ELISA or Enzyme-Linked ImmunoSorbent Assay
Can enzymes be used to determine if you are pregnant?
How can this plastic stick know if you are having a baby?
Human chorionic gonadotropin (HCG), is the hormone that appears in the blood and urine of pregnant women within days of fertilization. If you have this hormone your going to have a baby! How do you know if you have this hormone? Enzymes!
How Are Antibodies Made?
Scientists have learned to use the immune response of animals to make antibodies that can be used as tools to detect and diagnose diseases. The study of the immune system is called “immunology”. Animals such as chickens, goats, rabbits, and sheep can be injected with an antigen and, after a period of time, their serum will contain antibodies that specifically recognize that antigen. If the antigen was a disease agent, the antibodies can be used to develop diagnostic tests for the disease. In an immunoassay, the antibodies used to recognize antigens like disease agents are called primary antibodies; primary antibodies confer specificity to the assay.
Other kinds of antibody tools, called secondary antibodies, are made in the same way. In an immunoassay, secondary antibodies recognize and bind to the primary antibodies, which are antibodies from another species.
Secondary antibodies are prepared by injecting antibodies made in one species into another species. It turns out that antibodies from different species are different enough from each other that they will be recognized as foreign proteins and provoke an immune response. For example, to make a secondary antibody that will recognize a human primary antibody, human antibodies can be injected into an animal like a rabbit. After the rabbit mounts an immune response, the rabbit serum will contain antibodies that recognize and bind to human antibodies. The 2° antibodies used in this experiment are conjugated to the enzyme HRP which produces a blue color in the presence of its substrate, TMB. These antibody and enzyme tools are the basis for the ELISA.
Where Is ELISA Used in the Real World?
With its rapid test results, the ELISA has had a major impact on many aspects of medicine and agriculture. ELISA is used for such diverse purposes as pregnancy tests, disease detection in
people, animals, and plants, detecting illegal drug use, testing indoor air quality, and determining if food is labeled accurately. For new and emerging diseases like severe acute respiratory syndrome (SARS), one of the highest priorities of the US Centers for Disease Control (CDC) and the World Health Organization (WHO) has been to develop an ELISA that can quickly and easily verify whether patients have been exposed to the virus. Over-the-counter kits that are based on the same principles as the ELISA include home pregnancy and ovulation tests, and tests for the presence of illegal drugs like marijuana and cocaine.
Some tests give positive or negative results in a matter of minutes. For example, home pregnancy dipstick tests detect levels of human chorionic gonadotropin (hCG), a hormone that appears in the blood and urine of pregnant women within days of fertilization.
The wick area of the dipstick is coated with anti-hCG antibody labeled with a pink compound (step 1). When the strip is dipped in urine, if hCG is present it will bind to the pink antibody, and the pink hCG-antibody complex will migrate up the strip via capillary action (step 2). When the pink complex reaches the first test zone, a narrow strip containing an unlabeled,fixed anti-hCG antibody, the complex will bind and concentrate there, making a pink stripe (step 3). The
Step 1. dipsticks have a built-in control zone containing an unlabeled secondary antibody that binds unbound pink complex (present in both positive and negative results) in the second stripe (step 4). Thus, every valid Step 2. test will give a second pink stripe, but only a positive pregnancy test will give two pink stripes.
Before you conduct the real hands on lab visit these web sites
Conducting the virtual Immunology lab ELISA:
You are about to perform an ELISA or enzyme-linked immunosorbent assay, a test that detects antibodies in your blood to determine if you have been exposed to a disease or pregnant.
When you are exposed to a disease agent, your body mounts an immune response. Molecules that cause your body to mount an immune response are called antigens, and may include components of infectious agents like bacteria, viruses, and fungi. Within days, millions of antibodies — proteins that recognize the antigen and bind very tightly to it — are circulating in your bloodstream. Like magic bullets, antibodies seek out and attach themselves to their target antigens, flagging the invaders for destruction by other cells of the immune system. In fact, antibodies make up to 15% of your total blood serum protein. Antibodies are very specific; each antibody recognizes only a single antigen.
Step by Step:
1. Label your 12-well strip. On each strip label the first 3 wells with a “+” for the positive controls and the next 3 wells with a “–” for the negative controls
3. Use a fresh pipet tip to transfer 50 μl of the positive control (+) into the three “+” wells.
4. Use a fresh pipet tip to transfer 50 μl of the negative control (–) into the three “–” wells.
5. Transfer 50 μl of each of your team’s samples into the appropriately initialed three wells, using a fresh pipet tip for each sample.
6. Wait 5 minutes while the proteins in the samples bind to the plastic wells.
a. Tip the microplate strip upside down onto paper towels, and gently tap the strip a few times upside down. Make sure to avoid splashing samples back into wells.
b. Discard the top paper towel.
c. Use your transfer pipet to fill each well with wash buffer, taking care not to spill over into neighboring wells.
Note: the same transfer pipet is used for all washing steps.
d. Tip the microplate strip upside down onto the paper towels and tap.
e. Discard the top 2–3 paper towels.
8. Repeat wash step 7.
9. Use a fresh pipet tip to transfer 50 μl of primary antibody into all 12 wells of the microplate strip.
10. Wait 5 minutes for the antibodies to bind to their targets.
11. Wash the unbound primary antibody out of the wells by repeating all of wash two times. So this means wash it twice.
12. Use a fresh pipet tip to transfer 50 μl of secondary antibody into all 12 wells of the microplate strip.
13. Wait 5 minutes for the antibodies to bind to their targets.
14. Wash the unbound secondary antibody out of the wells by repeating wash step three times. So this means wash it three times.
15. Use a fresh pipet tip to transfer 50 μl of enzyme substrate into all 12 wells of the microplate strip.
16. Wait 5 minutes. Observe and record the results.
If the primary antibody (hCG) was present in your serum sample, the wells will turn blue. This is a positive diagnosis. If the wells remain colorless, the primary antibody was not present in your serum sample, and the diagnosis is negative.
1. Did both student serums have levels of human chorionic gonadotropin (hCG), a hormone that appears in the blood and urine of pregnant women?
2. If you tested positive for hCG, does this mean that you are pregnant?
3. What reasons could there be for a positive test and negative test in the strip?
4. Why did you test your samples in triplicate?
5. When you added serum samples to the wells, what happened to the serum antibodies if the sample was positive? What if it was negative?
6. Why did you need to wash the wells after every step?
7. When you added secondary antibody, what happened if your serum sample was positive? What if it was negative?
8. What antibody-based tests can you buy at your local pharmacy?
After completing the experiment. You and your team mates will construct an instructive iMovie that will describe that methods of the ELISA experiment. It could include still images as well as video of the procedure. You and you team will narrate the movie and it then will be saved to You Tube so that future students can view this instructional video before they do their experiment.