Biology

BIO 102 Lab 04: ELISA and Immunology

Instructions: Submit, pages 6 and 8 of this document. Print, complete both lab activities and answer the questions. Scan your lab pages using the free phone app AdobeScan, and upload your PDF to Canvas. Please be sure to write your name on your first page of work.

Background

The human immune system contains several layers of defense, but before explaining them in depth, we must review some terminology. Starting with the answer sheet in this lab, and using your book, fill in the definitions from question #1 before continuing to read the remainder of this lab. You may also find it helpful to review diagrams in the book relating to antigen binding.

The broadest part of the immune system responds in the same manner to every antigen (ie., invader) it encounters. It is called the nonspecific immune system. It includes things like our skin, mucus membranes, ear wax, stomach acid, sweat, tears, vaginal secretions, antimicrobial proteins, and internal cellular defenses. The nonspecific immune system will mount the same response regardless of the nature of the antigen. It has no memory and doesn’t have the ability to recognize that a specific defense may have been ineffective against a pathogen in the past. The nonspecific immune system’s primary function is to prevent us from getting sick by attacking anything foreign and if that fails, to contain the pathogen until our adaptive immune system is activated.

The adaptive immune system “studies” each pathogen and learns how to effectively kill it. It also has memory of the pathogens it has faced in the past and will improve its effectiveness every time it encounters the same pathogen, meaning that the person doesn’t get sick from later encounters with the same pathogen, provided that pathogen is recognized. It includes two types of white blood cells (all types of white blood cells are called leukocytes), the B & T cells (B & T cells are also called B lymphocytes and T lymphocytes respectively). The B cells will begin releasing antibodies into the interstitial fluid and blood after they encounter a specific antigen. Each antibody is specific not for just that antigen, but a single epitope on the surface of the antigen. Once a B cell begins producing antibodies, they can remain present in the body for years. Vaccines stimulate our B cells into making antibodies so we don’t get sick if we encounter certain viruses. Some vaccines only require a few administrations and the immunity lasts for life, while others need to be reminded about that antigen, hence the need for vaccine “boosters.”

In this lab, we will investigate an important test called ELISA. ELISA stands for enzyme linked immunosorbent assay. It is used in many different ways, from diagnostic lab tests used by doctors to measure a patient’s exposure to a virus, to research lab investigations that and separate a specific protein among many. Though very specific and powerful, it can be easily performed and is a commonly found experiment in college biology courses. ELISA functions detect the presence of antibodies or a pathogen. ELISA has several varieties, but two of the most common are: direct ELISA, which uses artificially made antibodies to bind to the antigen, while indirect ELISA uses the person’s antibodies against a specific pathogen to determine if a person has encountered the disease before. In indirect ELISA the artificial antibodies bind to a person’s antibodies against a specific disease. In both direct and indirect ELISA, the artificial antibodies are engineered to change color when added to a special chemical. Direct ELISA is explained in the image on the next page.

Direct ELISA

Image from Wikipedia.org, Reteived 6/12/19

BIO 102 Lab 04: ELISA and Immunology 7

In indirect ELISA parts of the suspected pathogen are anchored to the sides of a well plate (a well plate is a small circular clear plastic dish). The person’s blood serum is allowed to sit in the plate long enough for any antibodies present (if the person has encountered that pathogen recently) to stick to the antigen that is part of the well plate. In the diagram to the left, the middle representation shows a green antibody attached to a purple enzyme cluster; the antibody is bound to the red viral antigen. The serum is then drained away, but the attached antibodies will remain, stuck. Then, artificial antibodies are added, which will only stick if the person’s antibodies are attached to the antigen in the well plate. A color change will occur if artificial antibodies remain stuck (the bottom diagram to the left shows the blue colored molecules which contain the dye and the artificial antibody). Thus, a clear solution means the person is not sick and a color change means the person has encountered the disease before.

The Live Lab ELISA Procedure (Performed When Meeting in Lab) – READ ONLY

Read through the procedure below. This document includes sample ELISA data. You will use these results to answer the questions.

Every person gets 1 “serum” sample tube (we will avoid using real human bodily fluids in lab). One of these samples contains the antibodies against a sexually transmitted or blood borne disease.

Everyone will then share “fluids” three times. DO NOT start a round of sharing until instructed to do so!!! Each fluid sharing will be done by transferring ½ of the contents from one person’s tube into the other person’s tube. The cap off the receiving tube & swirl, then ½ of the receiving tube’s fluid is transferred back to the original tube, thus each tube is roughly ½ original and ½ new sample. Every time you change serum solutions you need to change pipette tips or you will contaminate the samples.

Record your tube # here____________________

Round of fluid transfer Partner’s name Partner’s tube #
1
2
3

Again, no one should have a 2nd (or 3rd) partner until their instructor tells them to find one. At each round you only share with 1 other person!

Everyone should record their information on the spreadsheet on the projector/board while doing the ELISA test.

Now that everyone has done the fluid transfer it is time to find out who has the disease & see if you can figure out where the disease started.

Using a pen, mark one row of the well plate with +, the second with -, and then each person at your table gets a row with their initials. Each row should have 3 wells.

1. Transfer 1/3 of your serum sample into each of the well plates with your initials. The positive control goes into the 3 wells with the “+”, and the negative control goes into the plate with the “-”.

2. Allow the samples to remain in the well plate depressions for 3 minutes

3. Empty the plate in the sink and wash the sample depressions 3 times with ELISA wash solution, tap plate against a paper towel on the counter each time. Be careful not to allow fluid to spill from one well to another while washing & rinsing!

