Antibody Response Evaluation for Malaria Vaccine

Mice sera were collected from all groups 4 weeks after prime, 1st boost immunization, and 2nd boost immunization using a retro-orbital plexus puncture. Sera from naïve mice were used as a negative control. Antibody responses against P. berghei antigen, AMA1 rVV, and MIC rVV were determined by enzyme-linked immunosorbent assay (ELISA), as described previously [17 (link)]. Briefly, 96-well immunoplates were coated with 100 μL of P. berghei antigen, AMA1 rVV, or MIC rVV at a final concentration of 2, 0.5, and 0.5 μg/mL, each respectively in 0.05 M, pH 9.6 carbonate bicarbonate buffer per well at 4 °C overnight. Afterwards, 100 μL of serially diluted serum samples were added into the respective wells and incubated at 37 °C for 1.5 h. HRP-conjugated goat anti-mouse IgG (100 μL/well, diluted 1:2000 in PBS) was used to determine the P. berghei-specific IgG antibody response. O-phenylenediamine was dissolved in citrate substrate buffer, and OD₄₉₂ was measured using an EZ Read 400 microplate reader (Biochrom Ltd., Cambridge, UK).