04 - Put the M into STEM: Quantitative Techniques for Biotechnology
PERFORMING AN ELISA
The following reagents are necessary to perform the ELISA. • Test Samples. For many medical tests, this will be blood, or urine, or saliva from the patient. This experiment is a simulation – there are no live virus or human samples involved. • Control Samples. These are lab prepared samples that we know how the assay should react. A positive control will give a positive result, and a negative control a negative result. If our controls fail, or if our assay does not give the correct results, it lets us know the experiment is invalid. • Primary Antibody. An antibody generated to recognize and bind directly to the antigen of interest. So, for a COVID test, we’re looking for antibodies to SARS-CoV-2, the virus that causes COVID-19. • Enzyme-linked Secondary Antibody. This antibody binds to the primary antibody and lets us detects the presence of our antigen in patent samples. It is connected to an enzyme that turns over a particular substrate, producing either color or light, which we can visualize. • Buffer. This is generally used to dilute antibodies and wash wells. • Microtiter plate. This is a thin piece of plastic with multiple little wells, each of which will provide a separate mini test tube that is in parallel with the other reactions. • Pipette. A lab device that allow us to transfer samples and reagents into the wells. There are a few ways we can do this. One is a plastic transfer pipet. The other is a micropipette, which is a precision tool used to make measurements of very small samples. To simplify today’s demonstration, I’m going to use the transfer pipets. So, we can run this experiment without any special equipment.
The basic ELISA follows a few simple steps:
1. The sample is added to the wells of the microtiter plate, where it adheres to the plastic through hydrophobic and electrostatic interactions. If the sample includes antigens, they will adhere to the plate. 2. After washing away any excess sample, the wells are “blocked” with a protein-containing buffer to prevent non-specific interactions. 3. The primary antibody is added to the wells, where it recognizes the antigen and binds through electrostatic interactions. This forms the antibody-antigen complex. Excess antibody is washed out of the wells.
A.
B.
Product
Substrate
ADD SAMPLE
ANTIGEN BINDS BIND PRIMARY
HRP Enzyme Secondary Antibody
Primary Antibody
Antigen
BIND SECONDARY ADD SUBSTRATE
READ
Figure 2: Optimized ELISA workflow
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