Immunology Lab Worksheet

Virtual Lab The Immunology Virtual Lab Student Worksheet IMMUNOLOGY VIRTUAL LAB WORKSHEET INTRODUCTION DIAGNOSIS 1. Where are antibodies found? Plasma of the blood 2. How can they be used in the laboratory? determine wether a sample carries a disease 3. What does ELISA stand for? enzyme-linked immunosorbent assay 4. What are ELISA assays used for in labs? to measure the concentration of antibodies or antigens 5. What are the three important limitations of an ELISA? Explain each. Limitation A positive result Explanation confirming a presence of an antibody but it not necessarily making the patient sick A false negative where the amount of antibodies is too low to be measured A positive result may occur if an unrelated antibody reacts with the antigen nonspecifically Virtual Lab The Immunology Virtual Lab Student Worksheet BACKGROUND 1. What test can be used to determine whether a patient has an infectious or autoimmune disease? the glisa test 2. What does a positive result indicate? the antibody is there and implies that the person has encountered a particular disease. 3. The watery fluid of the blood is called serum . 4. What is allowed to react with the target antigen? a portion of the serum containing the antibody 5. Detection is possible when a second antibody is added . 6. Once isolated, the secondary antibody can be chemically linked to a system that can produce a detectable signal 7. What is the signaling system? consists of an enzyme attached to the second antibody 8. What happens when the appropriate chemical (substrate) is added? the enzyme converts to a colored substance that can be measured. 9. How is the test quantified? how much enzyme is present by the amount of color produced. 10. What does the amount of color reflect? the amount of antigen initially present. Virtual Lab The Immunology Virtual Lab Student Worksheet 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)? an autoimmune disease where antibodies fight the bodies own cell 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? precipitate the blood cells and obtain the serum 4. What are you preparing in step 2? Why are there three different solutions? preparing serial dilutions. 3 solutions to make diff dilutions 5. In steps 3 and 4, you prepare an ELISA plate. What has the ELISA plate been pretreated with? Why? 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 a immune assay that affects the pr Virtual Lab The Immunology Virtual Lab Student Worksheet c. What is the negative control? (Step 4) buffer d. Why is it necessary to have a positive and a negative control? (Step 4) the elisa isnt always conducted under appropriate condition so if either test gives unexpected results the assay cant be trusted 6. Why incubate the plate in step 5? ensures that the antibodi present in the sample will interact correctly with the antigen 7. Next, in step 6, the plate is washed. Why wash the plate? helps remove any antibodies that dont react with the sle antigens 8. In step 7, a secondary antibody is added. What is a secondary antibody? Please define. an antigen from a diff species which is used in an immunoassay that detects primary antibody 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) substate abts, produces a yellow solution 9. How can the yellow color be quantitatively measured? At what wavelength? (Step 10, in "why") can be estimated by the eye or 4/4 nanometers. Virtual Lab The Immunology Virtual Lab Student Worksheet 10. Record your results. Indicate on this page and on the computer which boxes turned color. A C Yellow yellow yellow yellow yellow 1:100 + (positive) Yellow 1:2 1:10 B Yellow – (negative) yellow 11. Did you complete the ELISA correctly? (Yes/No) yes If yes, proceed to #12 and #14. If no, proceed to #13 and #14. 12. What do the results indicate about: Patient A: likely to have sle Patient B: Might have it but further testing is required Patient C: Does not have it 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. Virtual Lab The Immunology Virtual Lab Student Worksheet 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? ELISA is the most frequently used type of test to monitor for HIV infection because of its moderately straightforward methodology and innate high sensitivity. A general attribute of all varieties of ELISA is the use of enzyme conjugates that bind to specific HIV antibody, and chromogens that produce color in a reaction catalyzed by the enzyme conjugate. Therefore, for both diseases ELISA is used for diagnosis. If the results for HIV test are same then either patients are HIV positive (suffering from HIV) or HIV negative (not suffering from HIV).