Day 5 - Oh No, Another Medical Acronym: ELISA

ELISA stands for enzyme-linked immunosorbant assay.  Okay, so what does that really mean?  Basically, it is a test that detects antibodies.  If the test is positive, there are antibodies and that person has been exposed to a disease.  ELISA techniques are also used in pregnancy tests.


Answers to the Pre-Lab Focus Questions:
1.  The immune system protects us from disease by making antibodies against the disease's antigen.
2.  Doctor's use vaccines to use the immune system to protect us from disease.
3.  HIV is an example of a disease that attacks the human immune system.
4.  T-cell defects and autoimmune diseases can prevent the immune system from working properly.
5.  It is important to be able to detect antibodies in people who don't appear sick to be able to treat an illness before it takes over their bodies.  This can also help identify carriers of diseases.
6.  ELISA stands for enzyme-linked immunosorbant assay.
7.  The enzymes are used in this immunoassay to indicate the presence of antibodies.  The enzymes conjugate to the secondary antibody to produce a blue color.  Since the secondary antibody can only remain present in the microplate wells by binding to the antibody, which has bound to the antigen, the presence of the secondary antibody indicates the presence of antibodies.  Thus, the enzymes, which both bind to the secondary antibody and produce a blue color when doing so, indicate a positive result for antibodies.
8.  We need to assay positive and negative control samples as well as our experimental samples to make sure we don't get a false positive or a false negative result.

ELISA test materials:
Yellow tube of patient #10 serum sample
Green tube of purified antigen
Violet tube of positive control
Blue tube of negative control
Orange tube of secondary antibody
Brown tube of substrate
12-well microplate strip, of which we only used 9 wells
Pipet that transfers 50 microliters
Yellow tips
Transfer pipet for wash buffer
70-80 ml wash buffer in beaker
Large stack of paper towels
Marking pencil


Steps of an ELISA antibody test:


1.  Label the outsides of the microplate wells: "+" on three of the wells for the positive control, "-" on three of the wells for the negative control, and "10" on three of the wells for the serum sample.

2.  Using the pipet with a sterile, disposable tip, add 50 microliters of a purified antigen to each of 9 wells of a microplate strip.  Let it sit for 5 minutes, so that the antigens can bind to the walls of the strip.  This happens through hydrophobic interactions.  Dispose of the pipet tip in the biohazard waste bag.

3.  Wash out the excess purified antigen by dumping the fluid onto paper towels, filling the wells with a wash solution, and dumping the wash solution onto paper towels.  Dispose of the paper towels in the biohazard waste bag. 


4.  Add 50 microliters of the serum samples, positive control, and negative control to each of the appropriately labeled microplate wells.  Get a new, sterile pipet tip after every different solution.  We used patient serum sample #10.  Let it sit for 5 minutes to give it time to bind to the antibodies.  Dispose of the pipet tips in the biohazard waste bag.


5.  Wash out the excess purified antigen by dumping the fluid onto paper towels, filling the wells with a wash solution, and dumping the wash solution onto paper towels.  Dispose of the paper towels in the biohazard waste bag. 


6.  Add 50 microliters of the secondary antibody to each of the wells.  Let it sit for 5 minutes to give it time to bind to the antibodies.  Dispose of the pipet tip in the biohazard waste bag.


7.  Wash out the wells as described above, in step 5, but do it TWICE.

8.  Add 50 microliters of the enzyme substrate to each of the wells.  Let it sit for 5 minutes to give it time to bind to the antibodies.  Observe for the appearance of a blue color.  This indicates a positive diagnosis.  Dispose of the pipet tip in the biohazard waste bag.  The "+" wells should turn blue, and the "-" wells should remain colorless.

9.  Dispose of the microplate wells in the biohazard waste bag.




Result:  Our sample was a positive result.  The two "+" wells turned blue, the three "-" wells remained colorless, and the three "10" wells with our sample turned blue.  Thus, patient serum sample #10


Answers to the Post-Lab Focus Questions:
1.  Our serum did have antibodies to the disease, because it turned blue, thus indicating a positive result.
2.  If we tested positive for antibodies, it means that we have been exposed to the disease.
3.  There could be several reasons for a positive test when we do not actually have the disease: we are carriers, we are immune even though we are exposed, or the ELISA test was faulty or contaminated.
4.  We assayed our sample in triplicate to ensure that the results were accurate.  Multiple positive results ensured the reliability of the results.
5.  When we added the positive serum samples to the wells, the serum antibodies conjugated with the antigens on the sides of the wells.  If the serum had been negative, there would be no antibodies to stick to the antigens on the sides of the wells.
6.  We needed to wash the wells after every step to get rid of excess antigens or antibodies that would remain in solution and mess up results.  We wanted only the antigens that adhered to the wells' walls and the antibodies that conjugated with them to remain.
7.  When we added the secondary antibody to our positive serum sample, it adhered to the antigen-antibody complex that had already formed between the antigen on the well's walls and the primary antibody.  If our serum had been negative, the secondary antibody would not have adhered to anything, because it could only adhere to the antigen-antibody complex.  Then antigen-antibody complex would not be present if the serum were negative, since there would be no primary antibodies.  Thus, with a negative serum sample, the secondary antibody would just be washed away with the wash buffer.
8.  At our local pharmacy, we can buy pregnancy tests, illegal drug tests, and air quality tests, among others.