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.

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

Stability and reproducibility of proteomic profiles measured with an aptamer-based platform

The feasibility of SOMAscan, a multiplex, high sensitivity proteomics platform, for use in studies using archived plasma samples has not yet been assessed. We quantified 1,305 proteins from plasma samples donated by 16 Nurses’ Health Study (NHS) participants, 40 NHSII participants, and 12 local volunteers. We assessed assay reproducibility using coefficients of variation (CV) from duplicate samples and intra-class correlation coefficients (ICC) and Spearman correlation coefficients (r) of samples processed (i.e., centrifuged and aliquoted into separate components) immediately, 24, and 48 hours after collection, as well as those of samples collected from the same individuals 1 year apart. CVs were <20% for 99% of proteins overall and <10% for 92% of proteins in heparin samples compared to 66% for EDTA samples. We observed ICC or Spearman r (comparing immediate vs. 24-hour delayed processing) ≥0.75 for 61% of proteins, with some variation by anticoagulant (56% for heparin and 70% for EDTA) and protein class (ranging from 49% among kinases to 83% among hormones). Within-person stability over 1 year was good (ICC or Spearman r ≥ 0.4) for 91% of proteins. These results demonstrate the feasibility of SOMAscan for analyses of archived plasma samples

Reproducibility of Circulating MicroRNAs in Stored Plasma Samples

Background Most studies of microRNA (miRNA) and disease have examined tissue-specific expression in limited numbers of samples. The presence of circulating miRNAs in plasma samples provides the opportunity to examine prospective associations between miRNA expression and disease in initially healthy individuals. However, little data exist on the reproducibility of miRNAs in stored plasma. Methods We used Real-Time PCR to measure 61 pre-selected microRNA candidates in stored plasma. Coefficients of variation (CVs) were used to assess inter-assay reliability (n = 15) and within-person stability over one year (n = 80). Intraclass correlation coefficients (ICCs) and polychoric correlation coefficients were used to assess within-person stability and delayed processing reproducibility (whole blood stored at 4°C for 0, 24 and 48 hours; n = 12 samples). Results Of 61 selected miRNAs, 23 were detected in at least 50% of samples and had average CVs below 20% for inter-assay reproducibility and 31 for delayed processing reproducibility. Ten miRNAs were detected in at least 50% of samples, had average CVs below 20% and had ICCs above 0.4 for within-person stability over 1–2 years, six of which satisfied criteria for both interassay reproducibility and short-term within-person stability (miR-17-5p, -191-5p, -26a-5p, -27b-3p, -320a, and -375) and two all three types of reproducibility (miR-27b-3p and -26a-5p). However, many miRNAs with acceptable average CVs had high maximum CVs, most had low expression levels, and several had low ICCs with delayed processing. Conclusions About a tenth of miRNAs plausibly related to chronic disease were reliably detected in stored samples of healthy adults

Summary of reproducibility of 15 CVD-related miRNAs with CVs below 20% in duplicate samples, samples with processing delayed up to 48 hours, and samples collected 1–2 years apart.

<p>Summary of reproducibility of 15 CVD-related miRNAs with CVs below 20% in duplicate samples, samples with processing delayed up to 48 hours, and samples collected 1–2 years apart.</p

Expression levels of 12 miRNAs* with delayed processing of 0, 24, and 48 hours at 4°C.

<p>*12 miRNAs detectable in ≥ 50% of samples and with CVs below 20% for inter-assay reproducibility, delayed processing reproducibility, and short-term within-person stability, and ICCs ≥ 0.3 for short-term within-person stability.</p

Average and maximum pair CVs for standardized values of 61 miRNAs measured in 15 duplicate (split) samples.

<p>Bold indicates acceptable results and italics indicates unacceptable results. Avg CV = average CV; Max CV = maximum CV; % Detected = % samples with detectable levels; NC = not calculated.</p><p>Average and maximum pair CVs for standardized values of 61 miRNAs measured in 15 duplicate (split) samples.</p

ICCs, average pair CVs, and correlation coefficients (r) using standardized values for 61 miRNAs measured in 80 participants one year apart.

<p>Bold indicates acceptable results and italics indicates unacceptable results, NC = not calculated.</p><p>ICCs, average pair CVs, and correlation coefficients (r) using standardized values for 61 miRNAs measured in 80 participants one year apart.</p

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

<p>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).</p

Comparison of serum anti-Neu5Gc antibody levels between coronary artery disease cases and controls matched for age and sex in the EPIC-Norfolk cohort.

<p>Comparison of serum anti-Neu5Gc antibody levels between coronary artery disease cases and controls matched for age and sex in the EPIC-Norfolk cohort.</p