4. Add antibody (AB) to the washed out sample depressions and allow to sit for 3 minutes

5. Repeat #2

6. Add color substrate (CS) and allow to sit for 3-5 minutes

7. Positive reaction is blue, negative reaction is clear

8. Record who was “sick” on the projector/board.

9. Determine from the sharing & who was sick, what couple started off the disease.

10. Answer questions 2-5 on the answer sheet.

Sample ELISA results:

Each of the circular discs is a well, this would be called a 24-well plate because it contains 24 wells and a different sample can be run in each well. The blue color changes indicates a positive result, the clear (ie., see through) indicates a negative result.

Use the data in this table to answer the ELISA questions on the worksheet.

Sample Class ELISA Results Data Table
Patient’s Sample # Patient’s Name + or – First Partner’s # Second Partner’s # Third Partner’s #
1 Cary 5 14 24
2 Chris 4 12 15
3 Ryan + 6 10 14
4 Bo 2 9 23
5 Tim 1 11 13
6 Lei + 3 7 19
7 Vashti + 10 6 22
8 Geeta 11 13 21
9 Vijaya 12 4 11
10 Xin + 7 3 17
11 Jacob 8 5 9
12 Fred 9 2 16
13 Diane 14 8 5
14 Tiffany + 13 1 3
15 Thy 16 24 2
16 Yukti 15 18 12
17 Mary + 18 23 10
18 Michel 17 16 20
19 Vincent + 24 22 6
20 Yan yan 23 21 18
21 Beatrice 22 20 8
22 Swati + 21 19 7
23 Kirsten 20 17 4
24 Alex 19 15 1

*Disclaimer: the names are randomly chosen from instructor names at NVCC, spring 2020 semester. No matches or +/- results are actually true, all data was randomly selected and assigned.

ELISA Worksheet

1) Define the following terms dealing with the immune system

Antigen: _________________________________________

Pathogen: ________________________________________

Epitope: _________________________________________

2) Why did you run both positive & negative controls?

3) What is a false positive? How do you think one could come about using a test like the ELISA?

4) At the end of 3 rounds of fluid transfers, what percentage of the class had the disease?

5) You can narrow the initial outbreak down to 2 people, who are they?

6) Describe how an organization like the CDC could use results like those you obtained to track down “patient zero” for a disease outbreak.

7) Do you think this method shows a direct or indirect ELISA procedure? Why?

8) With an indirect ELISA, it tests not for the antigen, but antibodies against the antigen, does a positive test then mean the person currently has the disease in question? If not, what does a positive

Immunity & Vaccinations

Background

Vaccinations are given to stimulate the production of antibodies without the animal actually getting the sick with a disease. Vaccinations come in many different forms, but the most common types are inactivated viruses or bacteria (also called attenuated bacteria or viruses). Attenuated means the pathogen has been disabled so it can’t cause the full-blown illness, but is still figuratively “alive” so the animal may get a very mild form of the illness. You can think of it as a recognizable, but harmless form of the pathogen. Inactivated vaccines, which are “dead,” often contain fragments of the original pathogen which are attached to another particle to attract B cells. As the body removes the attenuated (= inactivated) pathogen, the adaptive immune system learns, with an eye toward remembering this encounter with the “pathogen” (note that your immune system does not “know” that it only encountered a harmless form of the pathogen). It remembers by making antibodies against that disease which should prevent future infections.

In the last decade, a movement has started in the U.S., supported by Hollywood celebrities among others, who claim that vaccines are unnecessary. Some even claim that vaccines even cause autism. The claim that vaccines cause autism has been conclusively proven false through many studies conducted all over the world. One initial study, which caused the initial concern, was filled with many mistakes and after close examination, was determined to be invalid by many different scientists. See https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2831678/ and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3136032/ for more information on this.

As to the first claim that vaccines are unnecessary against certain diseases, we will closely examine exactly how vaccines protect many people not just those who receive the vaccine.

In every population there will be people whose immune system is weakened. It could be weakened for many reasons: genetic (missing or damaged genes); the person is elderly, very young, allergic; or undergoing chemo or radiation therapy for cancer (which can cripple the immune system). These people often can’t receive vaccinations or if given, they won’t produce antibodies or sufficient numbers of antibodies to prevent them from getting sick.

Procedure

Visit https://fred.publichealth.pitt.edu/measles

This is a program that simulates the spread of the measles. On the left side of the screen will assume that only 80% of the people are vaccinated. The right side will assume that 95% of the people are vaccinated.

1. Select District of Columbia & for the city, select Washington D.C. Answer questions 1 & 2 about this simulation.

2. Now change the state to North Carolina. For the city select Hickory. Run the simulation again. Answer question 4 on the answer sheet.

3. Now change the state to New York & the city to New York. Run the simulation again. Answer question 5 on the answer sheet.

Disease Spread Simulation Answer Sheet

1) How long did it take for the disease to disappear on the vaccinated side for D.C.?

2) Make a hypothesis on why the 80% vaccinated side of D.C. Eventually saw the disease begin to slow down? About how many days did it take to slow down?

3) How does each side compare to D.C.?

4) Hickory has a population of about 40,000 people & D.C. Has about 630,000 people. What does this tell you about how population density affects disease spread?

5) How does New York city compare to D.C & Hickory? Explain why you think that is the case.

6) Based on these simulations and given that both measles and COVID-19 are both airborne proximity diseases, why are all the governors capping the size of gatherings?

7) For a blood borne (or STD/STI) based disease (like that simulated with the ELISA part of this lab), would limiting the size of gatherings be as effective at stopping the spread of those diseases? Explain.

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