Goat anti mouse igg2a hrp

Lipofectamine RNAimax (#13778150) and Lipofectamine 2000 (#11668019) were from Thermofisher Scientific. Optimem was purchased from Gibco (#31985-070). Cell-Titer Glo was from Promega (#G7570). Western blot antibodies were from the following sources: anti-EIF5A (ABclonal #A2016); anti-PES1 (#PA564020) and anti-POLR2E (#MA525454) were purchased from Thermo Fisher Scientific; anti-β-actin antibody (#sc-47778) was purchased from Santa Cruz. The secondary antibodies goat anti-rabbit IgG-HRP (#4030-05) and goat anti-mouse IgG2a-HRP (#1081-05) were from Southern Biotech.

ELISA plates (MaxiSorp for IgG and Immulon 4HBX for IgE) were coated with 5 μg/mL recombinant protein or 40 μg/mL crude JRC extract overnight and then blocked with 2% BSA in PBS. Serum samples were diluted 1 : 100- or 1 : 1000-fold in 1% BSA in PBS and then a 1 : 3 serial dilution was made. To detect IgE, sera were treated with Agarose-Protein G (Thermo Fisher Scientific, Rockford, IL) for 50 minutes and then 1 : 20 diluted samples were loaded to ELISA plates. Samples were detected with goat anti-mouse IgG1-HRP, goat anti-mouse IgG2a-HRP (Southern Biotech, Birmingham, AL), or rat anti-mouse-IgE-biotin (R35-118, BD Pharmingen, San Jose, CA) followed by Pierce Streptavidin-HRP (Thermo Fisher Scientific, Rockford, IL). Reaction was developed with SureBlue TMB Substrate (KPL, Gaithersburg, MD) and stopped with TMB Stop Solution (KPL, Gaithersburg, MD). Plates were read (OD₄₅₀) by using Epoch ELISA reader (BioTek, Winooski, VT). Average background (PBS only) was calculated, and samples which have OD₄₅₀ value more than 2 ∗ average background are considered as positive. The dilutions of such samples are determined as the endpoint titers.

Five micrograms of GST-tagged HPV16-E6E7 protein suspended in PBS buffer was coated on the Corning ELISA plates at 4 °C overnight. Mouse plasma was obtained at 21 days after immunization. The serum was separated and incubated with the plates after blocking with 1 × ELISAPOT Diluent (Invitrogen, cat. 00–4202-56) for 2 h at room temperature (RT) with a 1:100 dilution overnight at 4 °C. After five times washings, the goat anti-mouse IgG2a-HRP (Southern Biotech, cat. 1081–05) were added into the plates with a 1:5000 dilution for 1 h at RT. Finally, after washing the plates, TMB 1-Component Peroxidase Substrate (Invitrogen, cat. 00–4201-56) was used to indicate the reaction which was stopped using a 2 M HCl solution. The absorbance at 450 nm was determined within 30 min using a Synergy HTX instrument (BioTek Instruments, Highland Park, VT).