Anti murine igg2a

Serum samples were analyzed by ELISA according to the general protocol described in section 2.4. Recombinant gp120IIIB (1 µg/ml) was added to Nunc MaxiSorp 96 well plates (50 µl/well) and incubated overnight at room temperature. Serum samples were diluted in ELISA-buffer and added to the 96 well plates in dilution 1∶50 followed by 3-fold or 5-fold dilution. As secondary reagents, we used alkaline phosphatase-conjugated anti-murine IgG (Fc-specific, cat. no. A2429, Sigma) or biotinylated IgG subclass-specific antibodies (anti-murine IgG1 (clone 10.9), anti-murine IgG2a (clone 8.3), or anti-murine IgG2b (clone R12-3). Threshold for end point titers was set to 2x the absorbance value of the negative controls (sera from mice given NaCl).

ELISAs to evaluate the secretion of vaccibodies from transfected cells were performed using either an anti-mCherry antibody (clone 1 or clone 2 denoted a-mCherry.1 or a-mCherry.2) [32] (link) or anti-human CH3 (clone A57H) or NIP-BSA (NIP conjugated to BSA) (1 µg/ml) for capture. As detection antibodies, either biotinylated anti-mCherry (a-mCherry.1-bio or a-mCherry.2-bio) [32] (link) or biotinylated anti-human CH3 (a-hCH3-bio, clone HP6017) were used, followed by incubation with alkaline phosphatase-conjugated streptavidin and p-nitrophenyl phosphate substrate. For detection of anti-mCherry antibodies in sera of immunized mice, microtiter plates were coated with purified recombinant mCherry. Serum samples were serially diluted and incubated overnight, followed by incubation with alkaline phosphatase-conjugated anti-murine IgG (Fc-specific, cat. no. A2429, Sigma) or biotinylated IgG subclass-specific antibodies (anti-murine IgG1 (clone 10.9), anti-murine IgG2a (clone 8.3)). Threshold for end point titers was set to 2× the absorbance value of the negative controls (sera from mice given PBS).