Polyclonal human antibodies against glycans bearing red meat-derived non-human sialic acid N-glycolylneuraminic acid are stable, reproducible, complex and vary between individuals: Total antibody levels are associated with colorectal cancer risk

Abstract

Background: N-glycolylneuraminic acid (Neu5Gc) is a non-human red-meat-derived sialic acid immunogenic to humans. Neu5Gc can be metabolically incorporated into glycan chains on human endothelial and epithelial surfaces. This represents the first example of a "xeno-autoantigen", against which circulating human "xeno-autoantibodies" can react. The resulting inflammation ("xenosialitis") has been demonstrated in human-like Neu5Gc-deficient mice and contributed to carcinoma progression via antibody-mediated inflammation. Anti-Neu5Gc antibodies have potential as biomarkers for diseases associated with red meat consumption such as carcinomas, atherosclerosis, and type 2 diabetes.

Methods: ELISA assays measured antibodies against Neu5Gc or Neu5Gc-glycans in plasma or serum samples from the Nurses' Health Studies, the Health Professionals Follow-up Study, and the European Prospective Investigation into Cancer and Nutrition, including inter-assay reproducibility, stability with delayed sample processing, and within-person reproducibility over 1-3 years in archived samples. We also assessed associations between antibody levels and coronary artery disease risk (CAD) or red meat intake. A glycan microarray was used to detected antibodies against multiple Neu5Gc-glycan epitopes. A nested case-control study design assessed the association between total anti-Neu5Gc antibodies detected in the glycan array assay and the risk of colorectal cancer (CRC).

Results: ELISA assays showed a wide range of anti-Neu5Gc responses and good inter-assay reproducibility, stability with delayed sample processing, and within-person reproducibility over time, but these antibody levels did not correlate with CAD risk or red meat intake. Antibodies against Neu5Gc alone or against individual Neu5Gc-bearing epitopes were also not associated with colorectal cancer (CRC) risk. However, a sialoglycan microarray study demonstrated positive association with CRC risk when the total antibody responses against all Neu5Gc-glycans were combined. Individuals in the top quartile of total anti-Neu5Gc IgG antibody concentrations had nearly three times the risk compared to those in the bottom quartile (Multivariate Odds Ratio comparing top to bottom quartile: 2.98, 95% CI: 0.80, 11.1; P for trend = 0.02).

Conclusions: Further work harnessing the utility of these anti-Neu5Gc antibodies as biomarkers in red meat-associated diseases must consider diversity in individual antibody profiles against different Neu5Gc-bearing glycans. Traditional ELISA assays for antibodies directed against Neu5Gc alone, or against specific Neu5Gc-glycans may not be adequate to define risk associations. Our finding of a positive association of total anti-Neu5Gc antibodies with CRC risk also warrants confirmation in larger prospective studies.

Fig 1. Distribution of Anti-Neu5Gc IgG titers in EPIC-Norfolk cohort using defined ELISA target antigens.

Each dot represents a value for a single subject. (A) Levels of serum anti-Neu5Gc IgG quantified by ELISA using Neu5Gcα2-polyacrylamide (Neu5Gcα2-PAA) as the target antigen (N = 2716). The mean anti-Neu5Gc IgG titer was 8.6 μg/ml (SD±10.6). (B) Levels of serum anti-Neu5Gc IgG quantified by ELISA using Neu5Gcα2–6Lacβ-human serum albumin (HSA) as the target antigen (N = 2712). The mean anti-Neu5Gc IgG titer was 1.4 μg/ml (SD±1.2). (C) There was no correlation between anti-Neu5Gc IgG antibody levels directed against the two different chemically synthesized ELISA targets: Neu5Gcα2-PAA and Neu5Gcα2–6Lacβ-HSA.

Fig 2. Correlation between total and inhibitable anti-Neu5Gc IgG titers in the NHS cohort.

Plasma anti-Neu5Gc IgG levels were quantified by ELISA using wild type mouse serum that contains naturally occurring Neu5Gc-containing epitopes as the target antigen (N = 46). A strong correlation was observed between total and inhibitable anti-Neu5Gc IgG titers in the NHS cohort (Spearman r = 0.80).

Fig 3. Distribution of anti-Neu5Gc IgG titers against a mixture of epitopes in NHS II population.

Plasma total and inhibitable anti-Neu5Gc IgG levels were quantified by ELISA using wild-type mouse serum as a target that contains naturally occurring Neu5Gc containing sialoglycoproteins epitopes as antigens (N = 46; Table 1). Total anti-Neu5Gc IgG refers to the “raw” antibody level obtained against the ELISA target. To obtain a more specific titer, plasma was incubated with free Neu5Gcα2Me to block non-specific binding and the inhibitable anti-Neu5Gc IgG is this signal subtracted from the total signal. Plasma anti-Neu5Gc IgG antibodies in the NHS II population displayed a discontinuous distribution. While the majority of individuals have low levels, a minority of samples (represented by dots) exhibit very high levels of anti-Neu5Gc IgG.

Fig 4. Distribution of Anti-Neu5Gc IgG titers against a mixture of epitopes in the EPIC-Norfolk cohort.

(A) Serum anti-Neu5Gc IgG antibodies in the EPIC-Norfolk population assayed as in Fig 1. Serum total and inhibitable anti-Neu5Gc IgG levels were quantified by ELISA using wild type mouse serum that contains naturally occurring Neu5Gc containing epitopes as the target antigen (N = 1469). The mean total IgG was 1.7 μg/ml (SD±1.6) and the mean inhibitable IgG was 0.4 μg/ml (SD±1.2). Total and “inhibitable” anti-Neu5Gc IgG were defined as in Fig 1. (B) The distribution of levels of anti-Neu5Gc IgG was evaluated by dividing all the samples into 16 bins, in steps of 1 μg/ml each, and the frequency of number of tested samples in each bin described as bar charts for both total (black) and inhibitable (grey) IgG values.

Fig 5. Lack of Correlation between Anti-Neu5Gc IgG titers and red meat intake in the NHS II population.

Plasma anti-Neu5Gc IgG levels (total anti-Neu5Gc IgG levels, or inhibitable anti-Neu5Gc IgG levels) were quantified by ELISA using wild type mouse serum as a target that contains multiple naturally occurring Neu5Gc containing epitopes (N = 338). Individuals were sorted into categories based on red meat servings/day (s/d).

Fig 6. No correlation in heatmap matrix of antibodies against 31 individual Neu5Gc-glycans and red meat consumption in the EPIC-Norfolk samples studied for CRC Risk.

The top and left side of the figure represents the individual glycans on the microarray, with each number representing the glycan ID. Each colored dot represents the correlation between the anti-Neu5Gc IgG against the individual glycans and red meat consumption. Black represents a correlation of 1, shades of blue are between 0 and 1, while shades of red are correlations between 0 and -1.