This article discusses the process of eliciting an immune response through the interaction of antigens and antibodies on dendritic cells. It details the methodology for verifying the efficiency of antibody-antigen conjugation using ELISA.
To elicit an immune response, antigens bind to the antibodies that are specific for the endocytic receptors present on the surface of dendritic cells, or DCs — the antigen-presenting cells — which help in the uptake of antigens.
The formation of an antibody-antigen complex is a crucial step and can be accomplished in vitro by chemically coupling the antigen to a DC-targeting monoclonal antibody.
To verify the efficiency of the conjugation process, begin with an ELISA plate precoated with the captured antibodies. Treat the plate with a blocking solution to prevent the non-specific binding of the antigens to the coated surface.
Add chemically crosslinked, antibody-antigen conjugates to the plate, and incubate. The specific epitope of the antigen interacts with the captured antibody's paratope forming an antibody-antigen-antibody complex.
Next, overlay the plate with a third set of antibodies linked to an enzyme. These antibodies bind specifically to the primary antibodies present in the conjugate, enabling its detection.
Add a buffer containing the chromogenic substrate to visualize the conjugates. The enzyme attached to the antibody reacts with the substrate, generating the colored product.
Treat the plate with an acidified inactivation solution, which inactivates the enzyme and terminates the reaction. Analyze the plate spectrophotometrically. The presence of a colored solution verifies the presence of antibody-antigen conjugates and confirms successful conjugation.
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.
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