Profiling Human Serum Antibodies with a Carbohydrate Antigen Microarray

Laboratory of Medicinal Chemistry, National Cancer Institute, Frederick, MD 21702, USA.
Journal of Proteome Research (Impact Factor: 4.25). 08/2009; 8(9):4301-10. DOI: 10.1021/pr900515y
Source: PubMed


Carbohydrate antigen arrays (glycan arrays) have been recently developed for the high-throughput analysis of carbohydrate macromolecule interactions. When profiling serum, information about experimental variability, interindividual biological variability, and intraindividual temporal variability is critical. In this report, we describe the characterization of a carbohydrate antigen array and assay for profiling human serum. Through optimization of assay conditions and development of a normalization strategy, we obtain highly reproducible results with a within-experiment coefficient of variation (CV) of 10.8% and an overall CV (across multiple batches of slides and days) of 28.5%. We also report antibody profiles for 48 human subjects and evaluate for the first time the effects of age, race, sex, geographic location, and blood type on antibody profiles for a large set of carbohydrate antigens. We found significant dependence on age and blood type of antibody levels for a variety of carbohydrates. Finally, we conducted a longitudinal study with a separate group of 7 serum donors to evaluate the variation in anti-carbohydrate antibody levels within an individual over a period ranging from 3 to 13 weeks and found that, for nearly all antigens on our array, antibody levels are generally stable over this period. The results presented here provide the most comprehensive evaluation of experimental and biological variation reported to date for a glycan array and have significant implications for studies involving human serum profiling.

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    • "The remaining investigated clinical parameters were not significantly different in their anti-P 1 IgM antibody levels mentioning Grade (G1 vs G2/3, P ¼ 0.2883; t-test) or tumour origin (ovary vs tube vs peritoneum, P ¼ 0.322; ANOVA). An increasing age has previously been demonstrated to be associated with reduction of AGA in a cohort of 48 control plasma samples using glycopeptide arrays (Oyelaran et al, 2009). To investigate the influence of age on anti-P 1 antibodies (IgG and IgM), we have applied Pearson's correlation to suspension array data and found a moderate negative correlation (rho ¼ À 0.33, Po0.01) in the entire cohort (mean age 56.4 (28–87) years). "
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    Full-text · Article · Aug 2014 · British Journal of Cancer
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    • "Next, our attention was directed to the effect of O-glycosylation at the immunodominant PDTRP motif on the interaction of mAbs with MUC1 fragments. In the present study, modifications of two 23- mer peptides with Tn, T, and sialyl-T antigens were performed at five potential glycosylation sites, PDTRP [42] [45] [48] [54] [57] [60], GSTA [43] [44] [46] [47] [49] [50], and VTSA [52] [53] [55] [56] [58] [59], respectively. Microarray slides displaying compounds 41–60 were prepared by a standardized protocol illustrated in Fig. 1B and employed for assessing the binding profile of mAbs. "
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    Full-text · Article · Nov 2013 · Biochimica et Biophysica Acta
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    • "The detection of anti-glycan antibodies in serum is of mounting interest for the evaluation of carbohydrate-based vaccines and pathogen infection as well as for the detection of biomarkers in diseases like cancer. The profiling of human serum antibodies has shown that a substantial part of circulating antibodies is directed against carbohydrates [1]. The affinity of anti-carbohydrate antibodies towards their epitopes, demands a multivalent presentation of the carbohydrate-ligands and highly sensitive screening methods. "
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    ABSTRACT: Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or Streptococcus pneumoniae were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against S. pneumoniae respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers.
    Full-text · Article · Aug 2013 · PLoS ONE
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