Exploring Viral Infections and ELISA Laboratory Techniques

Part 1 Learning Objectives:
1.Explore how viruses infiltrate a human body and how they can spread.
2.Understand why certain parts of the body are more susceptible to infection over others.
3.Understand the epidemiological tracking of a viral pandemic.
Background Information on Viruses
A virus is an infectious biological agent made of genetic material packaged within a protein coat. A virus does not have any metabolic activity on its own and must infect a cell to replicate itself. Once it infects a susceptible cell a virus can direct the cell machinery to produce more viruses. Most viruses have either RNA or DNA as their genetic material and the nucleic acid may be single- or double-stranded. The group of cell types that a virus can infect is typically very narrow and so viruses tend to have very specific host ranges, meaning you won’t usually see a plant virus infecting a human. Human viruses are responsible for many diseases, including the common cold, flu, AIDS and the most recent pandemic COVID-19.

For more information about human viruses and pandemics, please read this excerpt from the Campbell Biology textbook (please see Canvas), then answer these next three questions:
1. What are three ways that viruses make us ill? Why do we recover completely from a cold but not from polio?

2. What tools are in the medical arsenal against human viral diseases? 
3. Emerging viruses such as HIV, Ebola, and SARS seem to burst upon the human scene. What are three processes that contribute to this sudden emergence? 
For the next three questions use your Marieb A&P textbook:
4. Describe the tissue types that make up the external surfaces of the following:

Skin:
Mouth:
Oropharynx:
Bronchi:
Alveoli:
Esophagus:
Stomach:
Small Intestine:
Urethra:
5. Describe 4 different “first-line” defensive mechanisms present in the tissue types mentioned above.
6. Given the above tissue types, what tissue type do you think a virus like nCov-19 would infect? Describe some of the secondary lines of defense in the body that would activate after a viral infection.
CDC Outbreak Computer Simulation “Midterm Revenge”
Please go to the CDC simulation website then click on “Level 1” and then scroll all the way to the right to access the simulation “Midterm Revenge” and then please answer the questions below. It is recommended you read all the questions prior to starting the simulation.
7. Even though inflammation is often necessary for your body to fight an infection, what are some of the ways that inflammation can have adverse effects on your digestive system? How about on your respiratory system?
8. Describe the 4 patterns about a disease outbreak that an epidemiologist can learn from plotting an Epi Curve. Look up the current Epi Curve for COVID-19, what is the total number of people infected in the world right now? What does “flattening the curve” mean?
9. In this simulation, what virus are the students getting infected with? What organ system does this virus attack? What would be a common route of infection for this virus?
10. What is the single easiest and best way to prevent most respiratory and gastrointestinal viral infections in humans?

Background Information on Viruses

Part 2 Learning Objectives:
1.Explain how an Enzyme-Linked Immunosorbent Assay (ELISA) works and how it can be used to detect antigens or antibodies from a patient’s blood.

