Hrp conjugated rabbit anti mouse igg2a

ELISA was performed to measure the titers of Ag-specific IgG1 and IgG2a antibodies in serum. Nunc 96-well MaxiSorp plates (Thermo Fischer Scientific) were coated with 100 µl 1 µg/ml unmodified LYS in carbonate buffer (pH 9.6) (SSI Diagnostica, Hillerød, Denmark). The plates were incubated at 4 °C overnight and washed with 0.2% Tween (Merck) in PBS (pH 7.2, Gibco Life Technologies™). The plates were blocked for 1.5 h with 2% BSA in PBS. To give a 10-fold dilution curve, 1% BSA in PBS was added to each well and the serum was added. The plates were incubated at RT for 2 h. After washing the plates, they were incubated for 1 h with either horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 (Invitrogen, Carlsbad, CA, USA) diluted 1:20,000 in 1% BSA in PBS or HRP-conjugated rabbit anti-mouse IgG2a (Invitrogen) diluted 1:5,000 in 1% BSA in PBS. The plates were developed with 100 μl/well 3,3′,5,5′-tetramethylbenzidine substrate (Kem-En-Tec Diagnostics, Taastrup, Denmark) for 10 min. The reaction was stopped by adding 0.2 M sulfuric acid (VWR, Radnor, PA, USA), and the plates were analyzed using a TECAN Sunrise™ ELISA reader (Tecan Trading) at OD₄₅₀ nm corrected at OD₆₂₀.