Using a Sandwich ELISA to Determine the Immunogenic Glycoprotein Content in a Vaccine

Begin with a passivated assay plate coated with rabies immunogenic glycoprotein-specific monoclonal antibodies and a blocking agent to prevent non-specific interactions.

Introduce serially diluted reference and test rabies vaccines with decreasing glycoprotein content. Seal the plate to prevent contamination.

During incubation, immunogenic glycoproteins interact with coated monoclonal antibodies via a specific antigenic site.

Post-incubation, remove the film, aspirate the supernatant, and wash the plate.

Overlay with enzyme-conjugated glycoprotein-specific monoclonal antibodies that bind to a different antigenic site on the glycoprotein, forming an immunocomplex.

Add a solution containing a substrate and a chromogen. When the substrate interacts with the enzyme, it causes the oxidation of the chromogen, changing the solution color.

Add a stopping solution to inactivate the enzymes.

Measure the absorbance and generate a reference curve using the reference vaccine values.

Identify the region where there is a linear relationship between absorbance and the glycoprotein content of the reference vaccine dilutions. Then, extrapolate the glycoprotein content of the test vaccine dilutions based on the absorbance that falls within this region.