OS042. Discovery and characterization of antibody biomarkers ofpre-eclampsia and reagents for their detection

The effective management of pre-eclampsia (PE), a complex condition affecting 5% of pregnancies [1], would benefit from a clear diagnostic assay. Due to this need, great interest lies in the development and validation of objective biomarkers, among which antibodies remain attractive given their amplification by the immune system, stability, and current clinical use. Although several recent studies lend support to the idea that autoantibodies against the angiotensin II AT1 receptor (AT1-AAs) could be involved in PE [1], their presence is not highly specific for PE as measured by cell-based assays for AT1 agonism [2]. Given these results, we hypothesized that additional antibody biomarkers may exist in PE, and that a small panel of such biomarkers could provide an effective diagnostic tool. Furthermore, development of a simple binding assay will facilitate AT1-AA detection for larger-scale studies, while identifying other antibody biomarker(s) would enhance understanding of the immune component to PE pathogenesis.

The primary objective of this study was to identify a panel of peptide reagents that preferentially bind PE patients' serum antibodies. In addition, we sought to measure the frequency of AT1-AAs in a new patient cohort using a simplified assay.

Plasma samples were obtained from normal-outcome pregnancies (n=28) as well as PE patients (n=25) and enriched for the antibody fraction. The seven amino acid AT1-AA epitope [1] was expressed on the surface of E. coli for reactivity analysis by flow cytometry. A bacteria-displayed linear peptide library was screened against the antibody repertoires using a unique method of fluorescence activated cell sorting (FACS) to isolate peptides that react with multiple patients and show little reactivity with normal-outcome pregnancies. FACS analysis was used to measure individual peptide reactivity, and statistical analyses included a Student's t-test and nonparametric tests to compare the means, medians, and normal scores.

Using this simple binding assay, AT1-AAs were detected in a majority of PE patients and more rarely in normal-outcome pregnancies. Among the isolated peptide mimics, several unique peptides demonstrated significantly (p<0.03) higher reactivity with PE patients than with control samples but showed no sequence homology to the known AT1 epitope. Not only did our peptides perform well with the original set of samples used for discovery, but these peptides also reacted with antibodies from a small set of new PE (n=10) sera but not from new normal-outcome (n=10) pregnancies.

Peptides distinct from the AT1 epitope and identified by library screening exhibited potential diagnostic utility for PE, suggesting that a panel of such peptides might provide a novel diagnostic test to distinguish PE from other conditions with similar symptoms. Also, this study demonstrated AT1-AA presence in an independent patient cohort with a simple binding assay, which can be easily expanded to evaluate larger cohorts.