Virtual Lab The Immunology Virtual Lab Student Worksheet IMMUNOLOGY VIRTUAL LAB WORKSHEET INTRODUCTION Go to http://www.hhmi.org/biointeractive/immunology-virtual-lab. Start the Virtual Lab and maximize the screen if you wish. Answer the following questions in the spaces provided. DIAGNOSIS 1. Where are antibodies found? - Antibodies are found in the section of blood called the gamma-globulin. 2. How can they be used in the laboratory? - They can be used in laboratories to help diagnose diseases caused by malfunction of the immune system. 3. What does ELISA stand for? - Enzyme-linked immunosorbent assay 4. What are ELISA assays used for in labs? - In labs, ELISA can be used to determine what specific antibody is present in a blood sample. 5. What are the three important limitations of an ELISA? Explain each. Limitation Explanation 1. A false negative where there are not enough antibodies to be measured 2. A positive result confirming an antibody that isn’t necessarily making them sick 3. A positive result that is an unrelated antibody reacting with the antigen non specifically BACKGROUND 1. What test can be used to determine whether a patient has an infectious or autoimmune disease? - The glisa test can be used. 2. What does a positive result indicate? - A positive result means there is an antibody present. 3. The watery fluid of the blood is called: 4. What is allowed to react with the target antigen? - The part of the serum which contains the antibody. 5. Detection is possible when: there is a second antibody added. 6. Once isolated, the secondary antibody can be: linked chemically to a system that produces a detectable signal. 7. What is the signaling system? - The signaling system is an enzyme attached to the second antibody. 8. What happens when the appropriate chemical (substrate) is added? - When added, the enzyme converts to a colored substance which can be measured. 9. How is the test quantified? - It is quantified by how much enzyme is present based on the amount of color produced. 10. What does the amount of color reflect? - This reflects the amount of antigen present initially. LAB NOTEBOOK Proceed through the entire lab simulation protocol. Be sure to read the captions below the pictures (left side) and the information in the lab notebook (right side). Be sure to "start over" to begin the lab. You CANNOT skip any steps. Answer the following questions as you proceed. 1. What is systemic lupus erythematosus (SLE)? - This is an autoimmune disease where antibodies fight the cells in the body. 2. From Figure 1 (click on it), what are the four steps of an ELISA protocol? A. Bind to sample support B. Add primary, wash C. Add secondary antibody enzyme conjugate D. Add substrate 3. In step 1, you centrifuge the samples. What does a centrifuge do? - The centrifuge precipitated the blood cells and obtained the serum. 4. What are you preparing in step 2? Why are there three different solutions? - We are preparing serial dilutions. There are three different solutions to make the different dilutions. 5. In steps 3 and 4, you prepare an ELISA plate. What has the ELISA plate been pretreated with? Why? - The ELISA plate was pretreated with sle antigen to get the antigens to bind to the plate. a. What is the positive control? (Step 4) - The anti-DNA primary antibody b. What is a primary antibody? Please define. - The second antibody used in an immunoassay that affects the pr. c. What is the negative control? (Step 4) - The buffer d. Why is it necessary to have a positive and a negative control? (Step 4) - This is important because the ELISAis not alway being used in appropriate conditions so it will be easier to know if the assay cannot be trusted by having two tests performed. 6. Why incubate the plate in step 5? - This is to ensure that the antibody present in the sample will interact with the antigen correctly. 7. Next, in step 6, the plate is washed. Why wash the plate? - This helps remove the antibodies that did not react with the sle antigens. 8. In step 7, a secondary antibody is added. What is a secondary antibody? Please define. - The second antibody is an antigen from a different species. This is used in an immunoassay which detects primary antibodies. a. What is the attached enzyme in this assay? (Step 7) - Hrp enzyme b. What is the specific substrate for HRP? What color does it produce? (Step 7) - The specific substrate for HRP is the substrate abts. This produces a yellow solution. 9. How can the yellow color be quantitatively measured? At what wavelength? (Step 10, in "why") - The yellow color can be determined by a 4/4 nanometer or by eye. 10. Record your results. Indicate on this page and on the computer which boxes turned color. - All of my boxes turned yellow. 11. Did you complete the ELISA correctly? (Yes/No) No If yes, proceed to #12 and #14. If no, proceed to #13 and #14. 13. Explain what you did wrong and what you will need to do next time. For more information, check your printable summary page. Did your incorrect procedure provide you any results? Explain what went wrong. - When doing the experiment, I did not wash the ELISA plate in step 6. In order for my results to come out correctly, I will need to wash it. 14. This virtual lab was testing for lupus. How is this same test used to test for the presence of HIV? If the results for an HIV test were the same as in this exercise, what would they indicate about the three patients? - This can be used for HIV because the ELISA test can detect the HIV antibody. If the test was done correctly, patient A would have HIV, patient B would need further testing, and patient C would not have HIV.