e. a detection pAb directly conjugated to ALP. PBMC from five healthy donors were stimulated with R848 + IL-2 and the number of IgG-producing cells was enumerated using antibodies from the two different protocols. The mAb-based system detected higher numbers of IgG-producing

cells in all five subjects, compared to the pAb-based system (Fig. 3), thus adding another parameter explaining the better sensitivity of the new protocol. After optimizing the new protocol, its functionality for the detection of vaccine-induced antigen-specific B-cell responses was evaluated. PBMC samples from the four healthy adults in cohort 1 vaccinated against pertussis, tetanus and diphtheria were assessed for antigen-specific B-cell responses to PT, FHA, PRN, TTd MK-8776 clinical trial and DT. Individual changes over time, after vaccination, in the memory B-cell population were observed (Fig. 4). The subjects’ response to the different antigens varied which is expected as the

Afatinib in vitro subjects differed in age as well as with regard to previous vaccinations and natural infections. They also differed in their peak response time point and in the magnitude of the response. The response was maintained over the 3-month test period after vaccination but with decreasing levels over time. Unstimulated cells also yielded detectable ASC, albeit fewer than found in the pre-activated memory B cells. The unstimulated ASC most likely represent active plasma blasts in vivo-induced by the vaccination and were generally only observed one to two weeks after vaccination. Yet another aspect of improving the new B-cell ELISpot protocol by using biotinylated antigens for detection was investigated. In the regular setup of the protocol, the antigen was coated and the detection of ASC was achieved by a biotinylated detection mAb. In the alternative protocol, coating

was done with capture mAbs and detection was achieved with a biotinylated antigen. Pilot tests had shown that coating with antigen required a concentration of 10 μg/ml, while only 1 μg/ml or even less was needed for the alternative protocol (data not shown). Three of the vaccinated adults from cohort 1 assays were tested using both protocol variants for the measurement of activator-induced ASC specific for TTd and DT. The results showed no difference in spot detection next even though the biotinylated detection system uses a ten times less antigen (Fig. 5). The homeostasis of the memory B-cell population and its contribution to the maintenance of humoral memory is still enigmatic. Little is known about why some pathogens evoke life-long memory whereas others evoke protection lasting only a decade or less (Amanna et al., 2007 and Amanna and Slifka, 2010). It is known that circulating memory B cells are responsible for the rapid and protective antibody response seen after a re-encounter with a pathogen (Tangye and Tarlinton, 2009).