ELISA Test Step-by-Step Guide
Hands-on ELISA testing is a great way to make molecular biology relevant to your students.
Two of the biggest epidemics in the last 40 years, epidemics all of your students will know about, have been the human immunodeficiency virus that causes AIDS, and the SARS-CoVirus 2, more commonly known as COVID-19. The ELISA amino acid assay has proven just as useful for testing for COVID-19 as it was in testing for HIV 40 years ago.
Your students may also be interested in knowing that ELISA has been adapted for home pregnancy tests. In the clinic, it’s used to test for prostate cancer and the antibodies generated in lupus, rheumatoid arthritis, and other autoimmune diseases they will encounter further in their studies.
Students will know what ELISA is used for, even if they couldn’t explain what the technique is, as in this sample answer for the study question, “What is ELISA?”
ELISA is a powerful method for detecting specific proteins in complex protein mixtures. It continues to be used more and more in medicine to detect antibodies produced in response to the presence of pathogens like those produced by HIV and the novel coronavirus. Just in the last few months of 2020, advances in ELISA testing have made a “spit test” for COVID-19 possible. A commercially available, serum-based enzyme-linked immunosorbent assay has made rapid testing for the virus possible with 100% specificity, in much the same vein as rapid ELISA tests for HIV that have been around for several years.
Teachers can motivate their students to pay attention with ELISA. And the steps for carrying out Modern Biology’s IND-03: ELISA immunoassay couldn’t be easier. In this experiment, students use a tried-and-true procedure to bind an antigen to the walls of the microplate, bind an enzyme-linked antibody to the antigen, and then use that enzyme to detect the presence of the antigen, indicating infection.
Polymerase Chain Reaction (PCR) testing has also been used as a detection method for COVID. Amplification of viral RNA makes it much easier to see the small quantities of DNA required for testing. Both PCR and ELISA are rapid tests your students can accomplish quickly, within a typical lab period. Later in your course, your students can work with the lab materials supplied by IND-07: Amplification of a Hemoglobin Gene by PCR to amplify a few nanograms of DNA into a few milligrams of DNA in just a few hours, and then IIND-21: Identifying Genomic and Plasmid DNA Sequences in E. coli by Colony PCR to amplify specific DNA sequences without DNA purification.
Steps for This Experiment
It is useful to explain to students that direct ELISA in medical labs is typically performed in a 96-well microtiter plate by a chemical reaction that results in the covalent attachment of antigen, which is most often a protein, through its free amino groups. The test then involves probing each coated well with an antibody specific for the target antigen, probing that bound antibody with a second antibody specific for the stable region of the first antibody, and using a variety of detection methods. Different enzymes result in different color changes that can be read automatically with a scanner in some lab settings, or in the case of this experiment, can be seen with the naked eye.
What’s involved in teaching these labs? Let’s consider the easy steps for teaching Modern Biology’s IND-03: ELISA immunoassay.
- In this lab, your students will be using a goat-anti-rabbit IgG antibody to study the specificity of an antibody antigen reaction, in order to compare the binding of this antibody serum to IgGs in serum from chicken, cow, horse, and rabbit. To aid in this, the anti-rabbit IgG has been covalently linked to peroxidase, which will catalyze a color producing reaction, so that your students can study the results without the aid of specialized equipment.
- Assign your students to work in pairs. The ELISA Immunoassay kit will include the reagents and testing materials necessary for the experiment to be performed twice by 8 groups, or once by 16 groups of students. Make sure that each group has read the guide included with your supplies before commencing the lab, and have all of the included materials, as outlined in the experiment guide.
- Your student groups will need to number the provided microtitration plate. These plates usually come in a size to accommodate 96 wells over their surface, but for the purposes of this experiment, they will be provided with a 1/4 section of a standard plate, resulting in a plate that contains 24 wells. These plates should be labeled so that across the top of the grid reads the numbers one through six, and down the left-hand side are the letters A to D for clear tracking of the experiments.
- Next, they will need to proceed with the absorption of the antigen, which will require the students to perform serial, ten-fold dilutions of the sera, after which the plate will need be left undisturbed for twenty minutes to provide adequate time for the proteins to be adsorbed to the well surfaces. Afterwards, your students will use TBS-gelatin to block sites on the plastic that are not bound to serum proteins. Once this process is complete, they will use pipettes to carefully discard the liquids in each well, preparing them for the antibody reaction.
- From here, your students will add the goat anti-rabbit IgG peroxidase to each well and rotate the plate to ensure that all surfaces at the bottom of each well comes into appropriate contact with the anti-body solution. The plate will again need to be left alone for 20 minutes, so that the antibody can properly bind to the immobilized IgG. After this, TBS-gelatin will again be utilized, and the excess solution discarded as done previously.
- The final step for your students will be to use the provided color development solution in each well, and then wait 15 minutes for change to occur. After this, the microtitration plate can be placed over a blank piece of white paper so that the intensity of the blue and yellow product can be examined and compared.
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