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Abstract
BackgroundMost 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.MethodsUsing 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.ResultsUsing 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.ConclusionsThese 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.
Highlights
The final discontinuous epitope sets were tested in a confirmatory cohort of celiac disease (CD) patients and controls, yielding 99% sensitivity and 100% specificity
These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests
Kang Bei, Hari Krishnamurthy, Tianhao Wang and Karthik Krishna are employed by Vibrant Sciences
Summary
Antibody detection is one of the main approaches for the diagnosis of many diseases, including autoimmune disorders, infectious diseases, and cancers.[1,2,3] the development of antibody-based assays has been intensively pursued for the diagnosis and treatment of many diseases; 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.[5,6,7] 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 computational algorithm to identify the discontinuous epitopes from native and modified gliadin peptides that glutamic acid was substituted for glutamine, which are highly reactive in patients with celiac disease (CD). 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
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