Measuring Immunoglobulin G Levels Against a Test Vaccine in a Mouse Serum

Begin with a multi-well plate coated with a test vaccine and block non-specific interaction sites.

Acquire a serum sample from a mouse injected with the test vaccine.

This serum contains immunoglobulin G, or IgG, antibodies that recognize the test vaccine.

To the vaccine-coated wells, add serially diluted serum samples.

The IgGs bind to the vaccine. Wash to remove unbound antibodies.

Add peroxidase-conjugated secondary antibodies that bind to IgGs. Remove unbound secondary antibodies.

Add a chromogenic substrate. The peroxidase interacts with the substrate, generating a blue-colored product.

Introduce an acid to stop the reaction. The change in the pH turns the solution yellow.

Using a spectrophotometer, measure the color intensity of each well to determine the concentration of IgG in the serum sample.

For antigen coating of the enzyme-linked immunosorbent assay, or ELISA plate, dilute HI protein antigen to 10 micrograms per milliliter with coating solution, and add 100 microliters of the diluted antigen per well in the ELISA plate. Incubate the plate at 4 degrees Celsius overnight. After incubation, wash the plate with 300 microliters of PBST per well.

Seal the wells with 300 micr