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