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 interpolated 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; stage I (n = 19), stage II (n = 40), stage III (n = 34) and stage IV (n = 6) was similar (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.

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), ribotype-specific 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.