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Although the term immunohistochemistry (IHC) is often used interchangeably with immunocytochemistry (ICC), there are significant differences between IHC and ICC in terms of the analyzed biological model. In simple terms, IHC is performed on tissue-derived samples, which are processed histologically into thin sections and the staining process exploits enzymes that catalyze the deposition of dye stain product at the antigenic sites in the sample. The ICC relies on the same enzymatic reactions as the IHC, but it is carried out on samples containing cells grown in the monolayer or in cells in suspension, deposited on the slide.
(Immunocytochemistry, I. 2016)
The Epstein–Barr virus, formally called Human gammaherpesvirus 4, is one of the nine known human herpesvirus types in the herpes family, and is one of the most common viruses in humans.
It is best known as the cause of infectious mononucleosis. It is also associated with various non-malignant, premalignant, and malignant Epstein–Barr virus-associated lymphoproliferative diseases such as Burkitt lymphoma, hemophagocyticlymphohistiocytosis, and Hodgkin's lymphoma; non-lymphoid malignancies such as gastric cancer and nasopharyngeal carcinoma; and conditions associated with human immunodeficiency virus such as hairy leukoplakia and central nervous system lymphomas.
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About 200,000 cancer cases per year are thought to be attributable to EBV.
Infection with EBV occurs by the oral transfer of saliva and genital secretions.
Most people become infected with EBV and gain adaptive immunity. Infants become susceptible to EBV as soon as maternal antibody protection disappears. When infection with EBV occurs during adolescence, it causes infectious mononucleosis 35 to 50% of the time.
EBV infects B cells of the immune system and epithelial cells. Once EBV's initial lytic infection is brought under control, EBV latency persists in the individual's B cells for the rest of their life. (Humans, I. 2017)
Signs and Symptoms
Children who contract EBV exhibit few symptoms or may even appear asymptomatic, but when EBV is contracted as an adolescent or adult, it may cause fatigue, fever, inflamed throat, swollen lymph nodes in the neck, enlarged spleen, swollen liver, or rash.
EBV that are made in the B cells have low numbers of gHgLgp42 complexes, because these three-part complexes interact with Human-leukocyte-antigen class II molecules present in B cells in the endoplasmicreticulum and are degraded. In contrast, EBV from epithelial cells are rich in the three-part complexes because these cells do not normally contain HLA class II molecules. As a consequence, EBV made from B cells are more infectious to epithelial cells, and EBV made from epithelial cells are more infectious to B cells.
EBV can infect both B cells and epithelial cells. The mechanisms for entering these two cells are different.
To enter B cells, viral glycoprotein gp350 binds to cellular receptor CD21 . Then, viral glycoprotein gp42 interacts with cellular MHC class II molecules. This triggers fusion of the viral envelope with the cell membrane, allowing EBV to enter the B cell.
To enter epithelial cells, viral protein BMRF-2 interacts with cellular β1 integrins. Then, viral protein gH/gL interacts with cellular αvβ6/αvβ8 integrins. This triggers fusion of the viral envelope with the epithelial cell membrane, allowing EBV to enter the epithelial cell..
Latent EBV expresses its genes in one of three patterns, known as latency programs. EBV can latently persist within B cells and epithelial cells, but different latency programs are possible in the two types of cell. (Humans, I. 2017)
The significance of detecting EBV is to avoid it from spreading s it’s a very contagious virus and spreads easily, as explained above, through saliva and other bodily fluids. It may cause mononucleosis or other illness such as types of cancer. As well as it might help in diagnosis, treatment and development evaluation of people with infection. (Humans, I. 2017)
Immunohistochemical method
Receipt of request has been collected. This is a form filled out by the pathologist requesting an IHC test.
Determination of tissue samples is necessary to maintain cell and tissue morphology during IHC use and during storage. It also prevents autolysis and necrosis of stimulated tissues, preserving antigenicity and increasing the refractive index of tissue components. The fixtures used are influenced by the target antigen as well as the desired detection technique; fluorescent or chromogenic. The tissue sample is then either embedded in paraffin or frozen.
Alcohol fixation has been used for this test. The sample has been mounted with APES-coated slides then air dried under airflow for 30 to 60 minutes. The sections has then been stored at -80 degrees. When it was ready it was left for 5 min at room temperate to let it warm up. The fixative has then been prepared and three slides have been used as follow; 1. Paraformaldehyde (0.3M glycine added in the blocking buffer before the antibody has been included), 2.acetone and 3 1;1 solution acetone:alcohol. Each one has been fixed for 15 min. (Abcam.com. 2019).
