Goat anti mouse igg2a

The humoral response of mice after immunization was characterized by the reactivity of IgG antibodies from diluted sera (1:100) with native Toxoplasma gondii antigen (TLA) and by the levels of chimeric antigen-specific IgG1 and IgG2a antibodies (in sera diluted from 1:100 to 1:204,800). The immunoenzymatic reactions were developed with goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, USA), goat anti-mouse IgG1 (Bio-Rad AbD Serotec GmbH, Puchheim, Germany) or goat anti-mouse IgG2a (Bio-Rad AbD Serotec GmbH, Puchheim, Germany) secondary antibodies conjugated with HRP, as described previously [19 (link)].

Two weeks and 6 months after the last boost immunization, immunoglobulin levels were determined in diluted mouse sera: 1:100 for IgG specific to TLA of the RH and Me49 strains and from 1:100 to 1:204,800 for IgG2a and IgG1 specific to vaccine antigens using an ELISA test (Enzyme-Linked Immunosorbent Assay). Assays were performed as described previously^{42 (link)}. Briefly, 96-well MaxiSorp plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight with 100 µl of TLA or recombinant antigens at a final concentration of 2 µg/well. The wells were then blocked with PBS supplemented with 10% of FBS (Merck KGaA, Darmstadt, Germany) and next diluted sera were added in duplicate. The reaction was developed with the secondary HRP-conjugated antibodies: goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), goat anti-mouse IgG1 (Bio-Rad, Hercules, CA, USA), goat anti-mouse IgG2a (Bio-Rad, Hercules, CA, USA) and the 2,2-azino-bis(3-ethyl-benzothiazoline6-sulfonic acid) diammonium salt (ABTS) (Merck KGaA, Darmstadt, Germany) serving as a chromogen. The absorbance was measured at 405 nm (Multiskan EX automatic ELISA reader) (Thermo Fisher Scientific, Waltham, MA, USA) and IgG1 and IgG2a titers were defined, as the highest serum dilution with OD > 0.3.

Plates were coated with mouse IgE F127, IgE(PNG), or Fel d 1. Serial dilution of sera (1:25, then serially 1:3) was added. HRP-labeled goat anti-mouse IgG (The Jackson Laboratory) antibodies were used to detect IgG (1:2’000). To detect IgG subclasses, HRP labeled rat anti-mouse IgG1 (BD Pharmingen), goat anti-mouse IgG2a (Bio-Rad), goat anti-mouse IgG2b (Invitrogen, Carlsbad, CA, USA), and goat anti-mouse IgG3 (SouthernBiotech, Birmingham, AL, USA) were used in a 1:2’000 dilution.