Eluates were used for ELISAs the next day. Serum Separator cards could be used with conventional spectrophotometric vibriocidal methods, and that they correlated with paired serum at a wide range of titers (r = 0.96, p<0.0001). Similarly, we used ELISA methods to show thatV.choleraeO-specific polysaccharide antibody responses from DBS eluates correlated with results from paired serum for IgG (r = 0.85, p = 0.00006), IgM (r = 0.79, p = 0.00049) and IgA (r = 0.73, p = 0.0019), highlighting its potential for use in determination of isotype-specific responses. Storage of DBS cards at a range of temperatures did not change antibody responses. == LDN-192960 Conclusion == In conclusion, we have developed and exhibited a proof-of-concept for assays utilizing DBS for assessing cholera-specific immune responses. == Author summary == Cholera remains a major public health issue among underprivileged populations in the developing world. Current methods of disease surveillance are inadequate for identifying key populations at highest risk of cholera. Serosurveillance can provide accurate measurements of an individual or populations exposure to cholera contamination or oral cholera vaccine (OCV) induced immunity, though they require venous blood draw and stringent processing needs. Dried blood spots (DBS) overcome these challenges, acting as a portable surveillance tool suitable for field use. We developed a drop-plate culture method for evaluating vibriocidal and cholera-specific isotype responses using DBS from OCV-immunized volunteers from South Sudan. Blood equivalent to only two drops were spotted on Whatman Protein Saver (WPS) DBS cards. Vibriocidal titers from WPS eluates determined by drop-plate culture methods correlated well with serum based assays. In addition, by using DBS cards capable of automatic separation of serum from blood, we demonstrate that vibriocidal titers andV.choleraepolysaccharide antibody responses could be measured by conventional spectrophotometric methods and that these responses are stable over a range of storage temperatures. In summary, we show that cholera-specific immune responses can be measured using DBS, providing a potential tool for large-scale serosurveillance field studies for cholera. == Introduction == Despite efforts to improve access to clean water and sanitation in resource-poor settings, cholera remains a significant public health threat globally. Identifying key populations at the highest risk of cholera is essential to prioritize public health interventions, including vaccination and efforts to improve water and sanitation. Furthermore, understanding the impact LDN-192960 of cholera control programs requires monitoring of evidence of cholera transmission. Current surveillance methods, largely based on health facility reports of acute watery diarrhea, with infrequent laboratory confirmation of the pathogen, are inadequate. Detection and quantification of immune responses in serum (serosurveillance) can provide a new avenue for rapid and accurate assessments of recent cholera exposure; and such an Mouse Monoclonal to Rabbit IgG approach could play a central role in estimating risk and transmission in a population [1]. However, serosurveys are limited by the need for venipuncture, refrigeration, and the processing of biohazardous samples, which may be difficult to achieve in the resource-poor settings where cholera occurs. Assays of antibody responses adapted to use dried blood spots (DBS) overcome the above challenges by 1) alleviating the need for venipuncture, 2) reducing the costs associated with blood collection, supplies, labor, transportation and storage, 3) being relatively stable at ambient temperatures for extended periods, and 4) reducing the amount of blood required. Although the first use of DBS dates back to 19thcentury, contemporary widespread use of DBS was pioneered by Robert Guthrie as a diagnostic tool for neonatal screening of phenylketonuria [2,3]. More recently, DBS have been used for screening of metabolic disorders, detection of pathogens, and drug testing, in various sample types including blood, saliva, breast milk and stool [46]. Several studies have exhibited the utility of DBS for serological and molecular detection of a number of infectious diseases, including tetanus [7], diphtheria [8], HIV [911], hepatitis A, B, LDN-192960 C and E [1215], dengue [16,17], polio [18] and measles [19]. The performance characteristics of DBS assessments have approached that of serum assays, and their low costs and convenience have resulted in widespread use and endorsement by the WHO [20]. Despite the extensive use of DBS in a wide range of settings, including its use for microbial detection ofVibrio choleraein stool [21], the use of DBS forV.cholerae-specific antibody responses has yet to be determined. Here we demonstrate the development and provide proof-of-concept of methods for the determination of cholera-specific immune responses, including vibriocidal titers, andV.choleraepolysaccharide-specific isotype antibodies.