Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates

To verify the success of the conjugation by ELISA, coat an appropriate 96-well ELISA plate with 100 microliters of the anti-OVA antibody per well, and serially dilute anti-DEC-205/OVA at a 1:2 ratio in blocking buffer, to obtain dilutions from 6 micrograms per milliliter to 93.8 nanograms per milliliter.

Add 100 microliters of each dilution to the appropriate wells of the antibody-coated plate for a one-hour incubation at room temperature. At the end of the incubation, add 100 microliters of horseradish peroxidase-conjugated antibody against the anti-DEC-205/OVA conjugate to the plate for a one-hour incubation at room temperature.

Then, add 50 microliters of horseradish peroxidase substrate to each well, to allow analysis of the color reaction by spectrophotometry.