Variability in specific antibody content between the different IVIg preparations examined in this study and that reported in an earlier study by Salcedoet al.14may be due to the different geographical regions from which the plasma samples were collected and/or differences in CD exposure. microarray and toxin neutralization assays, all IVIg preparations were analysed for specific B-Raf IN 1 binding and neutralizing antibodies (NAb) to CD antigensin vitroand the presence of antitoxin NAbsin vivofollowing IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multiisotype specific B-Raf IN 1 antibodies to a 7plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of antiCD antibodies and/or antitoxin neutralizing capacities prior to fractionation. Keywords:antibodies,Clostridium difficile, intravenous immunoglobulin == Introduction == Clostridium difficile(CD) is the leading cause of hospitalacquired infective diarrhoea, and is a global health problem1. The most prominent risk factor for disease development includes antibiotic use, which disrupts the gut microbiota, leading to loss of colonization resistance and subsequentC. difficileinfection (CDI). Other major risk factors are prolonged hospital stay, increasing age and underlying comorbidities1. CD exerts its major pathological effects through two proinflammatory and cytotoxic protein exotoxins, A and B. Some strains also produce a third protein toxin, known as binary toxin or CDT. Nontoxin virulence factors VPS15 such as surface layer proteins (SLPs) also appear to be involved in pathogenesis2,3,4,5. Several clinical studies have shown previously that antibodymediated immune responses to CD toxins A and B have an important role in asymptomatic carriage and predisposition to recurrent infection. Specifically, symptomless carriers of toxigenic CD and those who have had a single episode of CDI show more robust antitoxin immune responses than those with symptomatic and recurrent disease6,7,8,9,10. Circulating toxin A and Bspecific memory B cells have been detected after the development of CDI, strengthening the evidence for the importance of humoral immune responses against both toxins11. Early population prevalence studies also indicate that the majority of healthy adults have detectable antibodies to CD toxins A and B in their sera that are thought to arise from colonization in infancy or from repeated environmental exposure to CD in adulthood12,13. For this reason, polyclonal IVIg has been used offlabel to treat both recurrent and fulminant CDI. Human intravenous immunoglobulin (IVIg) consists of purified plasma immunoglobulins from hundreds to thousands of healthy blood donors. Although several encouraging case reports highlight the potential benefits of IVIg, its definitive clinical role is still unclear, due mainly to the lack of robust evidence from randomized controlled B-Raf IN 1 trials14,15,16,17. Contradictory results obtained in respect to its clinical efficacy may be ascribed, in part, to the poor characterization of commercial IVIg preparations in terms of their specific antibody content. The mode of action of IVIg remains poorly understood. While some attention has focused on the varying capacity of IVIg to treat recurrent CDI, presumably by neutralizing CD toxins A and B14, the full repertoire of CDassociated protein targets of these complex preparations remains illdefined, as do the subclass distribution of these specific antibodies. Furthermore, the exact prevalence, kinetics and individual variation of binding and neutralizing antibodies (NAb) against CD proteins in serum samples, including those exposed to IVIg, are poorly described. Microarray assays are a promising new tool for compositional bioanalysis of specific antibody content in patient sera and IVIg due to their high sensitivity, reproducibility and ease of use. The aims B-Raf IN 1 of this study were to investigate retrospectively the efficacy of three different commercial preparations of IVIg used in our institution for treating severe or recurrent CDI and to determine if these preparations possess specific binding and neutralizing antibodies to CD antigensin vitro. In a second cohort of patients receiving IVIg for multiple indications, we also aimed to demonstrate the presence of protective.