Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

Abstract

g 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. C lostridium difficile is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea, imposing a considerable financial burden on health service providers in both Europe and the United States (1-3). Infection causes a spectrum of clinical presentations ranging from an asymptomatic carrier state to severe fulminant colitis and death (4). Following successful treatment, an estimated 20% to 30% of patients with primary C. difficile infection (CDI) develop recurrence of symptoms, caused by either relapse of the original infection or reinfection with a new strain (5).This anaerobic and spore-forming bacterium exerts its major pathological effects through two proinflammatory and cytotoxic protein exotoxins, TcdA (toxin A) and TcdB (toxin B) (6). Nontoxin virulence factors, such as surface layer proteins (SLPs) and cell wall proteins, have also been described and may play a role in disease expression (7-9).The majority of healthy adults have detectable antibodies to C. difficile TcdA and TcdB in their sera that are thought to arise from colonization in infancy or from repeated exposure to C. difficile in adulthood from the environment (10, 11). Several clinical studies suggest that adaptive humoral immune responses, in particular to TcdA and TcdB, may influence clinical outcomes of CDI (12). Most notably, a landmark study in 2000 reported that a low IgG titer to TcdA, but not TcdB, at the time of infection is associated with development of symptomatic disease (13). More recently, the same group demonstrated an association between median IgG titers to TcdA and 30-day all-cause mortality (14). Several r...

Protective antibodies against Clostridium difficile are present in intravenous immunoglobulin and are retained in humans following its administration

“…Binding of antibodies within IVIg preparations and patient sera to specific CD antigens were determined by using a previously validated CD protein microarray . In brief, seven CD antigens, two positive controls: tetanus toxoid and lysates from Candida albicans , a negative control (printing buffer) and 10‐point twofold serial dilutions of human Ig (matching the tested antibody isotype) were spotted onto aminosilane slides (Schott, Mainz, Germany) in quadruplicate using a MicroGridII arrayer (Digilab, Marlborough, MA, USA) and a silicon contact pin (Parallel Synthesis Technologies, Santa Clara, CA, USA).…”

High prevalence of subclass-specific binding and neutralizing antibodies against <em>Clostridium difficile</em> toxins in adult cystic fibrosis sera: possible mode of immunoprotection against symptomatic <em>C. difficile</em> infection

“…Total specific IgA and IgG binding antibodies against the toxins were determined using a previously validated C. difficile protein microarray assay. 14 Prior to study commencement, we optimized both the dilution of the serum samples and concentration of printed antigens. The purity of all antigens tested in the current study was also evaluated using a silver stain (data not shown).…”

A Multi-Factorial Observational Study on Sequential Fecal Microbiota Transplant in Patients with Medically Refractory Clostridioides difficile Infection

“…We have previously established and validated a multiplex protein microarray system to measure antibodies to specific C. difficile antigens in human sera [32][33][34]. Briefly and unless otherwise stated, all target antigens and protein homogenates used in this procedure were diluted to 100 µg/mL in sterile print buffer (1× PBS containing 50 mM trehalose, 0.01% Tween 20) prior to application to the antigen microarrays.…”