Ap conjugated anti mouse igg

Serum titers of immunoglobulin subclasses were determined by specific ELISA kits (SouthernBiotech) according to the manufacturer's protocol. To detect anti-dsDNA autoantibodies in sera, high-binding ELISA plates were coated overnight with 2 μg/ml dsDNA from calf thymus (Sigma-Aldrich). Coated plates were blocked with 1% BSA and 0.5% gelatin in TBS for 2 h at room temperature, and diluted samples were incubated overnight at 4°C in TBS with 1% BSA. Bound anti-dsDNA antibodies were detected with AP-conjugated anti-mouse IgG (Jackson ImmunoResearch) and streptavidin-HRP (Bioresearch) followed by TMB substrate solution (eBioscience). Absorbance was measured at 450 nm. Serum titers of anti-ANA antibodies were determined by ANA Hep Screen ELISA kit (Demeditec) according to the manufacturer's protocol.

Quantification and antigenic analysis using ELISA was performed according to a previous report [39 (link)]. Rabbit serum immunized with the JEV Nakayama strain was coated onto Nunc Maxisorp ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) to quantify VLPs. Next, the VLPs from plasmid-transfected 293T cells, anti-JEV mouse polyclonal antibody, alkaline phosphatase (AP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and p-nitrophenyl phosphate (PNPP) (Nacalai Tesque, Kyoto, Japan) were serially incubated, and the absorbance was measured at 415 nm. The ELISA diluent comprised PBS containing 1% bovine serum albumin and 0.05% Tween 20. The number of VLPs was expressed as the optical density (OD) of mutant VLP relative to that of wild-type VLP (OD of mutant VLP/OD of wild-type VLP).
For antigenic analysis, the same ELISA procedure was followed, but using 3H12 as the detector antibody. Additionally, the reactivity of the VLPs was expressed as the OD of mutant VLP relative to that of wild-type VLP (OD of mutant VLP/OD of wild-type VLP).

Immunoglobulin subclasses in plasma were detected by specific ELISA kits (SouthernBiotech 5300-4B) according to the manufacturer’s protocol. To detect anti-dsDNA autoantibodies in plasma, high-binding ELISA plates were coated with 2 μg/ml dsDNA (Sigma-Aldrich, D4522) from calf thymus. Coated plates were blocked, and subsequently incubated with diluted samples overnight at 4 °C in TBS with 1% BSA. Bound anti-dsDNA antibodies were detected with AP-conjugated anti-mouse IgG (Jackson ImmunoResearch, 115-055-146) and streptavidin-HRP (BioTechne, DY998) followed by TMB substrate solution (eBioscience 00-4201-56). Absorbance was measured at 450 nm.