Hrp conjugated goat anti mouse igg antibody

Ninety-six-well immunoplates were coated overnight with 100 µl of 100 µg/ml LPS from S. flexneri 2a strain 301 diluted in carbonate coating buffer (50 mM Na₂CO₃-NaHCO₃, pH 9.6) at 4°C. The wells were then washed with 200 µl of wash buffer (1× PBS with 0.05% Tween 20) three times, and the plates were patted dry. Then, 200 µl of ELISA blocking buffer (1× PBS with 5% milk) was added to each well, and the plates were incubated at 37°C for 2 h. After washing and drying, 100 µl of immune serum, diluted to different concentrations in dilution buffer (1× PBS with 0.5% milk), was added to each well and incubated for 1 h at 37°C. After another washing and drying step, 100 µl of HRP-conjugated goat anti-mouse IgG antibodies (Abcam) diluted 1:50,000 in dilution buffer was added to each well, and the plates were incubated for 1 h at 37°C. After each well was washed five times and dried, 100 µl of color solution (o-phenylenediamine [OPD]–H₂O₂ solution) was added, and the samples were left to react in the dark for 15 min at room temperature. The reaction was stopped with 50 µl of ELISA stop solution (2 mol/liter H₂SO₄). A microplate reader was used to measure the OD₄₉₀.

Alpha-linolenic acid (ALA), Human Beta Amyloid 1-42 (Aβ_1-42), All-trans retinoic acid (RA), Insulin, Human Insulin ELISA Kit, Human IGF-I ELISA Kit, 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), and acridine orange (AO) were purchased from Sigma Aldrich, Saint Louis, MO, USA. Insulin Degrading Enzyme (IDE) was purchased from Abcam Inc., USA. Antibodies to the following targets were used: rabbit anti-TOMM20 antibody (Abcam Inc.), mouse anti-PARKIN (Abcam Inc.), rabbit anti-Synaptophysin (GeneTex, Inc., Alton Pkwy Irvine, CA, USA), mouse anti-β3-Tubulin (TUJ 1; Santa Cruz Biotechnology, Dallas, CO, USA), horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG antibodies (Abcam Inc.), HRP-conjugated goat anti-mouse IgG antibodies (Abcam Inc.), Alexa Fluor™488-labeled chicken anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor™568-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific).

The wells of 96-well microplates were coated with WT VLPs, hybrid HPV L1 VLPs, HPV pentamers (100 ng per well) or monoclonal antibodies (200 ng per well). The wells were blocked with blocking solution and incubated with 100 μL of 2-fold serially diluted anti-HPV WT L1, hybrid L1 mAbs or antigen (pentamer, VLPs) with the start concentration of 1 μg/mL. The wells were washed and then incubated with HRP-conjugated goat anti-mouse IgG antibody (Abcam; Cambridge, UK; 1:5000 dilution) or the secondary mAb diluted 1:5000 in HS-PBS. Following this, the wells were incubated with 50 μL 3, 3′, 5, 5′-tetramethylbenzidine for 10 min at 37 °C. The reactions were quenched with 50 μL of 2 mol/L H₂SO₄, and the absorbance at 450 nm (620 nm reference) was recorded using an automated ELISA reader (TECAN, Männedorf, Switzerland). The cut-off value for positive titers was set to an absorbance of 0.1. The reactivities of various VLPs were represented by EC₅₀ values, which reflects the antibody concentration required to achieve 50% of the maximal signal.