ELISA (Video transcript)

ELISA, or Enzyme-Linked Immunosorbent Assay is one of the most commonly-used techniques in immunology.

ELISA is used to determine if a patient has been exposed to a disease. It does this by detecting the presence of disease-specific antibodies in a patient’s serum. This is the basis of current COVID-19 antibody tests.

We will conduct this test using a strip-well tube, which has spaces – or “wells” as we call them, for twelve samples. The experiment could be conducted in a 96-well plate if we had more samples.

The wells are made of plastic – which has a charged surface.

The charged surface allows proteins, like antigens or antibodies to stick to the surface of the wells in a non-specific way.

REAGENTS:

In this experiment, we will use the following reagents:

A disease-specific antigen - which we will use to coat the to the wells of the tubes. This will be the basis of our experiment.

We will use four serum samples:
A positive control that contains the disease specific antibody
A negative control that lacks these antibodies
And two patient sera – whose disease status is unknown - which we will label A and B

We also will use a secondary antibody – which has an enzyme, horse-radish peroxidase – attached.

We will also use a substrate that the enzyme horse-radish peroxidase can act upon. The substrate is called tetramethylbenzadine, or TMB.

Finally, we have a wash solution – which consists of normal saline and a detergent.

We will perform these experiments in triplicate – using three samples per experimental condition to increase the likelihood that our results are valid.

We begin by labelling our strip well tubes with plusses for the positive control wells where we will add a verified disease antibody, With minus for the negative control wells in which no disease antibodies will be added and with As and Bs for the wells where we will add the two patient serum samples.

We start by adding a purified disease-specific antigen to the wells. We dispose our pipet tip in the sharp waste container and allow 5 minutes for the antigen to adhere (stick) to the wells. This forms the basis of our ELISA test.

After 5 minutes have elapsed we will wash the wells by adding wash buffer, using a 1-ml plastic transfer pipette.

We will wash the wells between all steps in the ELISA procedure. Washing, simply involves rinsing the wells with a buffer solution, several times. Our buffer contains a blocking agent – a detergent, which will bind to any charged sites remaining on the plastic wells. This critical step helps to avoid non-specific interactions that might produce false-positive results. Washing also removes any antigens that did not adhere to the wells.

Once all the wells are filled with wash buffer, we empty them by rapidly inverting the strip-well tubes over a waste dish.

We then firmly rap the tubes on a clean paper towel.

The wash step is repeated three times.

STEP 2: We will now add the positive and negative controls to their labeled wells.

Note that we will change our pipet tip between each of these reagents so we don’t cause any cross contamination between our wells. Such cross contamination could lead to false-positive results.We now add the patient sera to their labeled wells.

We allow five minutes for any disease-specific antibodies to adhere to the antigens that are fixed in the wells. In our ELISA procedure, the disease-specific antibody is termed the primary antibody.

We will then wash the wells 3 times with buffer to remove any unbound primary antibodies using the procedure previously described.

STEP 3: We now add the secondary antibody to the wells. The secondary antibody binds to the Fc portion of the primary antibody. The secondary antibody has an enzyme attached to it called Horse-radish peroxidase.

We will allow five minutes for the secondary antibody to bind to any primary antibodies that may be present in the wells.

We will wash the wells 3 times with wash buffer to remove any unbound secondary antibody, using the procedure previously described.

STEP 4: Finally, we will add substrate for the enzyme horse-radish peroxidase. This substrate is called TMB or Tetramethylbenzidine.

We will allow five minutes for the reaction to proceed, after which, we can read our results.

TMB is colorless, but in the presence of the enzyme horse-radish peroxidase it will be cleaved and produce a blue color. Wells that remain colorless are negative and wells that turn blue are positive. The presence of the blue color confirms that there was a specific interaction between the disease-specific antigen we affixed to the wells and antibodies in our patient serum.

In more sophisticated applications of ELISA we could quantify the amount of blue product in the wells to measure the amount of antibody in the serum.