Hrp conjugated goat anti mouse igg

FRNT was carried out in 48 multi-well plates, on Vero cells seeded at a density of 6.5x10⁴ cells/well 24h before infection. Sera samples were 2-fold serially diluted and incubated for 1.5h at 36^°C with an equal volume of DMEM serum-free medium containing 50 foci-forming units (FFU) of each virus. Vero cells were then infected in duplicate for 1h at 36°C, the inoculum was then removed and cells overlaid with 0.5 ml of DMEM containing 2% FCS and 3% carboxymethylcellulose (Sigma). After 3 days incubation at 36°C, cell monolayers were washed and fixed for 20min with PFA 3.7%, permeabilised with a 1% Triton solution in PBS for 10 min and treated with 0.3% H₂O₂ in methanol for 30 min. Infection foci were developed incubating with mAb 4G2 (1 ng/μl) for 1h at room temperature (RT), followed by incubation with HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, 1:1000). Foci were then stained with 100 μl True-Blue reagent (KPL, Gaithersburg, MA, USA) and counted. Neutralising antibody titres were expressed as the serum dilution yielding a 50% foci reduction when compared against pre-immune sera (FRNT₅₀).

Transfected COS-1 cells were harvested at 48 h post-transfection, and a fraction was used to determine the ratio of GFP-expressing cells via FACSCalibur™ flow cytometer (BD Japan, Tokyo, Japan). The remainder were lysed, and lysates with equal numbers of GFP-expressing cells were subjected to western blotting analysis as described previously [29 (link)]. Collected virus particles were also subjected to western blotting analysis. Subsequently, proteins were transferred to a polyvinylidene difluoride membrane filter (Immobilon; Merck Millipore, Burlington, MA, USA) and incubated with sera from BLV-infected cattle and BLV-uninfected cattle, anti-BLV gp51 monoclonal antibody (MAb) (BLV-2; VMRD, Pullman, WA, USA), anti-BLV p24 MAb (BLV-3; VMRD), or anti GFP MAb (1E4; MBL, Nagoya, Japan). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-bovine IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.). Densities of bands were analyzed using AlphaEaseFC^TM software (Alpha Innotech, San Leandro, CA, USA) and ImageJ software (National Institutes of Health, Bethesda, MD, USA).

ELISA plates were incubated overnight at room temperature with 5 μg/mL native H. pylori lysates. To detect specific IgG, antisera were collected and diluted 1:800 in PBS. To detect secretory IgA (sIgA), the supernatants from the homogenized stomach tissue or intestinal tissue were collected and diluted 1:5 in PBS. A HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) or a HRP-conjugated goat anti-mouse IgA (Jackson ImmunoResearch) was used as the secondary antibody.