Anti mouse igg2b

Nunc Maxisorp 96-well plates were coated with 10 μg/mL Ovalbumin (grade VI, SIGMA-ALDRICH), goat anti- mouse IgG (1 μg/mL) (Southern Biotech) and incubated overnight at 4°C. Plates were washed 3 times with 0.5% Tween-20 in PBS and blocked with 200 μl of 2% blotting-grade blocker (Biorad). Mouse serum samples were serially diluted in blocking buffer and incubated on blocked plates. Antigen-specific serum antibodies were detected using horseradish peroxidase (HRP)-conjugated antibodies (anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgE) (Southern Biotech) at 1:5000 dilution in blocking buffer. Incubation of serum samples or antibodies was conducted at room temperature. HRP activity was detected using 100 μL of tetramethylbenzidine (TMB) substrate (BD Biosciences) and stopped using 50 μL 2N H₂SO₄. Developed plates were recorded using BioRad spectrophotometer at 450 nm with correction at 595 nm by subtraction.

TNF-α and MCP-1 levels were measured using ELISA kits (R&D Systems, USA). The blood serum and cell supernatant were diluted and studied using a standard curve. All of the samples were measured in triplicate. The procedure was performed according to the manufacturer's instructions.
Anti-dsDNA antibody levels in the serum were determined by ELISA, as described previously (32 (link)). Briefly, 96-well microtiter plates (Costar) were pretreated with calf thymus dsDNA (Sigma-Aldrich) for 2 h at 37°C and then placed overnight at 4°C. After being washed with PBS containing 0.05% Tween-20 (PBST), the plates were blocked with 1% BSA for 1 h. After the plates were incubated with a 1:100 dilution of mouse serum, the levels of anti-dsDNA antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG2c, anti-mouse IgG3, anti-mouse IgM, anti-mouse IgA, and anti-mouse IgE (all from Southern Biotech). Tetramethylbenzidine (TMB) substrate was used for the development, and absorbance at 450 nm was measured on a Thermo Multiskan Spectrum 1500.

Micro-vinyl plates (96-well, Corning Costar, Corning, NY, USA) were coated with a standard curve of purified IgG1 (BD Pharminogen #554121, San Diego, CA, USA), IgG2b (eBioscience eBMG2b #14732-82, San Diego, CA, UAS), IgG2c (GeneTex #GTX35043, Irvine, CA, USA), or IgG3 (eBioscience #14-4742-82, San Diego, CA, USA) protein while remaining wells were coated with 0.5 μg/mL BaL gp120 recombinant protein (NIH AIDS reagent #4961, Germantown, MD, USA) in a sodium bicarbonate buffer and incubated overnight. Plates were washed with 0.05% PBST 3 times and blocked with 5% BSA in PBS. Mouse serum was diluted in PBS at 1:500 for IgG2b and IgG3 and 1:1000 for IgG1 and IgG2c, and incubated overnight at 4 °C. The following day, plates were washed 5 times with 0.05% PBST and one of the following secondary goat anti-mouse HRP antibodies were added for 2 h: anti-Mouse IgG1 (Southern Biotech 1070-05, Birmingham, AL, USA), anti-Mouse IgG2b (Southern Biotech 1090-05, Birmingham, AL, USA), anti-Mouse IgG2c (Southern Biotech 1079-05, Birmingham, AL, USA), or anti-Mouse IgG3 (Southern Biotech 1100-05, Birmingham, AL, USA). After washing 6 times with 0.05% PBST, 1-Step™ Turbo TMB-ELISA (Thermo Scientific #34022, Rockford, IL, USA) reagent was added and incubated for 20 min at room temperature. Sulfuric acid was used to stop the reaction. Plates were analyzed on a spectrophotometer at 450 nm.