Anti igg2a hrp

Serum specific antibody response (IgG₁ and IgG_2a) was determined by ELISA. Optimal assay conditions were determined in a checkerboard manner. Briefly, microtitre plates (Nunc, Roskilde, Denmark) were coated with 5 μg/ml ASE or SLA. Anti-mouse IgG₁-HRP was from Nordic Immunology (Eindhoven, Netherlands) and HRP anti-IgG_2a was from Southern Biotech (Birmingham, USA). Mice sera were diluted 1/800 and conjugate 1/2500 to determine IgG₁ against ASE; 1/50 sera dilution and 1/1000 conjugate dilution to estimate anti-SLA response. IgG_2a against ASE employed sera diluted 1/400 and 1/2000 dilution conjugate. Anti-Leishmania IgG_2a was determined with 1/25 sera dilution and 1/2000 conjugate.

Using DNA from calf thymus (Sigma, D1501), we made double-stranded DNA (dsDNA) using a protocol previously described [27 (link)]. High-binding ELISA plates (Costar, 3369) were then coated with a mixture containing 5μg/ml dsDNA overnight at 37°C. The plates were then blocked with PBS containing 1% BSA for 30 minutes. Following blocking, plates were washed several times with 0.05% tween-20 PBS. Serum was added at a 1:10 ratio in PBS containing 0.05% Tween and 1% BSA (PBS-T/BSA) and then diluted using 2-fold serial dilutions until 1:80. Plates were incubated at room temperature (RT) for 45 minutes and then washed. The HRP-conjugated secondary antibody (HRP-IgG or HRP- IgG2a, Southern Biotech) was then added at a 1:4000 ratio in PBS-T/BSA and incubated for 45 minutes at RT. Plates were then washed and 50μl of TMB Substrate (eBioscience) was added to each well. The reaction was stopped after 10 minutes using 50μl of 1M phosphoric acid and the plate was read at 450nm. Serum consisting of high titers of IgG and IgG2a autoantibodies was used as a positive control on each plate in addition to wells with no serum as negative controls. Plates were read by a Multiscan FC (Thermo Scientific, Waltham, MA) ELISA plate reader. HRP-Anti-IgG and HRP-anti-IgG2a were purchased from Southern Biotech (Birmingham, AL).