ELISA
ELISA (Enzyme-linked immunosorbent assay) is an immunoassay technique that detects and measures the amount of antigen or antibody using antigen-antibody interaction in vitro. In general, 96 microwell plates are used and there are direct ELISA, indirect ELISA, and sandwich ELISA methods.

Direct ELISA involves directly labeling the antibody. Direct ELISA is measuring the amount of antigen by the value of fluorescent from binding labeled antibody to the coated samples containing the target antigen in microwell plates. This method skips the second antibody binding procedure, making the experiment comparably faster and eliminates issues with cross-reactivity between reactants in the sample and the second antibody. However, because this method involves directly labeling the antibody, it creates problems with time and cost. Moreover, it also lacks the signal amplification option through the use of second antibody. On the other hand, indirect ELISA uses labeling of the secondary antibody, which is then bound to the primary antibody, allowing signal amplification and has the benefit of detecting signals in a small antigen sample. For this reason, the indirect method is used more often than the direct method.

Among the ELISA kits available, sandwich ELISA is widely used. This method uses purified antigen standard, which allows for the measurement of the antigen concentration in an unknown sample both efficiently and accurately. The two types of antibodies used must bind to two separate epitopes without overlap. These antibodies can be monoclonal antibodies that detect individual sites or polyclonal antibodies from a single batch that have been affinity purified.

Bind the capture antibodies to the bottom of the plate well and add the antigen to form a complex with the antibody. Remove the components that did not bind and bind detection antibody against the antigen’s another epitope to the antigen and add the labeled secondary antibody to bind to the detection antibody. This method has the added benefit of being extremely specific and simple as the antigen does not need to be purified. However, this method cannot be used will all antibodies and require two antibodies “matched pair” that can detect two separate epitopes.

Here, we will be discussing Indirect ELISA and Sandwich ELISA methods.

Indirect ELISA
  1. Dilute the antigen to 250ng/50µl with coating buffer and aliquot 50µ in individual wells. Let it coat at 4℃ overnight or at 37℃ for 2 hours.
  2. Discard the coating solution.
  3. Pipette 200µl 2% Skim milk/TBS-T and block at 37℃ for 1 hour.
  4. Wash once with TBS-T and pipette 50µl primary antibody into individual wells and let it react at 37℃for 2 hours.
  5. Wash 3 times with TBS-T and dilute the secondary antibody in 1:5,000 ratio and pipette 50µl into individual wells. Let it react for 1 hour at 37℃. (Select an secondary antibody that matches with the primary antibody such as goat anti-mouse IgG(Fc) HRP, goat anti-rabbit IgG(Fc) HRP, etc.).
  6. Wash 5 times with TBS-T and pipette 50µl OPD or TMB (color reagents) in each well. If the color changes, add stop solution (1N H2SO4) to stop the reaction and measure the O.D. value at 495nm (450nm).

Coating buffer (500mL)

0.2M Na2CO3

80mL

0.2M NaHCO3

170mL

D.W

up to 500mL

1X TBS-T(pH7.4)

10mM Tris

0.15M NaCl

0.1% Tween 20

Blocking Solution

2% Non fat dry skim milk(or 2% Bovine serum albumin) in TBS-T

Sandwich ELISA

  1. Dilute the capture antibody to 1-10µg/mL with coating buffer and aliquot 100µl into polystyrene microtiter plate. Then, coat overnight at 4℃ or for 2 hours at 37℃.
  2. Discard the coating solution and wash with 300µl TBS-T. Repeat twice.
  3. Pipette 300µl 2% Skim milk/TBS-T and block at 37℃ for 1 hour.
  4. Wash once with TBS-T and pipette 100µl diluted sample in individual wells and let it react for 2 hours at room temperature. In order to obtain precise measurement, conduct standards and blank on the same plate.
  5. Wash 3 times with 300µl TBS-T. Then, pipette 100µl of detection antibody into individual wells and let it react for 2 hours at room temperature.
  6. Wash 3 times with 300µl TBS-T and pipette 100µl conjugated antibody into individual wells. Let it react for 1-2 hours at room temperature.
    * Conjugated antibody must be diluted with blocking buffer before use.
  7. Wash 4 times with TBS-T and pipette 100µl OPD or TMB in individual wells and let it react for 15-30 minute. Then, add stop solution (2M H2SO4) to stop the reaction and measure the O.D. value at 495nm (or 450nm).
    * Draw a standard curve with the standard values from serial dilution and calculate the sample concentration from the curve.