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MCB 3020L Study Guide - Final Guide: Chlamydia Trachomatis, Immunodeficiency, Treponema Pallidum

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School
University of Florida
Department
Microbiology and Cell Science
Course
MCB 3020L
Professor
ashgar

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Document Summary

The purpose of this experiment is to learn the methods and principles behind the three types of elisa tests: direct elisa, indirect elisa, and sandwich elisa. We will then use the indirect elisa test in order to determine if our bodily fluids have been infected after direct contact with someone else. If the elisa test is performed correctly, and our bodily fluids have been mixed with someone who is infected , the test should indicate that we are too infected. Based on the fact that std"s tend to spread rapidly if not cared for, regardless of how many people begin infected, a majority will end up infected as well. Workflow of the elisa procedure: apply a sample of known antigen to a surface, often in the well of a microtiter plate. After this wash, only the antibody-antigen complexes remain attached to the well: the second antibodies, which will bind to any antigen-antibody complexes, are added to the wells.

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Related Questions

Medical testing: enzyme linked immunosorbent assay (ELISA) test for presence of antigen or antibody

EXPERIMENTAL PROTOCOL

In this laboratory, you will test seven different patients using a direct ELISA. We have access to serum samples from the main characters of the TV show The Big Bang Theory. You will test each of them for the presence of ZIKA virus and HIV, as well as perform a pregnancy test, using known antigens/antibodies for those diseases/test. Students will work in pairs. Serum for each character of the Big Bang Theory has been incubated overnight at 4°C in the wells of an ELISA plate. You are taking it from here.

1.) What basic principles of antibody-mediated immunity are utilized in an ELISA assay?

2.) What might have caused some positive results to be lighter in color than others?

3.) Why can some viruses not be targeted by antibodies for destruction? Is this a limitation for developing an ELISA-based protocol for detecting the disease?

4.) Postulate a hypothesis on how the characters infected with Zika contracted the disease (in time and space); consider that two of them live in the same apartment and the others visit them frequently.

SYMBIOTS LAB different from above ^

1. If you give a high temperature and high light treatment to Symbiodinium linchaea and Symbiodinium minutum, what results do you expect after 3 weeks of treatment?

I have recently peformed an ELISA (Enzyme-linked immunosorbentassay), but I still have some questions, let me first outline whatI did:

  • We had a number (20) of tubes containing fake 'bodily fluids' ,namely saliva. One of these tubes contained the Epstein-Barr virus,which on its turn causes Pfeiffer's disease, aka the 'kissingdisease' (so we basically conducted an experiment to see how fastthe virus would spread within a class if everybody started kissingeach other, if you don't mind the crudity).

  • Everybody mixed their saliva with the saliva of 3 other people,so at the end everybody had a tube with the saliva of 4 differentpeople in it.

  • Since we worked in pairs of 2, every duo got a microtiter platewith 12 wells in it: 3 wells for a positive control, 3 wells for anegative control, 3 wells for the fluids of 1 of the duo and 3wells for the fluids of the other one. You pippete in (is thatcorrect?) the fluids accordingly.

  • After a while you rinse and clean the wells with washbuffer.

  • Then you pippete in the primary antibody in each well.

  • After a while you rinse and clean the wells with washbuffer.

  • Following this you pippete in the secondary antibody (conjugatedwith the enzyme HRP).

  • After a while you rinse and clean the wells with washbuffer.

  • Finally, you add the enzyme substrate (TMB) to each well. Thiscauses a blue color if when in contact with HRP. The positivecontrols must come out blue, the negative must come out colorless,and the other wells can go either way, depending if you have thevirus.

I hope this is not to crudely explained, again. These are myquestions concerning the experiment:

  1. What do the components of the acronym 'ELISA' actually standfor? Does immunosorbent refer to the binding of the antigens to thewells? My English isn't too good, and these acronyms andabbreviations often seem foreign to me (nevermind that they areforeign since I'm not English, you get the point I hope).

  2. When you pippete in the saliva in the wells, what actuallysticks to the wells? All of the antigens in your saliva? Becauseit's hard to believe that they make special microtiter plates forevery single antigen seperately. I assume it is only the antigens,since the primary antibodies attach to the antigens only, so thereis no need for the microtiter plate itself to bind toantibodies.

  3. How would we get the primary and secondary antibody? This is myassumption: The primary antibody we get is by injecting someantigens of the Epstein-Barr Virus into a human and then taking aserum. The secondary antibody could be gotten by injecting a randomhuman primary antibody into ANY animal, irrelevant which anyimal,and then taking a serum. After that you conjugate an enzyme to thesecondary antibody. Is this correct?