An antigen with a primary antibody, a secondary antibody, is added to bind to the primary antibodies. An enzyme label is then added to the secondary antibody to bind it; this detection of bound antibodies is evident by a colorimetric reaction. (NovusBiologicals. 2017)
The chosen antibody is Epstein-Barr virus (EBV) [EBV01, 02 and 03] which is a mouse monoclonal antibody cocktails intended for laboratory use for qualitative identification of latent substances membrane protein 1 of Epstein-Barr virus (EBV) by immunodeficiency (IHC). This antibody reacts with 60 kD latent membrane protein 1 encoded by BNLF1 general of Epstein-Barr virus (EBV). (Biocare.net. 2017).
The tissue has been buffered with4% PFA and the blocks have been cut with a microtome to slides of 3-5 mm thick. Then it has been immersed in Citrate buffer at 4 degrees overnight. After that the tissue blocks have been placed at 90-100 degrees for 3-5 minutes followed by immediately placing it in cold 30% sucrose in PBS and incubated at 4 degrees until the blocks have sinked. Last but not least the tissue blocks have been immersed in an parrafin embedded medium and stored at -80 degrees. (NovusBiologicals. 2017)
Detection System and Inclusion of Appropriate Control
Identification of anti-viral capsid antigen (VCA) antibodies may indicate past or current EBV infection. In addition, high titres of anti-VCA antibodies are observed in EBV-associated neoplasmic disorders such as lymphoma in patients with AIDS. The purpose of this study is the development and optimization of P3HR1 cell slides for EBVserologic detection by indirect immunofluorescence (IIF) assay. P3 HR1 exponential growth culture cells were collected at different time points with phorbol-12-myristoyl-13-acetate, and used for slide preparation. The IIF assay was performed using antibody-positive serum in each slide as the primary antibody. An 11% increase in VCA expression was observed at 40 hours post stimulation.
The data was confirmed by Western blot and Pratirakshadman. Other members of the Herpesviridae family analyzed the intra- and inter-locase specificity developed for IgG and IgM antibodies using EBV-positive sera and positive samples. No incorrect-positive or false-negative results were found for [image: ndirectimmunofluorescence assay (IFA) of dengue 3 seruminoculated C6/36 ]EBV detection or cross-reactivity with other members of the Herpesviridae family with developed slides. In conclusion, the slides presented here are a useful tool for the diagnosis of EBV infection and serologic detection of IgG anti-VCA antibodies in EBV-related neoplastic disorders.
Frozen tissue blocks are useful for providing thin cryostat section for localization of antigens and where immunofluorescence will be used as in this experiment. The tissue must be frozen quickly to prevent the movement of antigens and for good structure preservation. Alcohol fixation has been used for this experiment as it has better preservation of antigenicity, has rapid fixation, easy and cheap to use. Paraformaldehyde has also been used as it fixes tissue by cross linking the proteins. Its effects are reversible by excess water and it avoids formalin fixation. The disadvantage is that it can distort nuclear and cytoplasmic detail. (Polak, J. and Van Noorden, S. 2003)
Monoclonal antibody has been used as they are antibodies generated from a single B cell clone which makes it recognize a single epitope. This makes it less likely to cross with other proteins and it will have a lower lot to lot variability as well as it has a lower background. The disadvantage of this is that it is quiet expensive to use and it requires much more time then polyclonal antibody. As well as indirect method has been used as it will give more clear results and is more sensitive. (NovusBiologicals. 2017)
Fluorescent labelling provides an instantly visible label with excellent contract when seen against dark. It is usually used on frozen section because formalin fixed tissue tends to auto fluorescent that’s why it was the preferred method to use for this experiment. In an enzyme labelled preparation, frozen sections show structural imperfections, but with immunofluorescence these can be ignored, as the background tissue should only be visible enough to set the specifically labelled structures in context. There is another disadvantage as well which is that it needs a special microscope which can be expensive. (Polak, J. and Van Noorden, S. 2003)
Regarding to health and safety while doing a immunohistochemistry experiment there should be caution with some staining products and other equipment’s like the microtome as they can be very dangerous. They should always wear a lab coat, safety googles and gloves to protect their self, so personal protective equipment(PPE) should always be used. There should always be a risk assessment taken before using any of the products
There are as well other types of methods that can be used to detect EBV virus, some of them are; precipitin ring test, oucterlonny assay, radial immunodifussion assay, flocculation assay, immunoelectrophoresis, ELISA and immunoblot assay. Those can all be used to test antigen-antibody reaction. (Courses.lumenlearning.com. 2012).
Developing an Immunohistochemistry Method to Identify Epstein Barr Virus in Tonsil Tissue. (2024, Feb 08). Retrieved from https://studymoose.com/document/developing-an-immunohistochemistry-method-to-identify-epstein-barr-virus-in-tonsil-tissue
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