HIV-1 Detection by Western Blot - Lab Report Example

HIV detection by Western Blot al Affiliation) Introduction The test of HIV using the western blot test involves the determination of the immunological impairment in the body through the agents such as the viruses (Kinter and Kinter, 2007). The test involves the transfer directly the protein bands that are in the polyacrylamide gel to where there is a charged nylon membrane that leads to the analysis of the results to determine that availability of the 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 (Kinter and Kinter, 2007). The transfer between the bonds is achieved in chambers that are specially designed through capillary flow or by application of vacuum. The total protein transferred can then be observed through the staining the membrane with protein dyes. Observing a specific protein within a mixture of proteins is most of the times detected by immunochemical methods (Libman and Makadon, 2003). The observation of the protein in a mixture cannot be detected by protein staining because the amount may be too low and the banding of the protein mixture may block it from view by the person that does the viewing. For immunological detection of specific protein, the unstained membrane is placed in barrier that blocks it and the barrier is that which contains detergent and protein that bind to all unoccupied sites on the nylon membrane (Kinter and Kinter, 2007). The membrane that is used in the making of the observation is then incubated in barrier that contains antibody to one or that which is more of the blotted proteins (Tsang, 1986). The antibody impasses to the adsorbed protein as an antigen and subsequent washings removes antibodies that are not bound. After all the treatment that occurs to the membrane, it is usually washed to remove the unbound secondary antibody-enzyme complex and is then incubated in a solution containing substrates that are of phosphates or those of peroxidase (Libman and Makadon, 2003). The products of the reactions that occur in the enzymes brings about chromogenic products that have increased visibility on the nylon membrane. In the preparation for the preparation of electrophoresis buffer that is used in the experiment and agarose gel, safety is always much considered in the laboratory through the putting on of gloves and googles to prevent harm in the laboratory (Tsang, 1986). The preparations starts from the dilution of the buffer gel and undergo through several steps until it is heated in the microwave for some time to dissolve the agarose powder. After the solidification of the agarose, the solution that comes out is that that remains clear. In the last stages of the test the membrane that was being prepared is removed from the distilled water and compared with the samples from the patients in the positive and the negative controls (Tsang, 1986). The western blot test is mainly performed and recommended when one is much exposed to HIV or the risks of contracting the virus. It can also be done to one that is doubtful about the HIV status. Results of the western blot test The molecular weight standards from the test Lane Sample Mol. Wt. (daltons) 1 A Positive control 120,000 2 B Negative control — 3 C Patient # 1 - positive 120,000 4 D Patient # 2 - negative 5 E Patient # 3 - positive 120,000 6 F Standard dye markers B-1 (Blue 1) 140,000 B-2 (Blue 2) 110,000 P (Purple) 90,000 R (Red) 70,000 The results obtained from the test can be represented in diagrams as follows In the results the patients showed different results which is shown by the dyes. The results that was obtained from the patients are then compared to come up with the diagnosis that bring one being diagnosed with HIV. The patients where the sample was taken showed using the dies. In the first patient the dyes remained red all through with a mass of 70000. The second patient showed red and green in the different spots. This differs in patients C and D where the dyes are also varied. The comparison can be done between patients B, C and D for diagnosis. Determination of the molecular weight of the viral protein In the determination of the weight the instance o 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. 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). Diagnosis The diagnosis using the method is done after a test is done to the anti-HIV antibodies that are in the body of an individual. In test the diagnosis is done in the infected cells are blotted on a membrane (Libman and Makadon, 2003). In the incubation step, the serum obtained from the body for test is applied with the free antibody in the sample being washed away. When the anti-human antibody is added and the bands stain, it indicates whether the person from which the sample was obtained from has the HIV. Picture of microarray The picture shows the step that the experiment undergoes in the determination of the proteins Two color microarrays 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). 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. 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