Hrp conjugated goat anti mouse secondary antibody

Surviving rabbits were executed on day 10 after the challenge. Lung, liver, and spleen tissues were collected from rabbits that died of infection or were euthanized at necropsy and were evaluated histopathologically (Prieto et al., 2000 (link)). Tissues were fixed, dehydrated, paraffin-embedded, sectioned, and immunohistochemically examined using mouse monoclonal primary antibody 4D5 and horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (ZSGB-Bio, China).

Ninety-six ELISA plates were coated with 100 ng/well of purified pGSDMD protein diluted in 100 μL pH 9.5 carbonate buffer solution. The plates were incubated at 37 °C for 3 h. After washing five times with PBST (PBS containing 0.05% Tween-20), 100 μL/well of 5% skim milk diluted in PBST was added to block the wells for 2 h at 37 °C. After another washing step, 100 μL/well of hybridoma cell culture supernatants or diluted mAbs was added to the plates, along with positive control (serum of the hyperimmune mouse), negative control (serum of the non-immunized mouse), and blank control. The plates were incubated at 37 °C for 1 h. After washing five times with PBST, 100 μL/well of HRP-conjugated goat anti-mouse secondary antibody (ZSGB-BIO, China) at 1:5000 dilution was added to the plates and incubated at 37 °C for 30 min. After a final wash, 100 µL/well of tetramethylbenzidine (TMB) substrate (Solarbio, China) was added to the plates for a 10-min chromogenic reaction at room temperature in the dark. The chromogenic reaction was stopped by adding 50 µL/well of 5M H₂SO₄. The optical density at 450 nm (OD_450nm) was determined using a microplate reader (TECAN, Switzerland). The testing results were considered positive for anti-pGSDMD antibodies if the OD_450nm value was 2.1-fold higher than that of the negative control.