HIV-1 Detection by Western Blot - Lab Report Example

HIV detection by Western Blot al Affiliation) Introduction Western blot and DNA/RNA microarrays have been used in the detection of HIV. In western blot the method involves the separation of the proteins of an individual in accordance to the sizes through the use of a polyacrylamide gel (Wikipedia, 2013). The method that is used makes the viral proteins that are obtained from the individual to be reacted with the patient’s serum. The reaction is done when the viral protein are transferred to the nitrocellulose paper. The detection of the HIV virus through the process is detected through the antihuman immunoglobulin antibody. The antibody when exposed to the enzyme with the substrate will lead to the production of a band that is colored (Luttmann, Bratke, Kupper and Myrtek, 2006). The test is done several times in the positive and the negative control serum to bring the clear identification of the viral protein. In the carrying out the test, there is the consideration of the nine HIV specific bands. When the bands are exposed to the serum of the individual the reaction that occurs in the bands and the patterns is what results to the diagnosis of the individual with the HIV virus. In the experiment to determine the availability of the virus, the polyacrylamide gel is extracted from the tray and the nylon that is used in the experiment is place directly onto the gel. After the nylon membrane is placed on the gel, bands of protein are transferred to the surface of the nylon membrane which makes them to be absorbed by the nylon membrane through the hydrophobic bonds (Wikipedia, 2013). The transfer between the bonds is achieved in chambers that are specially designed through capillary flow or by application of vacuum. DNA and RNA microarrays have been used in the determination of the infection with HIV since they are the key ways in the understanding of the expression of the genes and the method also allow for the rapid quantification of many of the genes once in the given cell that is focused. The microarrays have focused on the determination of the effect of the HIV on the single viral proteins in the individual in the line of lymphoid cells. The microarrays are used in the determination of the HIV due to the instance that the expression of the genes in an individual are always altered during the time of infection with the HIV (Luttmann, Bratke, Kupper and Myrtek, 2006). Infection in the individual is confirmed when the microarrays multiplex leading to the detection and also differentiate the sources that has brought the infection in the individual. In the carrying out of the experiment based on the microarrays, there are the representations by the colors that come about after the reactions (Kurien and Scofield, 2009). The colors that are obtained from the microarrays include black, yellow, green and red. The colors occur in spots in the sample that are taken from the individuals. In the indications, black shows that there is no expression in the sample. The other colors shows the normal level, up regulated and down regulation. The analysis diagnoses the individual to have HIV. Results from the experiment The microarray that was obtained from the experiment is shown below The molecular weight that was moved against the molecular weight of the proteins that were obtained from the patients are as shown below Distance moved by the band Molecular weight of the proteins (Mol. Wt. (Daltons) 140000 120000 110000 120000 90000 120000 70000 120000 The graph that is derived from the results that were obtained from the experiments are done in the representation of a graph as follows. The graph has the y-axis being the distance that is travelled and the x-axis being the mass of the viral protein. Determination of the molecular weight of the viral protein In the determination of the weight the instance of each of the bands that were traced in the experiment on the standard dye makers and the positive viral samples are measured. The measures are taken from the well to the bottom of each of the bands in the experiment (Luttmann, Bratke, Kupper and Myrtek, 2006). The dye makers are in terms of the colors blue 1, blue 2, purple and red. A semi log graph is then plotted with the distance that was travelled by each of the bands on the x-axis and the molecular weight of the bands on the y-axis. The molecular weight of the viral protein is obtained from the graph through the extrapolation from the standard curve (Wikipedia, 2013). The diagnosis The results that are obtained from the microarray can be used in the diagnosis of the patients. The bands that were obtained from the first patient varies severally with the yellow and black color being dormant (Kurien and Scofield, 2009). This makes the patient to be normal. The second patient in the experiment is up regulated. The third and the fourth patients also portrayed the same behaviors of being up regulated. The letters E-H represents the genes of the individuals that were used in the experiment. In the experiment, the two microarrays are used where two samples