The ELISA protocols for detection of the antibody binding to an antigen-coated microtitre plate are standard laboratory techniques and will not be described here. We will just mention that most recombinant antibody fragments are typically detected using monoclonal antibodies directed against a peptidic tag engineered at the C-terminal extremity of the recombinant antibody. In certain experimental conditions, such peptidic tags may undergo proteolytic cleavage, thereby lowering the sensitivity of antibody detection. Reagents that bind to the antibody molecule without impairing antigen binding (e.g., protein A or protein L) may therefore be preferable. Alternatively, the experimental scheme described below can be performed in a similar fashion, using radiolabeled antibodies and radioacitve detection of antibody-mediated antigen binding. The concentration of purified antibody preparations is typically determined spectrophotometrically (1 mg/ml antibody solution absorbs 1.4 absorption units at 280 nm). If necessary (for example when using supernatants), the concentration of active antibody can be detected with a straightforward ELISA adaptation of the protocol mentioned above for the determination of antibody concentration by band-shift assay.
- Coat with antigen (in identical fashion) an appropriate number of wells of two microtitre plates. Preblock the wells with 3% MPBS for 2 hours at room temperature, then wash with PBS.
- In parallel tubes, incubate an antibody solution (at concentrations below Kd, e.g. 0.5 nM) with increasing concentrations of antigen (e.g., ranging between 0.1 nM and 1 ÁM) in PBS [total volume of each reaction: 100 Ál].
- After 30 minutes incubation at room temperature, apply 90 Ál of the reaction mixtures to the wells of the first antigen-coated microtitre plate (perform the experiment in duplicate or triplicate), containing 30 Ál 10% MPBS.
- Incubate the reaction mixture on the antigen-coated plate for a suitably short time (e.g., 10 min.).
- After incubation, transfer the reaction mixtures to the second antigen-coated microtitre plate. The ELISA assay using this second plate will now be performed exactly as for the first microtitre plate. The purpose of the second ELISA assay is to check that only a small fraction of the free antibody is captured on the first microtitre plate and, therefore, no readjustment of the equilibrium occurred during the first capture step.
- Wash extensively the first ELISA plate and perform the remaining steps of an ELISA procedure, aimed at the determination of the antibody binding to the coated antigen.
- Develop the ELISA with a suitable chromogenic, fluorogenic or chemiluminescent substrate, and measure the individual wells with an appropriate ELISA plate reader. The highest ELISA signal should be observed at low concentrations of antigen. No ELISA signal should be observed at high concentrations of antigen. The concentration of antigen at which the half-maximal ELISA signal is detected corresponds to the dissociation constant Kd. Alternatively, the Kd value can be obtained by fitting the ELISA signal of the individual wells to the equation: Kd = [A][B]/[AB] .