COMPETITION ELISA
The ELISA protocols for detection of the antibody binding to anantigen-coated microtitre plate are standard laboratory techniquesand will not be described here. We will just mention that mostrecombinant antibody fragments are typically detected usingmonoclonal antibodies directed against a peptidic tag engineered atthe C-terminal extremity of the recombinant antibody. In certainexperimental conditions, such peptidic tags may undergo proteolyticcleavage, thereby lowering the sensitivity of antibody detection.Reagents that bind to the antibody molecule without impairingantigen binding (e.g., protein A or protein L) may therefore bepreferable. Alternatively, the experimental scheme described belowcan be performed in a similar fashion, using radiolabeledantibodies and radioacitve detection of antibody-mediated antigenbinding. The concentration of purified antibody preparations istypically determined spectrophotometrically (1 mg/ml antibodysolution absorbs 1.4 absorption units at 280 nm). If necessary (forexample when using supernatants), the concentration of activeantibody can be detected with a straightforward ELISA adaptation ofthe protocol mentioned above for the determination of antibodyconcentration by band-shift assay.
1. Coat with antigen (in identical fashion) an appropriatenumber of wells of two microtitre plates. Preblock the wells with3% MPBS for 2 hours at room temperature, then wash with PBS.
2. In parallel tubes, incubate an antibody solution (atconcentrations below Kd, e.g. 0.5 nM) with increasingconcentrations of antigen (e.g., ranging between 0.1 nM and 1 µM)in PBS [total volume of each reaction: 100 µl].
3. After 30 minutes incubation at room temperature, apply90 µl of the reaction mixtures to the wells of the firstantigen-coated microtitre plate (perform the experiment induplicate or triplicate), containing 30 µl 10% MPBS.
4. Incubate the reaction mixture on the antigen-coatedplate for a suitably short time (e.g., 10 min.).
5. After incubation, transfer the reaction mixtures to thesecond antigen-coated microtitre plate. The ELISA assay using thissecond plate will now be performed exactly as for the firstmicrotitre plate. The purpose of the second ELISA assay is to checkthat only a small fraction of the free antibody is captured on thefirst microtitre plate and, therefore, no readjustment of theequilibrium occurred during the first capture step.
6. Wash extensively the first ELISA plate and perform theremaining steps of an ELISA procedure, aimed at the determinationof the antibody binding to the coated antigen.
7. Develop the ELISA with a suitable chromogenic,fluorogenic or chemiluminescent substrate, and measure theindividual wells with an appropriate ELISA plate reader. Thehighest ELISA signal should be observed at low concentrations ofantigen. No ELISA signal should be observed at high concentrationsof antigen. The concentration of antigen at which the half-maximalELISA signal is detected corresponds to the dissociation constantKd. Alternatively, the Kd value can be obtained by fitting theELISA signal of the individual wells to the equation: Kd =[A][B]/[AB] .