2.Explain the difference between a direct ELISA and indirect ELISA

What is an Enzyme Linked Immunosorbent Assay or ELISA?
The Enzyme Linked Immunosorbent Assay is a medical laboratory diagnostic tool that can be used to detect and quantify the amount of hormones, peptides, antigens, proteins and antibodies present in a patient’s blood sample or other body fluid. The laboratory test operates and gives results based on the principle that antibodies are very specific in binding to antigens. Every human antibody generated is designed to bind to one and only one specific antigen. ELISA’s are typically performed in polystyrene plates (see Figure 1). An immunologist can look for either specific antigens or can look for specific antibodies from patient samples. Depending on which one the immunologist choses to test for determines the type of ELISA assay that will be conducted. 
A Direct ELISA (See Figure 2 A) will determine if a patient is infected with a specific antigen. Examples include strain specific E. coli and Staph aureus antigens. Prior to testing the patient’s blood the plate is pretreated with an antibody against the specific antigen the immunologist is looking for. After the antigen attaches to the plate and patient serum samples are added to the well. If the specific antigen is present it will bind to the antibody and antigens present in the serum that do not match the antibody will not bind. For the reaction to be visualized and quantified by the immunologist a second antibody is added. This antibody is special. The antibody also can specifically recognize the antigen but an enzyme is also attached to the antibody. This special antibody is called an enzyme-linked antibody. A colorless substrate that the enzyme will catabolize will cause a color to develop which the immunologist can visualize and quantitate. While advance computer will quantitate the amount of color developed can be assessed qualitatively. A darker color indicates the antigen is highly present in the patient’s sample, a light color means the antigen is present but in smaller and if the solution remains clear then the antigen is likely not present in the patient’s sample. 
An Indirect ELISA (see Figure 2B) will survey the patient’s sample for the presence of a specific antibody instead of an antigen. In this assay the plate is pretreated with a specific antigen. Patient serums are then added to the well. If a patient has an antigen against that specific antigen the antibody will bind to the antigen coated on the plate. Common examples of this used in clinical laboratories include assessing for antibodies against common sexually transmitted diseases such as AIDS and Herpes, viruses like West Nile and people with allergies. People with allergies develop antibodies against antigens that are not normally dangerous such peanuts or eggs. To detect if a patient has an antibody against the specific the enzyme-linked antibody is added for the immunologist to quantify the amount of antigen/antibody complex formed. This particular enzyme-linked antibody is designed to recognize and bind to the Heavy chain constant region of human antibodies (Refer to Figure 21.14 of the textbook). The Indirect ELISA is the more commonly run clinical laboratory test.
Importance of Controls:
The most important concern in an ELISA assay are untrustworthy results such as false positives. A false positive would mean the immunologist would tell the patient they have a disease or illness they do not actually have. To eliminate untrustworthy results, ELISA assays are always preformed alongside control samples. What is a control and why is it important? Controls are standards of comparison to check results against and are a key component of the scientific method. They are important because it validates the test, ensures proper laboratory technique was used, that chemicals are working properly, eliminates bias of the scientist (falsification or the skewing of results) and that patients get accurate results about their health. ELISA assays utilize both negative and positive controls. Negative controls are samples added that are known not to contain the antigen or antibody. When the enzyme-linked antibody is added it will have nothing to bind to preventing the development of a colored product when the colorless substrate is added. Wash solutions or water are typically used as the negative control. Positive controls are samples that are going to lead to a positive result. Typically the antigen or antibody of interest is added which enables the enzyme-linked antibody to bind. A colored product will develop upon the addition of the colorless substrate because the enzyme-linked antibody will metabolize the colorless substrate. Both positive and negative controls show both negative and positive results for which patient samples can be referenced against to confirm the presence or absence of disease. Importance: If you are going to confirm the presence or absence of disease you need to be 100% sure and the controls allow enable an immunologist to be certain.
Laboratory: ELISA Computer Simulation
Directions: Read all instructions carefully before beginning the computer simulation. There are a series of questions below to answer about the ELISA Computer Simulation, general concepts about the ELISA method and immunology.

Questions to Answer Prior to Starting the Simulation:
11. Read the background information on the ELISA assay. Based on this reading explain what you think an ELISA assay is. 

12. According to the background information there are two types of ELISA assays: Indirect and Direct. Briefly explain the difference between the two.

13. ELISA assays are always run with controls. Why are controls important?

Questions to Answer during the Simulation – questions may not necessarily be in order. It is recommended you read all the questions prior to starting the simulation.
Go to the following website:
http://www.hhmi.org/biointeractive/immunology-virtual-lab
Important: If you get bad results you do not need to redo the simulation. Bad results will not affect your grade. Make sure you roll the mouse over the entire screen looking for things to click. Usually bad results are obtained because something in an image that needed to be clicked on wasn’t.
14. What disease is the ELISA computer simulation looking for?

15. What portion of the centrifuged blood samples do you take? Why are you taking from that portion of the centrifuged sample?

16. In steps 3 and 4 you are preparing the ELISA plate. What are you pretreating the ELISA plate with?

17. Based on what you have prepared so far; what type of ELISA assay are you preforming? Explain your answer.

18. In step 6 you wash the ELISA plate. Why is this step important for the assay?

19. What color is produced from the enzymatic reaction in the ELISA plate?

20. How is the color produced quantitated in the assay?

21. Did you complete the assay correctly? If not please explain from the result page what you did incorrectly?

22. What do the results indicated for patients A, B and C? If you did it wrong the simulation does show you what the correct results are supposed to be.

Post ELISA Computer Simulation Assay Questions:
23. Consider your answer in question 11. Do you have a better understanding of what an ELISA is now? Why or Why not? 

24. The ELISA assay simulation uses a known antigen in a specific disease. What are some other infectious antigens or diseases that could be assayed using the ELISA diagnostic method based on?