Antibody repertoire profiling using bacterial display random peptide libraries for biomarker discovery.

摘要

Antibody biomarkers have been used to help diagnose autoimmune, inflammatory, and infectious diseases. Serologic diagnostic assays are non-invasive and relatively inexpensive in comparison to common diagnostic tools. The problem is a majority of diseases do not have a known or reliable biomarker because we lack generally applicable biomarker discovery techniques. To address this problem, we developed a general strategy to identify serum antibody specificities associated with a given disease state and peptide reagents for their detection. To validate the novel antibody repertoire profiling technology, random peptide libraries were screened for peptides that react specifically with antibodies from individuals with celiac disease (CD), an autoimmune disease with well understood pathology. The libraries were screened by fluorescence-activated cell sorting for peptides that simultaneously cross-react with pooled CD patient antibodies present in two separate patient groups labeled with spectrally distinct fluorophores, thus affording a quantitative separation. A panel of six unique peptide sequences yielded 85% sensitivity and 91% specificity on a set of 60 samples not used for discovery, proving the success of the technique. The biomarker discovery technology was further optimized and applied to address an unmet diagnostic need in non-responsive celiac disease (NRCD). Since current assessments of CD recovery are limited to invasive and costly serial duodenal biopsies, we sought to identify novel antibody biomarkers for CD patients that do not respond to traditional therapy. The dominant consensus epitope sequence identified by unbiased library screening was highly similar to reported deamidated gliadin peptide (dGP) B-cell epitopes. Measurement of anti-dGP IgG titer by ELISA discriminated between NRCD and responsive CD patients with 87% sensitivity and 89% specificity. Importantly, dGP antibody titer correlated with the severity of mucosal damage indicating that IgG dGP titers may be useful to monitor intestinal mucosal recovery on a gluten-free diet. Finally, the technology is currently being applied to identify unique antibody specificities in schizophrenia, which is a mental illness that lacks objective diagnostic measurements. These results demonstrate that bacterial display provides a highly effective tool for the unbiased discovery of disease-associated antibody specificities and peptide reagents for their detection that may have broad utility for diagnostic development.