Lab 6 StudentIMMUNOLOGY VIRTUAL LAB WORKSHEET

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Apr 3, 2024

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Kiaraliz Roldan-Mendez Lab 6 February 18, 2024 IMMUNOLOGY VIRTUAL LAB WORKSHEET INTRODUCTION Go to https://media.hhmi.org/biointeractive/vlabs/immunology/index.html . 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? The antibodies are found in the liquid portion of blood. 2. How can they be used in the laboratory? They can be used in a laboratory-based assay to help diagnose diseases caused by malfunctions of the immune system or infections. 3. What does ELISA stand for? ELISA stands for Enzyme-linked immunosorbent assay. 4. What are ELISA assays used for in labs? ELISA are used for in labs to determine whether a particular antibody is present in a patient’s blood sample. 5. What are the three important limitations of an ELISA? Explain each. Limitatio n Explanation First Positive result correctly identifying the presence of an antibody in the body could mean the patient may have had the disease prior and recovered. The body can keep producing the antibodies. Second People may have interfering substances in their blood or be poor producers of antibodies. The antibody count could be too low to measure or go undetected in the test resulting in a false negative. Third Positive result could result if a unrelated antibody reacts with an nonspecific antigen. This would cause an false positive result do the antibody reacting with an antigen.
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). You CANNOT skip any steps. Answer the following questions as you proceed. Make sure you click on the anything you see in red in each frame on the right side. It will help you to answer the questions in this lab. 1. What is systemic lupus erythematosus (SLE)? It is believed to be an autoimmune disorder where the cause is not known yet. The immune system loses the ability to distinguish between its own body components and antigens. Instead of fighting the antigens, the antigens will mistakenly attack the body’s own cells. This will cause the body to make autoantibodies. 2. From Figure 1 (click on it), what are the four steps of an ELISA protocol? 1. Bind the sample to support 2. Add the primary antibody then wash 3. Add the secondary antibody-enzyme conjugate then wash 4. Add the substrate 3.Why is it necessary to have a positive and a negative control? (Step 4) It is necessary to have a positive and negative control because the ELISA is subject to many errors. The biological and chemical reagents used in ELISA can change with time as well as ELISA are not always produced under the appropriate conditions. If either of the sample controls fail to react as expected than we can assume that the results for the patient’s samples cannot be trusted, and the assay must be repeated. 4. Why do you have to incubate the plate in step 5? Why must it be at 37 C? Make sure you answer both of these questions below. Incubating the plate for 15 minutes will ensure that the antibody present will interact correctly with the antigen. If the timer was below 15 minutes; the proper. Reaction would not occur causing no color to be evident at the end giving false negatives. It has to proceed long enough so that the binding can occur. The temperature must be at 37 degrees Celsius because SLE is a disease of humans. The human body is usually around 37 degrees Celsius. If the temperature was set too low, it would cause the reaction to not be completed while the temperature being too high would cause the protein to be affected via denaturation. 5. In step 7, a secondary antibody is added. What is a secondary antibody? Please define. A secondary antibody is an antibody that detects the first antibody. This antibody has to be made from a different species than the first antibody such as horse, donkey, rabbit, etc. in order to recognize the primary antibody as ’foreign.’ a. What is the name of the enzyme attached to the secondary antibody in this assay? (Step 7) The name of the enzyme attached to the secondary antibody in this assay is horseradish peroxidase. 6. At what wavelength can the yellow color be quantitatively measured? (Step 10, in "why") The yellow color can be quantitatively measured in a spectrometer at 414 nanometers.
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