Horseradish peroxidase hrp conjugated goat anti mouse igg

The viral titer was determined according to the median tissue culture infectious dose (TCID₅₀) method and calculated by the Karber method as reported previously (23 (link)). BHK-21 or DEF cells were infected with rCQW1 or rMM 1775 at 300 TCID₅₀. After 24 or 48 h, the supernatant was removed, and the cells were harvested and analyzed by Western blotting. A mouse anti-DTMUV-NS3 polyclonal antibody (self-prepared) and a mouse anti-β-actin antibody (Ruiying Biological, China) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam, Shanghai, China) was used as the secondary antibody. The proteins were visualized using Clarity Western ECL Substrate (Bio-Rad, USA).

The head kidney was manually homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO, USA) and lysed on ice for 20 min. After centrifugation at 4 °C at 12000 rpm for 10 min, the supernatant was collected, and the protein concentration was quantified with the Enhanced BCA protein assay kit (Beyotime, Shanghai, China). Blood was drawn from the caudal vein of tongue sole, and serum was prepared as described previously [43 (link)]. Head kidney leukocyte protein (50 μg) and serum protein (100 μg) were mixed with SDS-PAGE loading buffer, boiled at 100 °C for 10 min and then separated by SDS-PAGE. The protein bands were transferred from gel onto a polyvinylidene fluoride (PVDF) membrane, and the PVDF membrane was soaked in blocking buffer (5% BSA and 0.05% Tween 20 of PBS, pH 7.2) at room temperature for 1 h. The membrane was then incubated with antibody against rCsCD209 (1:1000 dilution) at room temperature for 1 h followed by extensive washing. The membrane was further incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Abcam, Cambridge, Cambridgeshire, UK, 1:5000 dilution) at room temperature for 1 h. After extensive washing, the immune-reactive protein bands were visualized by using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA).

Bacillus subtilis 168 strain (BS168) was purchased from Wuhan Pujian Biotechnology Co., Ltd. PDG1661 plasmid was purchased from BioVector Plasmid Vector Strain Cell Gene Collection Center. HuNPB3 street strain, BHK cells, and NA cells were stored in our laboratory. Reference serum from the rabies reference Laboratory of the World Organization for Animal Health. A fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (mAb) against RABV N protein was purchased from Fujirebio, an anti-RABV G protein mAb was purchased from Millipore, Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Abcam. HRP-conjugated goat anti-mouse IgG1, IgG2a, and IgA were purchased from Southern Biotech Company. IFN-γ and IL-4 ELISpot plates were purchased from MABTECH Company. Chemiluminescent Imaging System were purchased from Tanon Company.

The plasmid pPG-LFCA-E was extracted using a plasmid extraction kit (TIANGEN, Beijing, China). The sequences of PCR primers were PCR forward 5′-CACACTTCTGAAAAAAGGAGGGGAGACCACAACGGTTTCCCACTAGAAATAATTTT-3′ and PCR reverse 5′-CCGCCAAAACAGCCAGATCTGTTATCACTTGTACAGCTCGTCCATGCCGAGAGTG-3′. Protein samples of LR-LFCA, MC LR-LFCA, and LR-CON were prepared as follows: The cells were collected by centrifugation, treated with lysozyme (10 mg/mL), and disrupted by sonication (sonication parameters: 400 W; 20 min; 4  s on, 6 s off). Homogenate was supplemented with sodium dodecyl sulfate (SDS) solution (1%, PBS with), and the samples were boiled for 10 min. The protein samples were analyzed by Tricine-SDS-polyacrylamide gel electrophoresis (15%, w/v). Western blot assay was performed using Myc-tag antibody (1:1000) as the primary antibody (Affinity Biosciences, USA). A horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000) (Abcam, Cambridge, UK) was used as the secondary antibody.

Approximately equal amounts of each recombinant protein were subjected to 12% SDS-PAGE. The gel was stained with Coomassie blue or transferred to polyvinylidene difluoride membranes (Milipore, San Diego, CA, USA). The membranes were blocked in 5% skim milk for 2 h at room temperature (RT) followed by incubation with anti-histidine monoclonal antibody (mAb) (1:5000 dilution, Abcam, Cambridge, UK) or anti-ASFV swine serum (1:300 dilution, China Institute of Veterinary Drug Control, Beijing, China) overnight at 4 °C. After washing five times with 0.05% Tween-20 in PBS (PBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000 dilution, Abcam) or HRP-conjugated goat anti-pig IgG (1:5000 dilution, Abcam) for 1 h at RT. After washing with PBST, the blots were developed with enhanced chemiluminescence reagent (Thermo Fisher Scientific).

Samples were mixed with 4× loading buffer and heated at 100°C for 10 min. Proteins in each sample were separated by 12% SDS-PAGE. After the gel was stained with Coomassie blue staining solution for 10 min, the bands were decolorized with decolorization solution and analyzed. For western blot analysis, protein samples were electro-transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, United States). After blocking with 5% skim milk powder at 37°C for 2 h, hybridoma cell supernatant dilution (1:10 dilution) was added and incubated overnight at 4°C. After washing three times with PBS-Tween (PBST), a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Abcam, Cambridge, United Kingdom) was added and incubated at 37°C for 1 h. After washing three times with PBST, enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Waltham, MA, United States) was added. Blots were scanned and analyzed using the Odyssey CLx imaging system (LI-COR, Lincoln, NE, United States).

The S1-specific antibody titer was measured by a standard indirect ELISA (iELISA) method. Briefly, 96-well ELISA plates (Nunc, Thermo) were coated overnight with 1 ng per well of the S1 protein in a coating buffer. The plates were then blocked with 200 μl of ELISA assay buffer (Thermo) and incubated at room temperature for 2 h. Each serum sample was tested in duplicate at a dilution from 1:100 to 1:640,000 twice in ELISA assay buffer (Thermo), in which 100 µl was then added into the wells of each plate for 2 h incubation at room temperature. The horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000, Abcam) was added for 1 h incubation at room temperature. Then, 100 µl TMB solution (Thermo) was added to each well. After 15 min incubation, 100 μl 2 M H₂SO₄ solution was added to stop the reaction. The absorbance of each well was read by the 450 nm wavelength (PerkinElmer). The threshold was defied as the double of the average absorbance in the untreated group. The last dilution of a sample that is just larger than the threshold is defined as the antibody titer of this sample.