Igg2a m32215

To determine the antigen‐specific antibody response to the ovalbumin, NUNC MaxiSorp 96‐well plates (Thermo Fisher) were coated with 10 μg/ml purified ovalbumin (Sigma‐Aldrich) for 24 hours at 4°C in coating buffer in PBS. Next, plates were washed extensively with PBS/Tween 0.05% and additionally blocked with 1% of PBS/BSA. A dilution range of serum, obtained from vaccinated mice (on day 7 after vaccination) through a heart puncture, was incubated over night at 4°C. After washing, samples were incubated with anti‐mouse IgG‐biotin (and anti‐IgG1, 2, 3 isotypes) antibodies for 1 hour at RT and after wash incubated with HRP‐conjugated streptavidin for 1 hour at RT. Then, after washing, the ELISA plate was developed using TMB substrate buffer. Reaction was stopped when properly developed using 2N H2SO4 and extinction was measured at 450 nm using an iMark microplate reader (Bio‐Rad). Serum dilution of 1:400 showed the most consistent and reproducable signal to noise ratio. All samples were normalized with PBS as blanco. Secondary antibodies (1:2000) used: IgG1 115‐065‐205 (Jackson ImmunoResearch), IgG2a m32215 (Invitrogen), IgG2b ab97248 (Abcam), IgG3 1100‐08 (ITK), IgM 62‐6840 (Zymed), IgG 315‐065‐006 (Dianova), Streptavidin‐HRP p0397 (Dako).