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Nida Rahman Professor Narayan BIO 229-10

Lab 6 Pre Lab ELISA immunoassay

Q1. How does the immune system protect us from disease? The immune system includes physical barriers, such as the skin and mucous membranes that prevent pathogens from entering the body, and cellular responses, such as circulating macrophages that respond to foreign invaders. Our acquired immune system mounts a specific antibody response when the body is exposed to a foreign invader, and our immune cells attack the invader. Q2. How do doctors use the immune response to protect you from disease? Doctors use the immune response when we are vaccinated against diseases. Our immune system remembers the pathogens to which we have been exposed, and the next time we are exposed to the pathogens our immune system attacks them more quickly and efficiently. Doctors take advantage of this priming effect by exposing us to inactivated pathogens (killed or weakened organisms that cannot make us sick) so that if we are later exposed to the live pathogen, our body will mount a strong and immediate antibody response, reducing or eliminating the chance that it will make us sick. Q3. How are the antibodies in your body made? Antibodies are produced by specialized white blood cells called B lymphocytes (or B cells). When an antigen binds to the B-cell surface, it stimulates the B cell to divide and mature into a group of identical cells called a clone. Q4. How are antibodies that are used in ELISA made? Generally, to make a monoclonal antibody, an animal such as a mouse or a rabbit (though there are other possibilities) is injected with the antigen that you want to make an antibody to detect, along with an adjuvant (a substance designed to promote a stronger immune reaction.) The animal mounts an immune response. B cells are removed from the spleen or lymph node and fused with tumor cells so they can grow in a culture dish (or an animal) indefinitely. pumping out antibodies. Each hybridoma (fused cell) is grown up separately from the others so that you get separate populations of identical cells that are clones of the original hybridoma they grew from (hence the term monoclonal), and the bunch of cells that makes the best antibodies is selected. There are also polyclonal antibodies that are not from the descendants of a single hybridoma.

Q5. Why is a rapid antigen detection test necessary? It is to make sure the patient is suffering from a bacterial cause. This is important so you know which type of antibiotic to prescribe, or whether to give one at all (you would not give and antibiotic for a viral infection). The ELISA has been used as a diagnostic tool in medicine and is used with the HIV test to find antigens that may be positive. Q6. What does ELISA stand for? Enzyme-linked immunosorbent assay. Q7. Why are enzymes used in this immunoassay? Enzymes provide a way to see whether the primary antibody has attached to its target (antigen) in the microplate well. Primary and secondary antibodies are invisible, so a detection method is necessary. The enzyme horseradish peroxidase (HRP) is linked to the secondary antibody. HRP reacts with a colorless substrate in a chemical reaction that turns blue. If the secondary antibody is present in the well, the color change indicates a positive result. Q8. Why do you need to assay positive and negative control samples as well as your experimental samples? Controls are needed to make sure the experiment worked. If there are no positive controls and the sample is negative, we can't know if the sample was truly negative or if the assay didn't work. Conversely, without a negative control, there is no way of knowing if all samples (positive or not) would have given a positive result.

Nida Rahman BIO 229-01 Professor Narayan Lab 6: ELISA immunoassay

Post-Lab Questions 1. Did your sample contain the antigen? Yes, it did contain the antigen. 2. The samples that you added to the microplate strip contain many proteins and may or may not contain the disease antigen. What happened to the proteins in the plastic well if the sample contained the antigen? What happened if it did not contain the antigen? In either case, all of the proteins present in the sample bind to the plastic wells. There was a color change. 3. Why do you need to wash the wells after every step? It was important to wash the wells after every step, sometimes multiple times, in order to remove any proteins and antibodies that are unbound. This ensures that there will be no false positive results. 4. When you added the primary antibody to the wells, what happened if your sample contained the antigen? What if it did not contain the antigen? If it was negative for the antigen, then the primary antibody was flushed out through washing. If it was positive, then the primary antibody bound to the antigen and was not washed away during the washing step. 5. When you added secondary antibody to the wells, what happened if your sample contained the antigen? What if it did not contain the antigen? The secondary antibody bound to the primary antibodies if it was positive for the antigen. If it did not contain the antigen, the secondary antibody was unbound and washed away during washing. 6. If the sample gave a negative result for the disease-causing agent, does this mean you do not have the disease? What are reasons for there to be a negative result when you actually do have the disease? It is possible that a negative outcome could have been false. There may have been human error when performing certain steps of the experiment such as forgetting for wash and cross-contaminating the wells that were next to each other when adding the substances.

7. Why did you assay your samples in triplicates? It was important to assay the samples in triplicate to double check for possible error. If the result of all of the wells were not consistent with each other, then it is easy to spot a potential error because all three wells should contain the same result if the experiment was done properly. 8. Determine the concentration of antigen in your unknown sample using the standard curve. 9. What antibody-based tests can you buy at your local pharmacy Test kits that are based on the same principles as the ELISA include home pregnancy tests, ovulation tests, and tests for illegal drugs like marijuana and cocaine. 10. Draw the structure of antibody and label the parts.

11. Compare and contrast the different types of IG.

12. Why antibodies are referred to as immunoglobulins? An immunoglobulins test is done to measure the level of immunoglobulins, also known as antibodies, in your blood. Antibodies are substances made by the body's immune system camera.gif in response to bacteria, viruses, fungus, animal dander, or cancer cells. Antibodies attach to the foreign substances so the immune system can destroy them. Antibodies are specific to each type of foreign substance. For example, antibodies made in response to a tuberculosis infection attach only to tuberculosis bacteria. Antibodies also work in allergic reactions. Occasionally, antibodies may be made against your own tissues. This is called an autoimmune disease.

13. Name the two major serum proteins and draw the electrophoretic pattern of these proteins. The serum protein electrophoresis (SPEP) test measures specific proteins in the blood to help identify some diseases. Proteins are substances made up of smaller building blocks called amino acids. Proteins carry a positive or a negative electrical charge, and they move in fluid when placed in an electrical field. Serum protein electrophoresis uses an electrical field to separate the proteins in the blood serum into groups of similar size, shape, and charge. Blood serum contains two major protein groups: albumin and globulin. Both albumin and globulin carry substances through the bloodstream.

14. How many Y-shaped antigen molecules each y shaped structure of immunoglobulin can bind to? The Y shaped structure of the immunoglobulin can bind to two antigen molecules. 15. Which part of immunoglobulin binds to complement?

16. Which antibody is produced first in response any antigen? The first antibody to be produced in response to any antigen is the immunoglobulin M, IgM....