Table 1.
Description of experiments for inter-assay reproducibility, stability with processing delays, and short-term within-person stability.
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.
Table 2.
Comparison of serum anti-Neu5Gc antibody levels between coronary artery disease cases and controls matched for age and sex in the EPIC-Norfolk cohort.
Table 3.
Odds ratios for coronary heart disease by quintile of each analyte for 835 coronary artery disease cases and 1869 controls in the EPIC-Norfolk cohort.
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).
Table 4.
Inter-assay reproducibility, effects of processing delay and within-person reproducibility in the NHS cohort.
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).
Table 5.
Descriptive characteristics of the colorectal cancer case-control study for anti-Neu5Gc IgG levels, divided into quartiles in the EPIC-Norfolk cohort.
Table 6.
Matched logistic regressiona models for the sum of antibodies against all Neu5Gc-glycans measured in quartiles, and log transformed sum of antibodies against all Neu5Gc-glycans in the EPIC-Norfolk cohort.
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.
Table 7.
Linear regression models for the log transformed sum of antibodies against all Neu5Gc-glycans against food diary red meat for cases and controls separately in the EPIC-Norfolk cohort.