Igg2a

The titers of IgG antibodies were measured in the serum sample from each group of mice by an enzyme-linked immunosorbent assay (ELISA). Briefly, the recombinant GP and VP40 proteins were individually coated as the antigens at a concentration of 1 μg/ml in 96-well polystyrene microtiter plates for overnight at 4°C. After blocking with 5% milk in PBS buffer, the ELISA plates were incubated with serial dilutions of serum samples at 37°C for 2 h. The titer of total IgG was detected with horseradish peroxidase (HRP)-conjugated goat against mouse IgG (1:2000 dilution, Santa Cruz Biotechnology). IgG subtype antibodies including IgG1, IgG2a, and IgG2b (1:2000 dilution, Santa Cruz Biotechnology) were further detected individually. The immune complex was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma). The reaction was stopped with 2 mol/L H₂SO₄, and the plates were read with the absorbance at 450 nm in a microplate reader (Bio-Rad). All samples were detected in triplicate wells.

IHC was performed on all suspected toxoplasmosis cases: cases with intralesional zoites seen by HE stain, or cases with histopathological lesions compatible with T. gondii infection as previously described [2 (link),6 (link)]. FFPE tissue sections were deparaffinized, hydrated, and subjected to antigen retrieval in a pressure cooker at pH 9.0 buffer (Envision). Endogenous peroxidase activity was blocked with 3.5% hydrogen peroxide and to block non-specific protein bindings, slides were incubated in 6% powdered skim milk. A mouse primary monoclonal antibody (IgG2a) targeting p30 membrane of T. gondii tachyzoite (clone sc-52255; Santa Cruz Biotechnology) was used at a dilution of 1:100. First, slides were incubated in the primary antibody, overnight, at 4°C and then, incubated with an indirect peroxidase polymeric detection kit (Envision) for 30 minutes at room temperature, followed by revelation with 3,3′-Diaminobenzidine (DAB) solution. Slides were counterstained with hematoxylin. Positive controls included sections of liver from a NWP with confirmed T. gondii infection [2 (link)]. Negative controls had the primary antibody replaced by wash buffer.

Serum FMDV-specific IgG and IgG isotypes were measured by an indirect ELISA mainly as described by Zhang et al. [13 (link)]. Additionally, 96-well microtiter plates were loaded with 50 µL of rabbit anti-FMDV antibody (Lanzhou Veterinary Research Institute, Lanzhou, China) diluted in 0.05 M carbonate/bicarbonate buffer (pH 9.6, 1:10) per well. Then, it was incubated at 4 °C overnight. After washing with phosphate buffer saline containing 0.05% Tween-20 (PBST), the wells were blocked with 5% skimmed milk and incubated for 2 h at 37 °C. Plates were then washed with PBST, and 50 µL of the FMDV O antigen was added (1:10) with an incubation for 2 h at 4 °C. After that, 50 µL of serum (diluted 1:200 in PBS) was added and incubated for 1 h at 37 °C. Then, 50 µL of goat anti-mouse IgG (1:1000) or IgG1 (1:2000) and IgG2a (1:2000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was added and incubated for 1 h at 37 °C. After washing, 50 µL of tetramethyl-benzidine (TMB) reagent was added to each well and incubated for 10 to 15 min at 37 °C. The reaction was terminated by adding 50 µL of 2 M H₂SO₄ Optical density (OD) at 450 nm, which was measured by a microplate reader (Thermo-Multiskan FC, Shanghai, China).