Hrp conjugated goat anti mouse igg

Twelve days post-HAT selection, individual colonies were handpicked and transferred to 96-well plates containing medium E (Stem cell technologies). Four days later hybridoma supernatants were transferred to 96-well plates and fresh medium E was added to the cells. To test the specificity of hybridoma supernatants for MHCII and p:MHCII, a decoy screening approach was employed. ELISA plates were coated with 50 ng well⁻¹ either p63:IA^g7 or InsB_10–23:IA^g7 (for p63:IA^g7 antibodies), or 2W:IA^b or LLO:IA^b, and then blocked with 1% BSA in PBS for 1 h. Hybridoma supernatants were mixed 1:1 with ELISA wash buffer (PBS+0.05% Tween20) and added to the p:MHCII-coated plates and incubated at 37 °C for 2 h. Media alone was used as a negative control while anti-IA^g7 (clone 10–2.16 (ref. 28 (link)), BioXcell) or anti-IA^b (Y3P29 (link), ATCC) were used as a positive control. For antibody detection the wells were incubated with HRP-conjugated goat anti-mouse IgG (BioLegend) diluted to 1:2,000 at room temperature for 2 h followed by addition of ABTS substrate solution (KPL) and detection by absorbance at 405 nm. Antibodies reacting to both p:MHCII monomers were considered specific for MHCII independent of peptide, while antibodies reacting with only p63:IA^g7 or 2W:IA^b were considered p:MHCII specific.

Aβ40 or Aβ42 (Anaspec, USA) was coated on 96-well microplates overnight at 4 ℃, then blocked with blocking buffer (2% BSA + 5% goat serum in PBS) for 2 h at room temperature. The serum samples diluted into 1:1000 were added to the plates and incubated 2 h at room temperature. After washing, HRP-conjugated goat anti-mouse IgG (Biolegend) was added to the plates and incubated for 1 h. The reaction was developed by TMB substrate (Thermo Scientific, USA) and stopped with 0.1 N HCl. The microplate was read at 450 nm under a microplate reader (Bio-Tek, ELX800, USA). The antibody concentrations were calculated using a standard curve generated with known concentrations of anti-Aβ antibody.

Total and antigen-specific antibody levels in human and mouse sera were measured by ELISA. Sera were serially diluted in PBS containing 1% BSA and added to 96-well high-binding plates coated with recombinant proteins (10 μg/ml). Bound antibodies were detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or donkey anti-human IgG (BioLegend). Quantitative antibody measurements were performed using commercially purchased human and mouse IgG standards (ThermoFisher Scientific)

Ninety-six-well plates were coated with 1 μg/ml SARS-CoV-2 S1 protein (Sino Biological Inc.) or S1 RBD protein (Sino Biological Inc.) in PBS and incubated at 4°C overnight. After coating, plates were treated with 2% bovine serum albumin (BSA) (Sigma)/PBS for 2-5 h at 4°C. After blocking, serially diluted heat-inactivated sera or nasal washes (1%BSA/PBS) from the vaccinated or control mice were added to wells, and the plates were incubated for 1.5 h at RT, followed by washing 6 times with PBST and subsequent 1h incubation with an HRP-conjugated goat anti-mouse IgG (1:3,000 dilution; BioLegend) or goat anti-mouse IgA antibody (1:5,000 dilution; SouthernBiotech) in 1% BSA/PBS at RT. The wells were washed 6 times with PBST, and 100 μL of the TMB substrate set (BioLegend) was added to the immune complexes in each well. Plate development was then halted by the addition of 50 μL of 2N H₂SO₄ per well. The absorbance at 450 nm was recorded using a microplate reader. The ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >0.2. (Log₁₀ endpoint titers were given).

Protein samples were separated in polyacrylamide gels (7 to 12%, depending on the target protein) and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Western blotting (WB) was performed as described previously [56 (link)]. Primary antibodies used were anti-CD6 mAb (MEM98, EXBIO), anti-RasGAP mAb (B4F8, Santa Cruz Biotechnology), anti-p21 Ras mAb (Cytoskeleton, Inc), anti-α-tubulin mAb (B-5-1-2, Merck), anti-ERK 2 rabbit polyclonal (C14, Santa Cruz Biotechnology), and Phospho-p44/42 MAPK rabbit polyclonal (Cell Signaling Technology); secondary antibodies used were HRP-conjugated goat anti-mouse IgG (BioLegend), rat anti-mouse IgG (Abcam) for detection of CD6 in MEM98 immunoprecipitates, and mouse anti-rabbit IgG (Santa Cruz Biotechnology). Membranes were developed with Amersham ECL Select/Prime Western Blotting Detection Reagent (Cytiva). Data were acquired using the ChemiDoc XRS+ System (Bio-Rad) and analyzed by Fiji [57 (link)].