Goat anti mouse igg2a hrp

Serum levels of mouse MCPT-1 were evaluated by ELISA (ThermoFisher Scientific, Vienna, Austria and BD Biosciences, San Diego, CA, respectively). OVA-specific IgG1, IgG2a, and IgE in the serum were measured by ELISA, according to the following protocol. Plates were coated with OVA in BupH™ Carbonate-Bicarbonate buffer (ThermoFisher Scientific, Vienna, Austria), pH 9.4 and incubated at 4°C overnight. The plates were washed with 1x TBS 0.05% Tween-20 buffer and blocked with “Blocker Casein” in TBS (ThermoFisher Scientific, Vienna, Austria) at room temperature for 2 h. Serial dilutions of the mouse anti-ova IgG1, anti-ova IgG2a or anti-ova IgE standards (Chondrex, Woodinville, WA) were prepared. After dilution of experimental serum samples, the diluted standards and samples were added to the plate in duplicate and incubated for 1 h. Subsequently, the plates were washed and goat anti-mouse IgG1-horseradish peroxidase (HRP) (Abcam, Cambridge, UK) or goat anti-mouse IgG2a-HRP (Abcam, Cambridge, UK) or goat anti-mouse IgE-HRP (Bio-Rad, Hercules, CA) were added to appropriate wells. After incubation, TMB substrate was added to each well and incubated covered with foil until developed. Reactions were stopped with H₂SO₄, and the optical density of the reactions was measured on an ELISA plate reader at 450 nm.

The titers of OVA-specific IgG and IgG subtypes were measured using goat anti-mouse IgG H&L (HRP) (Abcam, Cambridge, UK), goat anti-mouse IgG1 (HRP) (Abcam, Cambridge, UK) and goat anti-mouse IgG2a (HRP) (Abcam, Cambridge, UK). OVA-specific IgG tracking was performed with the serial dilution method for analysis by ELISA; the titers were read as the UV absorbance signals, which twice exceeded the background value.

Serum antibody responses were analyzed by ELISA. High-binding plates (Greiner Bio-One, Germany) were coated overnight at 4 °C with 100 µL of 5 µg mL⁻¹ of soluble proteins, p*17 C and K4S2C, diluted in TBS buffer. The plates were blocked with 200 µL of 3% BSA in TBS buffer for 1 h at 25 °C. After three times washes with TBST, 100 µL of serially diluted mice serum samples (from 1:200 to 1:25, 600) was used as primary polyclonal antibodies and incubated for 1 h at 25 °C. Plates were washed three times with TBST before the incubation with the secondary antibodies, goat-anti-mouse IgG-HRP, goat-anti-mouse IgG1-HRP, goat-anti-mouse IgG2a-HRP, goat-anti-mouse IgG2b-HRP, and goat-anti-mouse IgG3-HRP (Abcam, United Kingdom), diluted 1:20,000 with TBST at 25 °C for 1 h. After washing three times with TBST, 100 μL of OPD (Sigma-Aldrich, USA) was added on plates and incubated for 15 min at 25 °C and the reaction was stopped using 50 µL of 0.5 N H₂SO₄. The results were measured at 490 nm using Elx808iu ultramicrotiter plate reader (Bio-Tek Instruments Inc., USA).