Profiling humoral immune responses to Clostridium difficile-specific antigens by protein microarray analysis

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotypespecific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated<10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost

The application of protein microarray assays in psychoneuroimmunology

Protein microarrays are miniaturized multiplex assays that exhibit many advantages over the commonly used enzyme-linked immunosorbent assay (ELISA). This article aims to introduce protein microarrays to readers of Brain, Behavior, and Immunity and demonstrate its utility and validity for use in psychoneuroimmunological research. As part of an ongoing investigation of psychological and behavioral influences on influenza vaccination responses, we optimized a novel protein microarray to quantify influenza-specific antibody levels in human sera. Reproducibility was assessed by calculating intra- and inter-assay coefficients of variance on serially diluted human IgG concentrations. A random selection of samples was analyzed by microarray and ELISA to establish validity of the assay. For IgG concentrations, intra-assay and inter-assay precision profiles demonstrated a mean coefficient of variance of 6.7% and 11.5% respectively. Significant correlations were observed between microarray and ELISA for all antigens, demonstrating the microarray is a valid alternative to ELISA. Protein microarrays are a highly robust, novel assay method that could be of significant benefit for researchers working in psychoneuroimmunology. They offer high throughput, fewer resources per analyte and can examine concurrent neuro-immune-endocrine mechanisms

Human blood autoantibodies in the detection of colorectal cancer

Colorectal cancer (CRC) is the second most common malignancy in the western world. Early detection and diagnosis of all cancer types is vital to improved prognosis by enabling early treatment when tumours should be both resectable and curable. Sera from 3 different cohorts; 42 sera (21 CRC and 21 matched controls) from New York, USA, 200 sera from Pittsburgh, USA (100 CRC and 100 controls) and 20 sera from Dundee, UK (10 CRC and 10 controls) were tested against a panel of multiple tumour-associated antigens (TAAs) using an optimised multiplex microarray system. TAA specific IgG responses were interpo- lated against the internal IgG standard curve for each sample. Individual TAA specific responses were examined in each cohort to determine cutoffs for a robust initial scoring method to establish sensitivity and specificity. Sensitivity and specificity of combinations of TAAs provided good discrimination between cancer-positive and normal serum. The overall sensitivity and specificity of the sample sets tested against a panel of 32 TAAs were 61.1% and 80.9% respectively for 6 antigens; p53, AFP, K RAS, Annexin, RAF1 and NY-CO16. Furthermore, the observed sensitivity in Pittsburgh sample set in different clinical stages of CRC;stageI(n=19),stageII(n=40),stageIII(n=34)andstageIV(n=6)wassimilar (73.6%, 75.0%, 73.5% and 83.3%, respectively), with similar levels of sensitivity for right and left sided CRC. We identified an antigen panel of sufficient sensitivity and specificity for early detection of CRC, based upon serum profiling of autoantibody response using a robust multiplex antigen microarray technology. This opens the possibility of a blood test for screening and detection of early colorectal cancer. However this panel will require further validation studies before they can be proposed for clinical practice

Internal quality control measures on MMA.

<p><b>A:</b> representative array image showing layout and inclusion of printed Human IgG standard curve (outlined in yellow) and 4 control antigens each as quadruplicate features, printed horizontally (outlined in white). The Human IgG standards are used to normalise all TAA features and arrays to a common scale. <b>B:</b> Overlay of 72 individual standard curves from 72 arrays probed with patient serum and one array probed with all detection reagents, except the anti-human polyclonal antibody (red diamonds). The dynamic range of the assay is shown in log2 scaled y axis. X axis represents the dilution of IgG (starting at 50ug/ml). <b>C</b>: Normalised signals to 4 control antigens obtained from 42 serum samples applied to optimised TAA arrays. Strong responses are seen to tetanus toxoid and <i>Candida albicans</i> antigen preparations.</p

Human Blood Autoantibodies in the Detection of Colorectal Cancer - Fig 5

<p><b>Sensitivity of of the Pittsburgh CRC sample set (n = 100) by stage (A) and site (B).</b> A: The percentage of CRC patients positive for AAb test (i.e sensitivity) by stage of disease at presentation for the Pittsburgh CRC samples. Cut-off point = 95th percentile of the control group. B: The percentage of CRC patients positive for AAb test (i.e sensitivity) by the site (left or right side of the colon).</p

A representative analysis of a selection of TAAs by Western blot (WB) and Silver stain.

<p>Samples were run on 4–20% precast polyacrylamide gels with a broad range protein markers. The 4 antigens (Gankyrin, H-Ras, RalA, and HCC1) were then either probed with anti-His antibody (<b>A</b>) or silver stained (<b>B</b>). Silver stain was used to check the purity of the antigens. The expected molecular weight of the examined antigens, Gankyrin, H-Ras, RalA, and HCC1 are 25, 40, 22 and 64 kDa respectively. The similar bands obtained before and after buffer exchange by both WB and silver stain indicate that the buffer exchange technique had no effect on the integrity of the antigens. <b>C</b>: Silver stain of 4 TAAs: Annexin, P53, KRAS and RAF1 (the expected molecular weight are 39, 53, 57 and 73 kDa respectively).</p

Representative antigen panels selected for all sample sets.

<p>The figure shows the autoantibody responses of the CRC patients (n = 131), healthy controls (n = 131), autoimmune (n = 21), IBD (n = 9) and allergy (n = 10) cohorts to a panel of TAA(s): data shows reactivity to AFP (A), Annexin (B), KRas (C), P53 (D), RAF1 (E) and NY-CO16 (F).</p

Technical development in microarray.

<p><b>A-C</b>: Demonstration of improvements in array background for a serum sample with high background signal when purified IgG (<b>B</b>) is used in place of whole serum (<b>A</b>). These arrays were processed at the same time and scanned under the same conditions. (<b>C</b>) displays the average background measurements taken from empty spots in both slides when the whole serum was used and when the purified IgG was used (n = 35). D-G: Effects of TAA denaturation of serum antibody binding. Representative antigens (<b>D</b>: AFP, <b>E</b>: APCN, <b>F</b>: SOX2 and <b>G</b>: P53) were printed as native protein or after heat-denaturation in the presence of 20mMDTT. Serum samples (n = 42) from a CRC cohort were used to examine alterations in autoantibody reactivity between the native and denatured TAAs. The mean of the signal from each TAA (4 replicates) is presented (n = 4).</p