2.5 Immunoglobulin isotyping ELISA
Isotype profile by indirect ELISA was evaluated to determine the IgG1, IgG2a, IgG2b and IgG3 subtypes in immunized mice at 40 days post vaccination (dpv) and the titer isotype of IgG1, IgG2 from sera of immunized cattle at 90 dpv and to determine IgA from nasal swabs at 6 days post challenge (dpc).
Greiner Microlon microtiter plates were coated with iBoHV-1 and dilutions were conducted as described previously. Bound antibodies in mice sera were detected with biotinylated goat anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Caltag Laboratories, San Francisco, CA). After incubation for 60 minutes, plates were washed with PBST and a dilution of streptavidin/alkaline phosphatase (SIGMA) was added to each plate. The results for IgG1, 2a, 2b, and IgG3 obtained in 1/50 sera dilution were expressed as OD at A492 nm.
For isotype detection in cattle, the same ELISA described in chapter 2.4 was used with modifications: anti-bovine IgG1, IgG2, or IgA mouse monoclonal antibodies (provided by Dr. S. Srikumaran, University of Nebraska, USA) were used, followed by incubation with horse radish peroxidase (HRP) conjugated anti-mouse Ig. Finally, the reaction was visualized, and the cut-off was established as described before for IgG1 and IgG2. The results for IgA 1/2 dilution in nasal swabs were expressed as OD at A492 nm.