Igg2a hrp

The serum levels of IgG_2a and IgG₁ were evaluated using HRP-conjugated antibodies against IgG2a and IgG1. To this aim, Maxi-Sorp plates (Greiner, Germany) were coated with 10 μg/ml L. major F/T Ag overnight and then blocked with 1% BSA at room temperature for 2 hours. After that, 100 μl (at a dilution of 1:50) of serum was added and incubated at 37°C for about 2 hours. The plates were washed, and anti-mouse IgG1-HRP or IgG2a-HRP (1: 10,000; Southern-Biotech, Canada) was added to each plate. After 2 h incubation at 37°C, the plates were rinsed again, and 20 μl of the TMB micro-well peroxidase substrate solution (KPL, Gaithersburg, MD) was added to each sample. Finally, the plates were incubated for 20 minutes at 37°C, and the reaction was blocked by adding 1% sodium dodecyl sulfate. The absorbance of each group was measured at a wavelength of 405 nm.

Serum samples were collected by tail bleeding, and specific IgG antibodies against DV1 or DV2 were determined by ELISA. Briefly, each well of the 96-well microtiter plate was coated with 2 μg of the concentrated DV1 or DV2 protein from the infected C6/36 cells, and followed by blocking with 2% bovine serum albumin. The plate was incubated with two-fold serial dilutions of the serum samples (started from 1:100), then antibody titers were detected with HRP-conjugated goat anti-mouse IgG (1:5,000, SANTA, USA), and substrate solution of orthophenylene diamine. The absorbance at 492 nm was measured using a microplate reader (Thermo, USA). The highest dilution, yielding an optical density (OD) greater than that of the negative control at the same dilution, was recorded as the end-point titer.
To determine the IgG subclasses, two weeks after the third immunization, antibody isotype ELISA was performed using serum samples (1:100) as the first antibody, and goat anti-mouse IgG1-HRP or IgG2a-HRP (1:4,000, SouthernBiotech, USA) was used as the secondary antibody.

To determine the titer of HSV-specific serum antibodies induced by vaccine, mice were immunized with vaccine virus or control supernatant. Blood was collected from the tail vein of mice 21 d after immunization. Serum was prepared by clot retraction and analyzed by ELISA as previously described [28 (link)]. Anti-mouse-IgG-biotin (R & D Systems, Minneapolis, MN, USA) was used as secondary antibody and detected using streptavidin-HRP followed by OPD substrate (Sigma-Aldrich, St. Louis, MO, USA). Alternatively, antimouse-IgG1-HRP and -IgG2a-HRP (SouthernBiotech, Birmingham, AL, USA) were used. Plates were read at 490 nm on a BioRad 680 reader. Antibody concentrations were determined by comparison to standard curves generated with serum containing known concentrations of IgG captured on plates coated with goat-anti-kappa light chain antibody (Caltag, Burlingame, CA, USA) as previously described [28 (link)].

AEAR was prepared in our lab [22 (link)]. 2014/2015 seasonal influenza virus split vaccine was purchased commercially from Shenzhen sanofi Pasteur biological products co., LTD (Shenzhen China). Goat anti-mouse peroxidase conjugates (IgG-HRP, IgG₁-HRP and IgG_2a-HRP) were purchased from Southern Biotech Inc. USA. Concanavalin A (ConA), lipopolysaccharide (LPS, from Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich Co. LLC. USA. Imject Alum Adjuvant (Alum) was from Thermo Scientific Pierce USA. CD11c-FITC, CD3-PE, CD4-APC, CD8a-FITC, CD44-PE, CD25-APC, Foxp3-PE, IFN-γ-PE, IL-4-PE, CD86-PE, CD40-APC, CD80-APC, MHC-II-PE, Cytofix/Cytoperm and Perm/Wash buffer were purchased from BD Bioscience USA. Carboxyfluorescein diacetate succinimidyl ester (CFSE), Treg staining kit were from eBioscience USA. All the other reagents were analytically purified in China.

To measure antigen‐specific antibody responses, enzyme‐linked immunosorbent assay (ELISA) Nunc MaxiSorp flat‐bottom plates (Invitrogen 44‐2404‐21) were coated with 2.5 µg mL⁻¹ with the mixture of four antigens in phosphate coating buffer (0.1 m Na2HPO4 in deionized water, pH 9.0) overnight at 4 °C. Next day, plates were blocked with 1% bovine serum albumin in phosphate buffered saline‐Tween (1X PBS, 0.05% Tween) for 2h at room temperature. Plates were then washed 4x with PBS‐T and incubated overnight at 4 °C with serially diluted sera collected from mice in PBS‐T. The next day, wells were washed 4x with PBS‐T and incubated for 1h at room temperature either with HRP‐IgG1 (SouthernBiotech 1070‐05) (1:4,000) or HRP‐IgG2a (SouthernBiotech 1080‐05) (1:4000). Wells were subsequently washed 4x with PBS‐T and developed with 2,2‐azinobis(3‐ethylbenzthiazolinesulfonic acid) (ABTS) (VWR 95059‐146) substrate for 5 min at room temperature. At the end of the incubation, the reaction was stopped with 10% SDS solution. Absorbance was measured at 405 nm to determine endpoint antibody titers.

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).