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

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 microliters of the sealing solution per well for two hours at 37 degrees Celsius. Then, dilute three microliters of the mice serum from each experimental group 100 times by adding 297 microliters of PBST, and vortex the diluted serum. Add 100 microliters of diluted serum per well in the coated ELISA plate for each serum sample. Similarly, dilute the positive and negative control serums 100 times with PBST, by adding 4 microliters of positive or negative control serum to 396 microliters of PBST followed by vortex mixing.

To perform double-ratio dilution, add 200 microliters of the previously diluted experimental serum to the first row of the coated ELISA plate. Then, add 100 microliters of PBST to all wells, except those in rows one and 12. Next, adjust the pipette gun to 100 microliters and transfer 100 microliters of serum from the first row to the second row containing PBST.

Mix the content in the second row 10 times, using the pipette gun. Similarly, perform 1:2 successive dilutions of the serum till row 11. Once done, discard 100 microliters of liquid from row 11.

Add 100 microliters of diluted negative and positive serum to each corresponding well in row 12, following the sample template. Seal the ELISA plate with plastic wrap and incubate at 37 degrees Celsius for one hour. Meanwhile, dilute secondary antibody anti-mouse IgG HRP 10,000 times by adding PBST. After the one-hour incubation, wash the ELISA plate with 300 microliters of PBST per well to clean the unbound antibodies. Then add 100 microliters of the diluted secondary antibody to each well, and incubate the plate at 37 degrees Celsius for 40 minutes.

Wash the plate with 300 microliters of PBST, per well. Then add 100 microliters of TMB color development solution to each well. Place the plates at 37 degrees Celsius and away from light for 10 minutes. After which, terminate the reaction immediately with 50 microliters of 2 molar sulfuric acid. Detect the optical density or OD values at 450 nanometers, using an enzyme marker within 15 minutes after termination.