Anti mouse igg1

Serum samples of immunized mice and naïve mice were gathered for cytokines and antibody levels detection 30 or 75‐days post‐infection. The effect of vaccine‐induced cytokine production was measured using mouse IFN‐γ, TNF‐α, IL‐12p70 and IL‐10 ELISA kits (BioLegend, San Diego, CA, USA) according to the manufacturer’s protocols. The levels of T. gondii specific total immunoglobulin G (IgG) and subclasses of IgG antibodies IgG2a and IgG1 as indicators of type 1T‐helper (Th1) and type 2T‐helper (Th2) responses, respectively, were analysed using ELISA assay as described previously (Wang et al., 2017a; Huang et al., 2019). A total of 100 μl soluble tachyzoite antigens (5 μg ml⁻¹) were coated in a 96‐well plate and incubated at 4°C overnight. Then, the plate was washed 3 times and blocked with 1% BSA at 37°C for 1 h. After washing 3 times, 100 μl serum samples were added to appropriate wells at 37°C for 45 min. Subsequently, 100 μl of HRP conjugated goat anti‐mouse IgG secondary antibodies (1:5000 dilutions, Beyotime Biotechnology, Beijing, China), anti‐mouse IgG1(1:10 000, Abcam, Cambridge, UK) and anti‐mouse IgG2a (1:10 000, Abcam) were added to each well and incubated at 37°C for 30 min. Finally, tetramethylbenzidine as substrate was incubated to visualize the immunoreaction, and the OD value was determined at 630 nm using a microplate reader.

To quantify the amount
of the RBD in solution, we developed a standard curve dot blot using
His-tagged sfGFP and the RBD. sfGFP-conjugated KLH or PGN microparticles
(of a known sfGFP concentration, as determined with a standard curve)
were dotted in duplicate on a nitrocellulose membrane (1.8 μL
dots, Thermo Fisher) in twofold dilutions. Unknown concentrations
of RBD-conjugated KLH or PGN microparticles were also dotted in duplicate.
In a final lane, unmodified PGN microparticles were dotted as a control.
An anti-his dot blot was conducted as follows. The blot was dried
for 15 min in a fume hood. Following drying, 10 mL of a 1× PBST
+ 5% blotting grade blocker (Bio-Rad) was added for 10 min. Two microliters
of the mouse anti-hexa His antibody (BioLegend) was added to the 10
mL sample and incubated for 1 h at room temperature. Blots were washed
16 times with 9 mL of PBST. Ten milliliters of the 1× PBST +
5% blotting grade blocker with 2 μL of anti-mouse IgG1 (Abcam)
was added and incubated for 1 h at room temperature. Blots were washed
16 times with 9 mL of PBST, developed using the Pierce ECL Western
blotting substrate and imaged using a GE Amersham imager 600. Dots
were quantified using the gel analysis protocol in Fiji (Version 1.0,
ImageJ), and curves were fitted, and unknown concentrations were evaluated
using a linear regression in GraphPad Prism 8.4.1.