Elisa for detection of HIV infection PDF

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ELISA for detection of HIV infection

Q1 Report on the result of screen, which individuals tested positive for HIV? Individuals who were tested positive for HIV were B, C, E, and G. The wells for these individuals turned yellow which indicated they were HIV positive. This is because these individuals had the serum antibodies against the viral antigens in the serum, which resulted in the binding of HIV antigens. Q2 Draw a diagram to show what was happening in the wells at each step with a sample which would result in a positive result. In the indirect anti-HIV ELISA, the wells were coated with purified HIV virus antigens, each individual sample was diluted of 1/400. The bound antigen-antibody complex is detected by adding an enzymeconjugated anti-human IgG antibody (horseradish peroxidase conjugate) into each well. The TMB substrate was then added into all the wells. The wells appeared blue in colouration, this indicated the presence of enzyme. In a HIV positive reaction, the well would turn yellow in colour, and if there is no sign of yellow colouration this will indicate HIV negative. This is because the patient’s serum does not contain the antibodies against the viral antigen in their serum, therefore no binding will occur due to no antigens being present. Therefore, no colouration will indicate HIV negative. However, in a positive reaction, the antigens attach to the antibody, this occurs by the antigens binding to the antibody binding site, figure 1 illustrates the binding of antigen to the antibody. After the binding the interaction of antibodies and antigens takes place where the antibodies are neutralised by the infectious particles. Figure 2 illustrates the how the antibodies are neutralising the infectious particles. Therefore, this means the individual has been exposed to HIV virus as their serum contained the antibodies against the viral antigen in their serum and the binding of HIV antigens is present on the surface of the wells, which results a yellow colour, indicating HIV positive.

Figure 1

Figure 2

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Q3 why is the ELISA designed to test for the presence of circulating anti-HIV antibodies in the serum, rather than the presence of the virus itself? ELISA is designed to test for the presence of circulating anti-HIV antibodies in the serum, rather than the presence of the virus, is because the virus is difficult to detect especially during the early stages of HIV infection. The test monitors the body’s reaction to the presence of virus and measures the amount of virus present in the individual’s blood stream. The body produces antibodies in reaction to the presence of a virus. Therefore, a HIV antibody test determines the presence of antibodies in response to the HIV virus. Q4 Why is the anti-HIV antibodies termed an indirect ELISA? Anti-HIV antibodies is termed indirect ELISA because it involves two binding processes, which include a primary and secondary antibody where the primary antibody is incubated with the antigen and the secondary antibody. Indirect ELISA is classed more sensitive when detecting substances. Q5 what was the purpose of the control wells? The purpose of control wells is to determine whether the test is interpreted correctly, control wells allow to help and compare results. Q6 Explain what is meant by a false positive test, name one experimental error that would result in a false positive test? A false positive is where a specific test is designed to detect medical conditions, a false positive means that the individual has been tested positive for a specific disease or condition, even though the person may not have it. For example, a false positive is referred to a certain test designed to detect HIV, tests positive for the disease, even though the person does not have the condition. A type I error is known as a false positive, this is the process of incorrectly rejecting the null hypothesis, in this case it referrers to stating the patient is not HIV positive. Q7 Explain what is meant by a false negative test, name one experimental error that would result in a false negative test? A false negative is a test result which indicates that a patient does not have a disease or condition, but a false negative test result means the patient does have it. A type II error is therefore the opposite to type I and is the false acceptance of the null hypothesis. Therefore, a type II error would imply the patient to be free from HIV when they are not. This indicates the patient is diagnosed with HIV. Q8 Explain why the window period is important in the diagnosis of HIV using this method The window period is the time from exposure of HIV infection to the point when the test can reliably detect the condition and give accurate results. It is important because within the window period the chances of a false negative result are a lot higher, which means that a test is not as reliable as it usually would be. A false negative is where a test reports the absence of the HIV virus when it is in fact presence. In this case is usually because there is not enough antibody for the HIV test to notice. A window period can detect the presence of HIV virus and the progression of the disease. Research suggests that the window period for 4th generation antigen and antibody test is four weeks. During this period 95% of infections are detected. The detection of HIV antibody response can be detected in early stages within two weeks and within other individual’s it can be detected in more than 99.9% by 12 weeks. The antibody test at 4 weeks can provide sufficient indication of HIV status, in week 4

the detection of HIV infections is 95%, but a test at 12 weeks after the exposure is considered in monitoring for HIV negative. Figure 3 shows the time of developing HIV antibodies

Figure 4 illustrates the timeline of HIV infection indicating immune responses and the window period for the test.

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References      

http://i-base.info/guides/testing/appendix-3-how-hiv-tests-work http://www.tccbiotech.org/files/documents/BioRad_ELISAmanual.pdf https://www.bio-rad-antibodies.com/elisa-types-direct-indirect-sandwich-competition-elisaformats.html https://www.cancer.gov/publications/dictionaries/cancer-terms?cdrid=340929 http://i-base.info/guides/testing/what-is-the-window-period https://www.aids.org.za/testing-and-treatment...