Hrp conjugated goat anti mouse igg antibody

Serum levels of IgA levels were measured through sandwich ELISA method as described previously (7 (link)). In brief, plates were coated overnight with 2.5 μg/ml goat F(ab’)₂ anti-mouse Ig (SouthernBiotech, 1012-01, 1:400) in sodium carbonate buffer (pH 9.6). After washing, the plates were blocked with 1% BSA for 2 hours. Then diluted serum samples and mouse IgA (SouthernBiotech, 0106-01, the first standard concentration is 100ng/ml), used as the standard, were added and incubated for 1 hour. Following another round of washing, the plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgA (SouthernBiotech, 1040-05, 1:5000, 2ug/ml) antibodies for 1 hour. To determine serum levels of IgA-IgG complexes, a cross-capture ELISA was performed. The 96-well plates were coated with 2.5 μg/ml of goat anti-mouse IgA (SouthernBiotech, 1040-01, 1:400) overnight. After washing and blocking, the diluted serum samples were added and allowed to bind for 1 hour. Then the HRP-conjugated goat anti-mouse IgG antibody (SouthernBiotech, 1030-05, 1:5000, 0.2ug/ml) was applied. All reactions were carried out at room temperature. Finally, the plates were developed using 3,3’,5,5’-tetramethylbenzidine (TMB) and the reactions were stopped with 1 M sulfuric acid. The results were analyzed using an ELISA reader (Bio-Rad 550) at 450 and 570 nm wavelengths.

ELISA plates (Nunc MaxiSorp 96-well) were coated with VP1uWT or VP1uAT peptides at 1 μg/mL in PBS at 0.1 mL per well overnight at 4 °C. Wells were blocked with SuperBlock (Pierce ThermoFisher Scientific, Waltham, MA, USA) for 1–2 h at 37 °C. The assay diluent was PBS containing 5% goat serum (GIBCO Invitrogen, Waltham, MA, USA) and 0.1% Tween 20. Serial dilutions were made with sera from mice immunized with VLPs. The monoclonal antibody MAB8293 was also used following serial dilution. Antibodies were incubated on plates for 2 h at 37 °C followed by addition of detection antibody (HRP conjugated goat anti-mouse IgG antibody, SouthernBiotech, Birmingham, AL, USA) for 1 h at 37 °C. After incubation with TMB substrate for 20 min at room temperature (RT) in the dark, 1 M phosphoric acid was added to the wells to stop the reactions. Washes between steps were with PBS, 0.1% Tween-20. Wells with no serum were included as blanks. Plates were read at 450 nm and fit to a 4-parameter curve (GraphPad Prism). The binding IgG titer was calculated as the serum dilution giving a net OD₄₅₀ of 0.5 after blank subtraction.

A monolayer of cells in 6-well plates were infected with WI/18 virus at the dilution that gives 10-20 plaques per well for each cell line. Cells were washed with PBS at 1 h post-infection. Cells were covered with an overlay consisting of MEM, 1.2% (w/w) colloidal microcrystalline cellulose, 2 mg mL⁻¹ L-Glutamine, 1 μg mL^-1 TPCK-trypsin and incubated for 3 days at 37 °C. After being fixed by 4% PFA and permeabilized with 0.1% Triton-X100, infected cells were stained with anti-NP antibody (1 μg/mL, H16-L10-4R5 (HB-65), BioXcell) and subsequently stained with a secondary HRP-conjugated goat anti-mouse IgG antibody (1:2000, Southern Biotech). Plaques were visualized with KPL TrueBlue (Seracare). Images were acquired with Keyence BZX700 Widefield fluorescence microscope and analyzed using image analysis software ilastik^{61 (link)} and Fiji^{62 (link)}.

Each well was coated with 100 μl of PT (5 μg/mL) in carbonate buffer (50 mM Na₂CO₃-NaHCO₃, pH 9.6) at 4°C overnight and blocked in 5% bovine serum albumin in PBS for 2 hours at 37°C. For the preparation of the loading samples, the PT sample (100 μL, Series of 10 4-fold dilutions) was first mixed with 100 μL of anti-PT standard serum and incubated for 2 hours at 37°C. The mixture was added to the ELISA plates and incubated for 2 hours at 37°C. The plates were washed three times with 1× PBS containing 0.05% Tween-20 (PBST), followed by the addition of 100 µL of HRP-conjugated goat anti-mouse IgG antibody (1:4000, Southern Biotech, USA). After incubation at 37°C for 2 hours, the plates were again washed five times with PBST. Then, the plates were developed with 3,3′,5,5′-tetramethytlbenzidine for 10 min at room temperature, and the reaction was stopped with 2 M H₂SO₄. The absorbance at 450 nm was measured by a microplate reader.

We coated 96-well ELISA plates (Thermo Fisher Scientific) overnight at 4°C with eight hemagglutination units (HAU) of virus in carbonate buffer. We used horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Southern Biotech) to detect binding of serum antibodies, followed by development with Super Aquablue ELISA substrate (eBiosciences). We measured absorbance at 405 nm on a microplate spectrophotometer (Bio-Rad). We analyzed serum samples starting at a top dilution of 1:20 (PBS controls and day 8 animals) or 1:1000 (all other samples), followed by 2-fold dilutions in 8 (PBS controls and day 8 animals) or 16 steps. We determined the end titer as the last dilution point with an OD value of > 2x the blank average OD value for each respective plate.

Antigen-specific antibody secreting cells present in the bone marrow were quantified using an ELISPOT assay. One day prior to assay initiation, Multiscreen ELISPOT plates (Millipore) were coated with 1μg of antigen/well, and incubated overnight. Blocked plates were washed three times with washing buffer (PBS + 0.5% Tween 20), blocked with collection medium for 2 h, and washed 3 times. Bone marrow was collected 21 days post-immunization in RPMI medium supplemented with 10% FBS, quantified using a Guava automated cell counter (Millipore) and resuspended to 1 × 10⁶ cells/mL. Cells were serially diluted 3-fold, added to plates, and incubated for 5 h at 37 °C. Secreted antibody was detected by addition of a 1:100 dilution of horse radish peroxidase (HRP) conjugated goat anti-mouse IgG antibody (Southern Biotech). Spots were visualized with AEC Peroxidase substrate kit (Vector Labs) according to manufacturer's instructions. Spots were quantitated on a CTL bioanalyzer.

WNV WN-80E specific antibody secreting cells present in the bone marrow were quantified using an ELISPOT assay. One day prior to assay initiation, Multiscreen ELISPOT plates (Millipore) were coated with 1ug of WN-80E/well, and incubated overnight. Blocked plates were washed three time with washing buffer (PBS + 0.5% Tween 20), blocked with collection medium for two hours, and washed 3 times. Bone marrow was collected 21 days post-immunization in RPMI medium supplemented with 10% fetal bovine serum (FBS), quantified using a Guava automated cell counter (Millipore) and resuspended to 1 X 10⁶ cells/mL. Cells were serially diluted 3-fold, added to plates, and incubated for 5 hours at 37°C. Secreted antibody was detected by addition of a 1:100 dilution of horse radish peroxidase (HRP) conjugated goat anti-mouse IgG antibody (Southern Biotech). Spots were visualized with an AEC Peroxidase substrate kit (Vector Labs) according to manufacturer’s instructions. Spots were quantitated on a CTL bioanalyzer.