Anti mouse igg2c

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.

Peripheral blood was collect and subsequent centrifugation at 10,621 x g for 5 min to isolate the serum. Serum titers against ID93 were then evaluated by antibody capture ELISA. Briefly, Corning high bind 384 well plates (VWR International) were coated overnight at 4 °C with 2 µg/ml ID93 in coating buffer (eBioscience). Then, plates were blocked with 1% BSA-PBS and serum samples serially diluted. Detection antibodies were anti-mouse IgG2c (Southern Biotech Cat# 1079-05) or IgG1 HRP (Southern Biotech Cat# 1070-05). Plates were analyzed at 450 nm (ELx808, Bio-Tek Instruments Inc.) and endpoints were set as the minimum dilution at which values were lesser or equal to an OD of 0.2.