COMPETITION ELISA
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 µM) in PBS [total volume of
each reaction: 100 µl].
- After 30 minutes incubation at room temperature, apply 90 µl of
the reaction mixtures to the wells of the first antigen-coated
microtitre plate (perform the experiment in duplicate or triplicate),
containing 30 µl 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]
