificity, and sensitivity, we aimed to produce a novel IgG antibody with high affinity and extensive applicability. Methods. An experimental murine IgG1 anti-αGal antibody (IgG-αGalomab) was developed by immunization of Ggta1 knockout (KO) mice, and its affinity was evaluated using ELISA, Western blot, flow cytometry, and immunohistochemistry/immunofluorescence. Results. Compared to IgM-M86, IgG-αGalomab demonstrated ~1200-fold higher binding potency and lower cross-reactivity with competitive molecules, i.e., bovine serum albumin, galactobiose, and lactose. Unlike IgM-M86, IgG-αGalomab showed an increasing affinity over time in the binding tests performed on xenogeneic tissues. Notably, high-affinity for αGal was detected by Western blot at high dilution [1:200,000] of IgG-αGalomab compared to IgM-M86 [1:1,000]. By flow cytometry, specificity and dose-dependent response were confirmed using in vitro cultures of porcine and human fibroblasts. Finally, αGal demonstrated to be detectable by IgG-αGalomab at the dilution of [1:1,000] while IgM-M86 at that of [1:100] in immunofluorescence and immunohistochemistry analysis. Conclusions. Altogether, our newly developed antibody showed high sensitivity and specificity for α-Gal in various applications. Based on its potential binding capacity, IgG-αGalomab could have important applications in precision medicine for predicting, treating, and preventing immune-mediated phenomena of patients in different medical areas.