Hrp conjugated goat anti mouse igg antibody

Both the class and subclass of the mAbs against Ag85A produced were determined using a mouse monoclonal antibody isotyping kit (Sigma-Aldrich, USA). Protocols were carried out according to the manufacturer’s instructions.
ELISA plates (Nunc, Denmark) were coated with purified rHis-Ag85A protein (1 µg/ml) and left overnight at 4°C. The next morning, plates were washed three times with PBST and blocked with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) at 37°C for 2 h. Following the blocking step, the plates were washed three times with PBST. Plates were then incubated with serially diluted culture supernatant and ascites. Plates were incubated with primary antibody for 1 h at 37°C. HRP-conjugated goat anti-mouse IgG antibody (1:8,000 dilution) (Sigma-Aldrich, USA) was then added to each well (100 µl/well) and incubated for 1 h at 37°C. Finally, the TMB substrate was then added to the plates and incubated for 10 min, after which the plates were read at 450 nm.

Plates were coated with 0.4 µg/well of H. parasuis lysates diluted in carbonate-bicarbonate buffer (pH 9.6) at 4°C overnight. The coated plates were blocked with 5% (w/v) skim milk in PBST (1× PBS with 0.05% Tween 20) and then incubated with the supernatant of hybridoma culture and HRP-conjugated goat anti-mouse IgG antibody (Sigma, USA) for 1 h at 37°C. TMB substrate (Tian Gen, China) was added for colorimetric detection. The results were analyzed using a spectrophotometer at an absorbance of 450 nm.

IPMA was performed as previously described in [25 (link)]. Briefly, Vero cells cultured in 24-well plates were infected with the isolated viruses. Eight hours post-inoculation, the cells were washed with PBS, fixed with ice-cold methanol for 20 min at −20 °C, and probed with a monoclonal antibody (mAb) against CEV-JL14-VP1 (1:500) for 1 h at 37 °C. After washing, the cells were further incubated with HRP-conjugated goat anti-mouse IgG antibody (1:1000, Sigma, St. Louis, MO, USA) for 45 min at 37 °C. The plates were then stained with 3-amino-9-ethylcarbazole (Amresco, Olympia, WA, USA) substrate and visualized using the Canon digital camera (Canon, Tokyo, Japan). Cells infected with the CEV-JL14 virus were used as positive controls. Non-infected cells were used as negative controls.

ELISA plates (Nunc, Denmark) were first treated with 5% glutaraldehyde (100 µl/well) and incubated at 37°C for 2 h. Plates were then washed three times with PBST. Following the washes, plates were coated with 1 × 10⁶ colony-forming units of BCG or rBCG:Ag85A bacteria (100 µl/well) and left overnight at 56°C. The plates were washed with PBST and blocked with PBS containing 2% BSA at 37°C for 1 h. Then, the plates were washed and incubated with the mAbs for 2 h at 37°C. HRP-conjugated goat anti-mouse IgG antibody (1:8,000 dilution) (Sigma-Aldrich, USA) was added to each well (100 µl/well) and incubated for 1 h at 37°C. Finally, the TMB substrate was then added to the plates and incubated for 10 min, after which the plates were read at 450 nm.

ELISA plates (Nunc, Denmark) were coated with MPT63, RpfE, CFP10-ESAT6, and Ag85A protein (1 µg/ml), respectively, and left overnight at 4°C. The next morning, plates were washed three times with PBST and blocked with PBS containing 2% BSA at 37°C for 2 h. Following the blocking step, the plates were washed three times with PBST. Plates were then incubated with the anti-Ag85A mAbs (1:2,000 dilution). Plates were incubated with primary antibody for 1 h at 37°C. HRP-conjugated goat anti-mouse IgG antibody (1:8,000 dilution) (Sigma-Aldrich, USA) was then added to each well (100 μl/well) and incubated for 1 h at 37°C. Finally, the TMB substrate was then added to the plates and incubated for 10 min, after which the plates were read at 450 nm.

Fractionated proteins in the gel were transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with 3% BSA in Tween 20-Tris-buffered saline (TTBS) (0.1% Tween-20, Tris-buffered saline of 20 mM Tris-HCl, 150 mM NaCl at pH 7.5) with 0.1% sodium azide for 1 hour at room temperature. After discarding the blocking buffer, the membrane was incubated with 500 times diluted monoclonal anti MSTN antibody or 200 times diluted egg yolk IgY (2.1 mg/mL) in TTBS overnight at 4°C. The monoclonal anti-MSTN antibody was from a previous study (Kim et al, 2006). The membrane was then rinsed three times, with TTBS, followed by incubation with diluted (1:10,000) HRP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich, MO, USA) or diluted (1:1,000) HRP-conjugated goat anti-chicken IgY (Abcam, MA, USA) in TTBS for 1 h at room temperature. The membrane was then rinsed three times with TTBS, and HRP activity was detected using SuperSignal^™ West Femto kit (Thermo Fisher Scientific, MA, USA) and images were captured by Invitrogen iBright^™ CL1500 Imaging System (Thermo Fischer Scientific, MA, USA).