Goat anti mouse igg1

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.

Catching ELISA was carried out to determine total IgA and IgM in spent medium from OFC. Briefly, 96 well microplates were coated with either affinity purified goat anti-pig IgA or goat anti-pig IgM (Bethyl Laboratories, Montgomery, Texas, USA). Serial dilutions of serum samples and reference standard were added to coated plates and incubated for 2 h at room temperature. Bound immunoglobulins were detected using isotype-specific monoclonal antibodies (anti-pig IgA K61.1B4, anti-pig IgM K52.1C3 from Biorad) followed by horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG₁. Concentrations of immunoglobulin subclasses were determined by interpolation of samples onto the reference standards.