Igg2b

The assessment of total serum IgG levels (anti-Toxocara) was performed by indirect immunoenzymatic assay (ELISA) using TES as the antigen at a concentration of 1 μg/ml and serum dilution of the primary antibody (1:50), as described by Avila et al. (2012) (link). For the secondary serum, horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody produced in goat (Sigma-Aldrich, St. Louis, Missouri, USA), a 1:5000 dilution in PBS-T, was used. Serum samples were examined individually and in duplicate, and readings were performed at a wavelength of 492 nm. The evaluated isotypes were IgG1, IgG2a, IgG2b and IgG3, following the recommendations of Monoclonal Mouse Antibody Isotyping Reagents (Sigma-Aldrich, St. Louis, Missouri, USA). The concentration of antigen used in sensitization and the dilution of the primary serum were the same as those used for the evaluation of total IgG. Each sample (pool of sera) was examined in triplicate to obtain the mean absorbance. The secondary antibodies anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Sigma-Aldrich, St. Louis, Missouri, USA) produced in goat were diluted 1:2000 in PBS-T and applied to the plates. After, the peroxidase-conjugated anti-goat antibody produced in rabbit (Sigma-Aldrich, St. Louis, Missouri, USA) was diluted 1:4000 in PBS-T was added.

ELISA plates were coated overnight at 4°C with either recombinant H7N9 HA (rHA) or polyclonal anti-mouse Fab2 IgG (Sigma). After blocking non-specific binding and subsequent washing, diluted sera or serial dilutions of purified murine IgG1, IgG2a or IgG2b (Sigma) were added to wells containing H7N9 rHA (Sino Biological Inc. Cat. no. 40103-V08B) or Fab2 IgG. Plates were incubated for 1 h at room temperature, and washed again, prior to further incubation for 1 h with IgG subclass-specific peroxidase-conjugated goat anti-mouse IgG antibodies. Bound IgG subclass antibodies were detected colorimetrically using TMB substrate.

The Ig subclass profile against NadA, fHbp and Men C CPS was analyzed by using an indirect ELISA as described above. Plates were coated as previously described. To detect the various Ig subclass, plates were incubated with the respective anti-IgG1, IgG2a, IgG2b, IgG3 and anti-IgM antibodies (derived from goat) (Sigma-Aldrich, St. Louis, MO) using a DF of 1: 2500. Plates were developed using the anti-goat IgG peroxidase conjugate and the respective substrate as described above. The results are expressed as (mean ± SD) of two replicas per subclass (see immunization schedule for proteinaceous antigens) or as (mean ± SEM) of 8 animals per subclass (see immunization schedule for conjugated vaccines).

Generation of the chicken anti‐EAAT2a (zebrafish) antibody is described elsewhere (Niklaus et al., 2017 (link)). Five dpf larvae were fixed in 4% PFA in phosphate buffered saline (PBS, pH 7.4) at room temperature for 40 min. Embryos were cryo‐protected in 30% sucrose in PBS at 4°C overnight, embedded in Tissue Freezing Medium TFMTM (Electron Microscopy Sciences), cryo‐sectioned at 14–16 μm and mounted onto Superfrost slides (Thermo Fisher Scientific). Immunohistochemistry was performed as described before (Niklaus et al., 2017 (link)). Chicken anti‐EAAT2a 1:500, mouse anti‐synaptic vesicle 2 (IgG1, 1:100, DSHB USA), mouse anti‐acetylated tubulin (IgG2b, 1:500, Sigma 7451) and mouse anti–glutamine synthetase (IgG2a, 1:200, EMD Millipore, MAB302) were used as primary antibodies. Secondary antibodies were goat anti‐chicken Alexa Fluor 488, goat anti‐mouse IgG2a Alexa Fluor 568, goat anti‐mouse IgG2b Alexa Fluor 647 and goat anti‐mouse IgG1 Alexa Fluor 647, all 1:500 (all from Invitrogen, Thermo Fisher Scientific). Slides were cover‐slipped using Mowiol (Polysciences) containing DABCO (Sigma‐Aldrich) and imaged with a TCS LSI confocal microscope (Leica Microsystems). Images were adjusted for brightness and contrast using Affinity Photo Version 1.8 and assembled in Affinity Designer Version 1.7.

Sera were tested for IgG antibody response by enzyme-linked immunosorbent assay (ELISA) using standard procedures^{15 (link)}. Briefly, ninety-six-well microtiter plates were coated with recombinant protein PA (BEI, NR-3780), LF (NR-4268), F1 (NR-44223), LcrV (NR-32875), or F1-LcrV monomer (NR-2563) at 1 µg/ml each, or heat-inactivated LVS (HI-LVS, 2 × 10⁷/ml) diluted in carbonate buffer overnight at 4 °C and afterwards processed at ambient temperature. The plates were washed three times with 0.05%Tween20-PBS, blocked in 3% BSA-PBS for 3 h, incubated with each serum sample serially diluted 2-fold twelve times at a starting dilution of 1:20 or 1:50 in 1% BSA-PBS for 90 min, and washed again. Bound antibody was detected by using alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO), subtypes IgG1 (Sigma), IgG2a (Abcam), IgG2b (sigma), or IgG3 (Sigma) diluted in 1% BSA-PBS and incubating for 90 min. Plates were developed with 100 µl of p-nitrophenylphosphate substrate (BioRad), and the A415 was read using a multiscan microplate reader (iMark, BioRad). The results are presented as the mean antibody endpoint titer and SE of the mean (SEM). Antibody endpoint titer is defined as the mean log dilution that yields an OD greater than the mean OD of Sham sera plus three standard deviations at the same serum dilution.

In order to characterize the IgG isotypes, microtiter plates were coated with F1–LcrV and F1–LcrV–HSP70(II) (0.1 μg/well) and blocked with BSA as mentioned above. IgG isotypes sera from animal groups viz; control, F1–LcrV and F1–LcrV–HSP70(II) collected after second booster, added at 1:1000 dilution in PBS in triplicate wells. Plates were incubated for 1 h at 37°C, washed and again incubated with goat anti-mouse isotype specific antibodies IgG1, IgG2a, IgG2b, and IgG3 (Sigma, USA) at 1:1000 dilutions for 1 h at 37°C. Detection of bound isotype specific antibodies was estimated with rabbit anti-goat IgG HRP conjugated (1:5000 dilutions). The reaction was stopped with 2N H₂SO₄ and optical density (OD) was measured at 490 nm.