Goat anti mouse igg hrp antibody

Serum samples collected from 5 C57BL/6 mice and 5 TACI -/- mice 71 days after P. yoelii infection were pooled. The avidity of the antibodies was evaluated using guanidine hydrochloride (GuHCl) as a dissociative agent (19 (link)). The ELISA plates were coated, blocked and serum titrations were prepared as described in ELISA method. Triplicate serum samples were added to each well. After 2 h of incubation and washing steps, “avidity samples” were incubated with 100 μl of 0.1 M GuHCl (Sigma, Darmstadt, Germany) while triplicate “control samples” were incubated in washing buffer. After incubation for 10 min and 5 washes, wells were exposed to goat anti-mouse IgG-HRP antibody (Southern Biotechnology Associates) for 1 h. Following washing steps, ABTS substrate was added and the plates were read on a VERSA max microplate reader (Molecular Devices). Antibody avidity was calculated using the method described by Perciani et al. (20 (link)). Optical densities from each titration were graphed using GraphPad Prism software (La Jolla, CA) and the area under the curve (AUC) was measured for both the GuHCl and control-treated samples for each serum pool. The formula (AUC of guanidine treated samples)/(AUC of control-treated samples) was used to calculate the avidity index ratio.

Serum samples were pooled or not from 3 C57BL/6 and 3 TACI -/- mice per time point at 8, 16, 22, 28, and 71 days post P. yoelii infection. Serum antibody levels against an extended version of the P. yoelii Merozoite Surface Protein 1(C-terminal 19-kDa fragment [rMSP-1₁₉]) (18 (link)) and whole P. yoelii 17XNL extract were measured by ELISA. ELISA plates were coated with 70 ng/well of rMSP-1₁₉ or sonicated P. yoelii infected RBCs in coating buffer (15 mM Na₂CO₃ and 35 mM NaHCO₃). After washing with PBS/0.05%Tween-20, plates were blocked with 5% milk/PBS. Next, 100 μl of 1:50 to 1:51200 titrated sera were added. After 2 h, plates were incubated with 1:3500 diluted goat anti-mouse IgG-HRP antibody (Southern Biotechnology Associates, Birmingham, AL). Plates were read on a VERSA max microplate reader after adding ABTS substrate (Molecular Devices, Sunnydale, CA).