, 2010). Crotty et al., for instance, developed a 6 day stimulation protocol using a combination of PWM + CpG + SAC; many labs have subsequently
adopted that protocol. In 2009, Pinna et al. evaluated methods for the activation of memory B cells and found that a 6 day protocol using the TLR7/TLR8 agonist R848 plus IL-2 was more efficient compared to PWM, CpG and SAC ( Pinna et al., 2009). Another aspect of the B-cell ELISpot protocol is the kinetics of the memory B-cell activation. Generally, cells are pre-stimulated for 5–6 days prior to being added to the ELISpot plate for the quantification of ASC ( Crotty et al., 2004, Buisman et al., 2009, Cao et al., 2010 and Weiss et al., 2012). The duration of the stimulation is likely to depend on the Buparlisib solubility dmso potency of the activation. Also, the differences between using PBMC and purified B cells can have an impact on the activating potency. ( Buisman et al., 2009). In the present study, a new IgG-specific B-cell ELISpot protocol utilizing R848 + IL-2 for the activation of memory B cells and new monoclonal antibodies directed towards IgG for detection was developed. The new protocol,
using a 72-hour pre-activation schedule, induced total IgG memory B-cell activation more efficiently than other various activator protocols including PWM + CpG + SAC. In comparison with an already established protocol utilizing CpG + IL-2 + IL-10 + antigen for a 5-day pre-activation, the new protocol yielded an increased detection sensitivity mafosfamide of antigen-specific memory B cells. The new protocol was TGF beta inhibitor subsequently used to assess vaccine-induced, antigen-specific antibody responses against
five different antigens (diphtheria toxin [DT], tetanus toxoid [TTd], pertussis toxin [PT], filamentous hemagglutinin [FHA] and pertactin [PRN]), and it successfully detected specific memory B-cell and plasma blast responses to all included antigens. However, due to the limited number of subjects included in this study no direct comparison with other vaccine studies was made. Additional optimization of the new protocol included the use of biotinylated anti-IgG or antigen for detection which further reduced the amount of antigen required for the analysis. For the initial work on optimizing the B-cell ELISpot protocol and the cross-comparison of protocols, cells were obtained from anonymous buffy coats from healthy blood donors (The Karolinska University Hospital, Solna, Sweden). For the assessment of vaccine-induced responses, blood was obtained from two different cohorts at different time points after vaccination; informed consent was given by all subjects. Cohort 1 consisted of four adults who were recruited for the evaluation of the new protocol’s functionality. These subjects were given one dose of the combined tetanus diphtheria and pertussis vaccine (COVAXIS® Td5ap, Sanofi Pasteur, North York, ON, Canada), containing ≥ 20 IU TTd, ≥ 2 IU DT, 2.5 μg PT, 5 μg FHA, 3 μg PRN and 3 μg fimbrial agglutinogens 2 + 3.
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