Ovalbumin-Induced In Vivo Antibody Production


The ultimate function of B cells is to produce antibodies, which bind antigens and trigger several effector functions of the immune system. To assess the function of B cells in mice, we offer the in vivo antibody production assay in response to immunization with an antigen. The mice are immunized with either chicken egg ovalbumin (OVA) or Keyhole Limpet Hemocyanin (KLH). Then the levels of IgM, IgG1, IgG2a, and IgG2b antibodies are detected by ELISA in the serum of these mice. The antibody levels may be assayed after primary and/or secondary stimulation with the antigens.


The mice were immunized by subcutaneous (s.c.) injection with 100µg OVA/CFA and challenged with 100µg OVA/IFA (s.c.) fourteen days later. The blood was collected fourteen days after the primary stimulation and again, ten days after the secondary immunization. The mice were bled from the retro-orbital sinuses. The blood samples were allowed to coagulate and then centrifuged at 1000g to isolate the serum. The serum was immediately frozen and stored at -80°C. The frozen serum samples, collected after the primary immunization and after the secondary stimulation, were thawed and analyzed in parallel. The measurements of IgG1, IgG2a and IgG2b were performed as follows: Serial dilutions of serum were prepared in phosphate-buffered saline (PBS). 96-well plates (Nunc) were coated with 100μl of 1 mg/ml of OVA in PBS overnight at 4°C. The plates were washed with PBS/0.05% Tween 20, blocked with 2% bovine serum albumin (Sigma) in PBS for 1 hr at 37°C, and washed again. 100μl of diluted serum was added to each well, and the samples were incubated overnight at 4°C. The plates were then washed, and peroxidase conjugated anti-isotype antibodies (Southern Biotechnologies) were added. The plates were rocked for 1 hour at room temperature and then washed four times with PBS/0.05% Tween 20. Tetramethylbenzidine peroxidase substrate (DAKO Corp) was added and the color development stopped with 1M H2SO4. The optical densities were measured at 450 nm. Combined serum from the control group was run on each plate as a standard and used to calculate the relative units for each isotype.

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