A dataset for the analysis of antibody response to glycan alpha-Gal in individuals with immune-mediated disorders

Patients and healthy individuals

A retrospective case-control study was conducted in patients suffering from COVID-19 admitted to the University General Hospital of Ciudad Real (HGUCR), Spain from March 1 to April 15, 2020. The infection by SARS-CoV-2 was confirmed in all patients included in the study by the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay from Abbott Laboratories (Abbott RealTime SARS-COV-2 assay, Abbott Park, Illinois, USA) from upper respiratory tract samples after hospital admission. Clinical features, as well as laboratory determinations were obtained from patient's medical records. The patients were grouped as hospital discharge, hospitalized and intensive care unit (Urra et al., 2021). Patients were hospitalized for developing a moderate-severe clinical condition with radiologically demonstrated pneumonia and failure in blood oxygen saturation. Patients with acute respiratory failure who needed mechanical ventilation support were admitted to a hospital ICU. The patients were discharged from the hospital due to the clinical and radiological improvement of pneumonia caused by the SARS-CoV-2, along with the normalization of analytical parameters indicative of inflammation, such as C-reactive protein (CRP), D-Dimer and blood cell count (Urra et al., 2021). Samples from asymptomatic COVID-19 cases with positive anti-SARS-CoV-2 IgG antibody titers but negative by RT-PCR were collected in May 22–29, 2020 and included in the dataset (Urra et al., 2021). Samples from healthy individuals (individuals without record of tick bites and allergic reactions) and patients diagnosed with tick-borne allergic reactions (AGS, anaphylaxis or urticaria) were collected prior to COVID-19 pandemic in April 2019 (Pacheco et al., 2021). The use of human peripheral blood serum samples from healthy individuals and patients diagnosed with tick-borne allergic reactions was done with their written informed consent in compliance with the Helsinki Declaration. Nursing personnel at the General University Hospital of Ciudad Real, Spain, extracted blood samples. Samples and data from patients with GBS included in this dataset were provided by the BioB-HVS, integrated into the Spanish National Biobanks Network. All samples were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees (Toledo Hospitable Complex 29012014-No17, University Hospital of Ciudad Real C-352 and SESCAM C-73).

Preparation of serum samples

For the preparation of serum samples, a sterile tube without anticoagulant was used to collect blood samples. The blood from each patient and the healthy individual was maintained in standing position at room temperature (RT) for clotting (20–30 min) and centrifuged at 1,500 × g for 20 min at RT. Serum was collected and conserved at -20°C until used for analysis.

Determination of antibody titers against α-Gal

For ELISA, high absorption capacity polystyrene microtiter plates were coated with 50 ng of BSA coated with α-Gal (Galα1-3Gal-BSA, 3 atom spacer, product code NGP0203, thereafter named α-Gal; Dextra, Shinfield, UK) per well in carbonate-bicarbonate buffer (Sigma-Aldrich, St. Louis, MO, USA). After an overnight incubation at 4°C, coated plates were washed one time with 100 µl/well PBS with 0.05% Tween 20 (PBST) (Sigma-Aldrich), blocked with 100 µl/well of 1% human serum albumin (HAS) in PBST (Sigma-Aldrich) for 1 h at RT and then washed four times with 100 µl/well of PBST. Human serum samples were diluted 1:100 in PBST with 1% HAS and 100 µl/well were added into the wells of the antigen-coated plates and incubated for 1 h at 37°C. Plates were washed four times with PBST and 100 µl/well of goat anti-human immunoglobulins-peroxidase IgG (FC specific) (Cat. No. I2136), IgM (µ-chain specific) (Cat. No. I1636), and IgE (ɛ-chain specific) (Cat. No. I6284) secondary antibodies (Sigma-Aldrich) diluted 1:1000, v/v in blocking solution were added and incubated for 1 h at RT. Plates were washed four times with 100 µl/well of PBST and 100 µl/well of 3,3,´5,5-tetramethylbenzidine TMB (Promega, Madison, WI, USA) were added and incubated for 20 min at RT. Finally, the reaction was stopped with 50 µl/well of 2 N H₂SO₄ and the O.D. was measured in a spectrophotometer at 450 nm. The average of two technical replicates per sample was used for analysis after background (coated wells incubated with PBS and secondary antibodies) subtraction.

Statistical analysis

Anti-α-Gal IgE, IgM and IgG antibody titers (O.D. at 450 nm values) were compared for each Ig by one-way ANOVA test (p < 0.05) (https://www.socscistatistics.com/tests/anova/default2.aspx) (Figure 1A and 1C). A Spearman Rho correlation analysis (p < 0.01; https://www.socscistatistics.com/tests/spearman/default2.aspx) was conducted between anti-α-Gal IgE, IgM and IgG antibody titers and age (Figure 1B).

Figure 1.

An example of the effect of certain factors such as (A) blood group, (B) age and (C) sex on the antibody response to α-Gal in healthy individuals. Anti-α-Gal IgE, IgM and IgG antibody titers were determined by ELISA. (A, C) The ELISA O.D. at 450 nm values were compared for each Ig by one-way ANOVA test (p < 0.05). (B) A Spearman Rho correlation analysis (p < 0.01) was conducted between anti-α-Gal IgE, IgM and IgG antibody titers and age. Correlation coefficient (R²) is shown. Please refer to Pacheco et al. (2021) for validation of the ELISA with ImmunoCAP Phadia 250 automated platform (Thermo Fisher Scientific, Uppsala, Sweden) with the commercial ImmunoCap α-Gal bovine Thyroglobulin kit according to the manufacturer’s instructions.

Dataset validation

The dataset (de la Fuente et al., 2020) was validated in studies reported by Urra et al. (2021), Pacheco et al. (2020) and Doncel-Pérez et al. (2020). A recent study correlated blood group with anti-α-Gal antibody response and SARS-CoV-2 infection (Hodžić et al., 2020b). Despite the presence of relatively high anti-α-Gal IgE levels in healthy individuals, factors such as tick bites or allergy correlate with higher IgE antibody titers against α-Gal (Pacheco et al., 2021). Additionally, a comparative analysis was conducted between the IgE+IgM+IgG antibody response to α-Gal and blood groups (Figure 1A), age (Figure 1B) and sex (Figure 1C) in healthy individuals (n = 75) to illustrate lower antibody titers in blood group B/AB individuals as previously reported (Cabezas-Cruz et al., 2017) but no differences regarding age and sex, which have been reported before as factors affecting the antibody response to α-Gal, infection and vaccination (Buonomano et al., 1999; Giefing-Kröll et al., 2015; Wang et al., 1995).

The main limitation of the dataset is sample size for some factors (i.e. age, sex or blood group), which were not disclosed by all individuals, and anti-α-Gal IgA antibody titers that could be considered in the analysis (Mateos-Hernández et al., 2020; Urra et al., 2021).

Underlying data

Harvard Dataverse: A dataset for the analysis of antibody response to glycan alpha-Gal in individuals with immune-mediated disorders. https://doi.org/10.7910/DVN/RBU2VR (de la Fuente et al., 2020).

This dataset contains characteristics and serum antibody levels of the individuals included in the study and was used in analyses reported in publications by Urra et al. (2021), Pacheco et al. (2021) and Doncel-Pérez et al. (2020).

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).