are prepared and the results that are obtained are compared. The comparison is done when the two samples are labelled with different dyes. In the diagnosis, the two dyes that were used differently in the comparison are mixed together to make a single microarray that is then scanned in the microarray scanner to observe them with a laser beam of wavelength (Kurien and Scofield, 2009). The intensity of the dyes are then used in the ratio-based analysis to identify the up and down regulated proteins. The analysis brings the diagnosis of an individual to be infected or the proteins containing the HIV element (Luttmann, Bratke, Kupper and Myrtek, 2006). The results that are obtained in the experiment are as follows and are used in the diagnosis of the patients with the HIV infection. Advantages and disadvantage of the methods used in the diagnosis Western blot in the diagnosis has come with advantages as compared to other methods such as ELISA. The method allows for the separation of the protein that is used in terms of conformation, charge and size. The method also allows for the obtaining of several targets as compared to the other methods. The method compares many proteins that is done by the other methods. Electrophoresis in the used in the proteins separates the protein into bands (Kurien and Scofield, 2009). The separation allows one to determine the size of the target protein. The method also allows one to determine the quantity of protein of interest through the running of an internal quantity standard with the samples that are used in the gel. In the method there is also probability of comparison in the samples that are obtained to bring a more analyzed diagnosis. The method can be used in the detection and characterizing of small amounts of proteins. The half-life of the small amounts of the proteins can be obtained by the method. Western blot can be used to detect the immunogenic response from the infectious agents which is not possible in many of the methods. The responses are difficult to detect since they cannot be separated from the samples obtained (Luttmann, Bratke, Kupper and Myrtek, 2006). The method do not only use antigens but also utilize the antisera which is widely used in the test for HIV presence. The method that is used is more specific on the antibodies in which they are used. The higher the specificity, the higher the independence of the method. The diagnosis method also has disadvantages. The carrying out of the experiment is time consuming. The time that the method takes is higher than other methods used in the diagnosis such as ELISA. The method has a high demand in terms of the experience in the person carrying out the experiment. The experiment also requires the optimization conditions that are to be maintained in the carrying out of the experiment. The conditions include the buffers, the concentration of the gel, the type of separation that is used experiment and also the protein isolation. In the use of the method, a protein that is not intended can bring the chance with the secondary antibody which can result in the labelling of an incorrect protein that was not intended. Incidents of oxidation can occur resulting to the appearance of the multiple bands in the experiment after the sample is processed (Luttmann, Bratke, Kupper and Myrtek, 2006). The method can also be of disadvantage due to the appearance of bubbles that are brought about by the transferring of the sample from the membrane and also in the incubation of the sample. Buffer is used in the method. When too much methanol is used in the transfer, the efficiency of the proteins is reduced. The transfer may not be inefficient making the higher molecular weight not being transferred. The use of the method is recommended since the method comes with many advantages in the diagnosis of HIV protein and also can be used in other diagnosis such that of cancer. References Kinter, M. and Kinter, C. (2007). Application of selected reaction monitoring to highly multiplexed targeted quantitative proteomics. Kurien, B. and Scofield, R. (2009). Protein blotting and detection. New York: Humana Press. Libman, H. and Makadon, H. (2003). HIV. Philadelphia: American College of Physicians. Lipman, M., Baker, R. and Johnson, M. (2004). An atlas of differential diagnosis in HIV disease. Boca Raton: Parthenon Pub. Group. Luttmann, W., Bratke, K., Kupper, M. and Myrtek, D. (2006). Immunology. Burlington: Elsevier Science. Ream, W. and Field, K. (1999). Molecular biology techniques. San Diego: Academic Press. Slonczewski, J., Foster, J. and Gillen, K. (2014). Microbiology. New York: W Norton. Tsang, V. (1986). Enzyme-linked immunoelectrotransfer blot technique (Western blot) for human T-lymphotropic virus type III/lymphadenopathy-associated virus. Atlanta, Ga.: U.S. Dept. of Health and Human Services, Public Health Service, Centers for Disease Control. White, J. (2004). The western blot! Parkville, Vic.: The Society. Farber, R. and Camille, M. (1992). I thought I had time. New York City: Artists Space. Wikipedia, Q. (2013). Elektrophorese. [S.l.]: University-Press Org. Read More