Hrp conjugated anti hamster total igg

Sera of immunized animals were analyzed by ELISA for antibody titers determination: total IgG and its subclasses IgG1 and IgG2/IgG3. ELISA plates were coated with 250 ng of each recombinant protein, and wells were blocked with PBS-T containing 10% nonfat dry milk and incubated with different dilutions of hamster sera, ranging from 1:200 to 1:409,600. Plates were washed and incubated either with HRP-conjugated anti-hamster total IgG (1:5,000, Sigma), anti-hamster IgG1, or anti-hamster IgG2/IgG3 (1:5,000, Southern Biotechnology). The wells were washed three times, and o-phenylenediamine (1 mg/ml) in citrate phosphate buffer (pH 5.0) plus 1 μl/ml H₂O₂ was added (100 μl per well). The reaction was allowed to proceed for 10 min and was interrupted by the addition of 50 μl of 2 M H₂SO₄ to the mixture. Readings were taken at 492 nm with a microplate reader (Multiskan EX; Thermo Fisher Scientific, Helsinki, Finland). The titer was considered the maximal dilution that showed an OD492 nm value above 0.1.

Sera of immunized animals were pooled from three animals and analyzed in triplicate by ELISA to determine the production of total IgG antibody and its subclasses IgG1 and IgG2/IgG3. ELISA plates were coated with 250 ng of each recombinant protein, and wells were blocked with PBS-T containing 10% non-fat dry milk and incubated with different dilutions of hamster sera, ranging from 1:200 to 1:12,800. Plates were washed and incubated with either HRP-conjugated anti-hamster total IgG (1:5,000, Sigma), anti-hamster IgG1, or anti-hamster IgG2/IgG3 (1:5,000, Southern Biotechnology). The wells were washed three times, and a solution 1 mg/ml o-phenylenediamine in citrate phosphate buffer (pH 5.0) plus 1 μl/ml H₂O₂ was added (100 μl per well). The reaction was allowed to continue for 10 min and was interrupted by the addition of 50 μl of 2 M H₂SO₄ to the reaction mixture. Readings were taken at 492 nm with a microplate reader (Multiskan EX; Thermo Fisher Scientific, Helsinki, Finland). The graphs were plotted with average absorbance values found at 200 times serum dilution.