ELISA was performed essentially as previously described (42 (link), 43 (link)). In brief, plates were coated with OVA protein (Invivogen Endofit™, 50 µL/well at 1 µg/mL). Dilutions of test sera (in triplicate) and a standard curve produced using serial dilutions of a reference serum pool from OVA-immune mice were added to the plate. Washing, secondary antibody binding, final washing and detection were all as previously described, with the exception that the secondary antibody used was alkaline-phosphatase conjugated goat anti-mouse IgG (Sigma). OD405 was quantified using a ELx800 plate reader (BioTek). Results were expressed in antibody units (AU), defined using the reference standard, by interpolation of OD405 readings on the standard curve.
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ELISA for Quantifying Antibody Levels
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Corresponding Organization : University of Oxford
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Variable analysis
independent variables
- Dilutions of test sera
- Antibody titer (expressed in antibody units, AU)
- Coating of plates with OVA protein (Invivogen Endofit™, 50 µL/well at 1 µg/mL)
- Use of a standard curve produced using serial dilutions of a reference serum pool from OVA-immune mice
- Washing, secondary antibody binding, final washing and detection procedures as previously described
- Use of alkaline-phosphatase conjugated goat anti-mouse IgG (Sigma) as the secondary antibody
- Positive control: Reference serum pool from OVA-immune mice
- Negative control: Not explicitly mentioned
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