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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Available from: Lisa M Mcshane, Oct 13, 2015
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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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    ABSTRACT: Background: The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. Methods: An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Results: Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1% and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. Conclusions: This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.
    British Journal of Cancer 08/2014; DOI:10.1038/bjc.2014.455 · 4.84 Impact Factor
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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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    ABSTRACT: Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear. A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies. Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats. We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption. General significance The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.
    Biochimica et Biophysica Acta 11/2013; 1840(3). DOI:10.1016/j.bbagen.2013.11.009 · 4.66 Impact Factor
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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.
    PLoS ONE 08/2013; 8(8):e73027. DOI:10.1371/journal.pone.0073027 · 3.23 Impact Factor
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