Antibody Profiling of Bipolar Disorder Using Escherichia coli Proteome Microarrays

To profile plasma antibodies of patients with bipolar disorder (BD), an E. coli proteome microarray comprising ca. 4200 proteins was used to analyze antibody differences between BD patients and mentally healthy controls (HCs). The plasmas of HCs and patients aged 18–45 years with bipolar I disorder (DSM-IV) in acute mania (BD-A) along with remission (BD-R) were collected. The initial samples consisting of 19 BD-A, 20 BD-R, and 20 HCs were probed with the microarrays. After selecting protein hits that recognized the antibody differences between BD and HC, the proteins were purified to construct BD focus arrays for training diagnosis committees and validation. Additional six BD-A, six BD-R, six HCs, and nine schizophrenic disorder (SZ, as another psychiatric control) samples were individually probed with the BD focus arrays. The trained diagnosis committee in BD-A versus HC combined top six proteins, including rpoA, thrA, flhB, yfcI, ycdU, and ydjL. However, the optimized committees in BD-R versus HC and BD-A versus BD-R were of low accuracy (< 0.6). In the single blind test using another four BD-A, four HC, and four SZ samples, the committee of BD-A versus HC was able to classify BD-A versus HC and SZ with 75% sensitivity and 80% specificity that both HC and SZ were regarded as negative controls. The consensus motif of the six proteins, which form the committee of BD-A versus HC, is [KE]DIL[AG]L[LV]I[NL][IC][SVKH]G[LV][VN][LV] by Gapped Local Alignment of Motifs. We demonstrated that the E. coli proteome microarray is capable of screening BD plasma antibody differences and the selected proteins committee was successfully used for BD diagnosis with 79% accuracy.The etiology and genetic contributions of bipolar disorder (BD)¹ largely remain unknown (1). Because of the presumed high level of etiologic heterogeneity and the overlap of dimensions across mood disorders and schizophrenia (2), the main difficulty in making an exact diagnosis for psychiatric disorder is the lack of pathological biochemical index (3). However, several lines of evidence support that various immunomodulatory factors, such as cytokine and soluble cytokine receptor, play an integral role in the pathophysiology of bipolar disorder (4–7). For example, several studies have reported that cell-mediated immunity cytokine abundance is correlated with mood state (8, 9). Our early works also found that higher levels of soluble interleukin-2 receptor (sIL-2R) (5, 10) and interleukin 1 receptor antagonist (IL-1Ra) (5, 11) are accompanied with bipolar mania. Furthermore, the abnormalities of total immunoglobulins levels in body fluid are observed in BD patients (12, 13).The possibility of biomarkers for assisting BD diagnosis has been recently highlighted (14–16). Tumor necrosis factor alpha (TNF-α), 3-nytrotrosine, interleukin-6, interleukin-10, and brain-derived neurotrophic factor in body fluids are potentially useful for classifying stages of BD (15). Nevertheless, they are not specific for distinguishing from other psychiatric diseases (17). Chronic inflammation exists in medicated bipolar patients displaying varied correlations with leptin, insulin, soluble TNF receptor-1 (sTNF-R1), and IL-1Ra (11). Notwithstanding, controversy exists as to whether these phenomena are state-dependent (5), normalize in remission (18), or represent trait markers exacerbated by the affective episodes (19). These discrepancies may be explained by heterogeneity in mood state, methodological differences, and not controlling for known confounds, such as obesity (6). In addition to inflammatory markers, increasing production of antibodies (20–22) and immunoglobulins (23, 24) may be implicated with BD.In recent years, proteomic technologies based on mass spectrometry have been increasingly used, especially in the search for diagnostic and prognostic biomarkers in neuropsychiatric disorders (25). Protein microarrays have been demonstrated as an effective high throughput platform for analysis of aberrant immune responses in diseases (26–29). It is hypothesized that the trait or state-dependent biomarkers of bipolar disorder may exist. We attempted to identify a committee of proteins for the diagnosis of BD through employing the ca. 4200 E. coli proteins in a microarray format. The two-phase strategy for identification and validation protein hits (30) was used in this study. Although the antigens on the microarray may not be directly associated with BD, this microarray provided hundreds of thousands of epitopes for analyzing antibody profiles of plasma samples in a high throughput fashion.