Peroxidase conjugated goat anti mouse igg secondary antibody

Reactivity of immune sera against chimeric antibodies was determined by solid-phase ELISA as previously described (27 (link)). Briefly, 96-well Maxisorp polystyrene plates (Nunc, Roskilde, Denmark) were coated with 10 μg/mL of chP3R99-LALA, ch1E10, or hR3 mAb and incubated overnight at 4°C. Diluted sera (1/1,000) were added and the plates were incubated for 1 h at 37°C. Peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used to determine the reactivity associated with IgG fraction using 0.4 mg/mL of ortho-phenylenediamine in citrate/phosphate buffer, pH 5 containing 0.2% of H₂O_2. Sera reactivity is expressed as OD values at 490 nm.

The presence of antibodies against CS was assessed by solid-phase ELISA, as previously described (27 (link)). Maxisorp polystyrene ELISA plates (Nunc) were coated with 10 μg/mL of CS and incubated overnight at 4°C. Mice sera (1/400) was added to the plates and incubated for 1 h at room temperature. Peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories) was added and the plates incubated for 1 h at room temperature. Colorimetric reaction was developed using 0.4 mg/mL of ortho-phenylenediamine in citrate/phosphate buffer, pH 5 containing 0.2% of H₂O₂. Sera reactivity is expressed as OD values at 490 nm.
The pattern of IgG subclasses developed in immunized mice was determined using biotinylated goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (BD Pharmingen, San Diego, CA, USA), followed by incubation with streptavidin-conjugated to alkaline phosphatase (Jackson ImmunoResearch Laboratories) for 30 min at 37°C. The reaction was developed using 1 mg/mL of para-nitrophenyl phosphate in diethanolamine buffer solution pH 9.
Antibody response induction was considered positive when hyperimmune serum OD was ≥0.2 and at least twofold the preimmune serum OD value. Assays were performed in triplicate for each sample and the coefficient of variation was <15% for all values.