Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays

PLOS ONE, Dec 2019

Background Most antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods. Methods Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes. Results Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity. Conclusions These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis.

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Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays

January Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays Rok Seon Choung 0 1 2 Eric V. Marietta 0 1 2 Carol T. Van Dyke 0 1 2 Tricia L. Brantner 0 1 2 John Rajasekaran 0 1 2 Pankaj J. Pasricha 0 1 2 Tianhao Wang 0 1 2 Kang Bei 0 1 2 Karthik Krishna 0 1 2 Hari K. Krishnamurthy 0 1 2 Melissa R. Snyder 0 1 2 Vasanth Jayaraman 0 1 2 Joseph A. Murray 0 1 2 JAM are joint senior authors on this work. 0 1 2 0 1 2 0 1 Division of Gastroenterology and Hepatology, Mayo Clinic , Rochester, MN , United States of America, 2 Vibrant Sciences LLC , San Carlos, CA , United States of America, 3 Center for Neurogastroenterology, Johns Hopkins University , Baltimore, MD , United States of America, 4 Division of Clinical Biochemistry and Immunology, Mayo Clinic , Rochester, MN , United States of America 1 Competing Interests: The authors have the following interests. John Rajasekaran , Vasanth 2 Editor: Massimo Pietropaolo, Baylor College of Medicine, UNITED STATES Most antibodies recognize conformational or discontinuous epitopes that have a specific 3dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods. - OPEN ACCESS Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: JAM and RSC were supported by the Mayo Foundation. Vibrant Sciences LLC provided support in the form of salaries for authors JR, VJ, KB, TW, HKK and KK, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Background Methods Results Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes. Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity. Conclusions These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultrahigh-density peptide microarray described here would be broadly useful to develop highfidelity diagnostic tests and explore pathogenesis. Jayaraman, Kang Bei, Hari Krishnamurthy, Tianhao Wang and Karthik Krishna are employed by Vibrant Sciences. Dr Pasricha's participation in this publication was as a paid consultant for Vibrant Sciences. All opinions expressed and implied in this publication are solely those of Dr Pasricha and do not represent or reflect the views of the Johns Hopkins University or the Johns Hopkins Health System. Material related to the subject matter discussed in the present article has previously been disclosed in the International Patent Application Nos. PCT/US2013/ 070207 and PCT/US2013/025190. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Introduction Antibody detection is one of the main approaches for the diagnosis of many diseases, including autoimmune disorders, infectious diseases, and cancers.[ 1–3 ] Indeed, the development of antibody-based assays has been intensively pursued for the diagnosis and treatment of many diseases; however, only a small number of biomarkers have been identified as effective.[ 1, 4 ] With peptide arrays, the overlapping synthetic peptide approach has been used as an effective way to map the epitope specificity of antibodies.[ 5–7 ] This method is especially effective for identifying linear antigenic epitopes derived from known target proteins, but has been restricted by the expense and logistics of acquiring and handling large numbers of peptides. Recent advances in semiconductor methods and the generation of high-throughput peptide microarrays using a combination of lithography and biochemistry for peptide synthesis have opened the door to a new era in the identification of novel biomarkers of disease.[ 5, 8, 9 ] Here, we describe a novel method for silicon-based peptide microarray with computa (...truncated)


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Rok Seon Choung, Eric V. Marietta, Carol T. Van Dyke, Tricia L. Brantner, John Rajasekaran, Pankaj J. Pasricha, Tianhao Wang, Kang Bei, Karthik Krishna, Hari K. Krishnamurthy, Melissa R. Snyder, Vasanth Jayaraman, Joseph A. Murray. Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays, PLOS ONE, 2016, 1, DOI: 10.1371/journal.pone.0147777