Immunohistochemistry for Identification of Epstein Barr Virus

This essay discusses the immunohistochemistry method for identifying Epstein Barr Virus (EBV) in human tonsil tissue samples. It covers the fixation process using Formalin-Fixed Paraffin Embedded (FFPE) technique, antigen retrieval methods, choice of primary antibody, and the detection system. The essay also emphasizes the importance of using monoclonal antibodies for greater specificity in the antigen-antibody reaction.

Introduction
Immunohistochemistry is an essential tool to detect specific antigens involved in a
disease of interest. Synthetic monoclonal and polyclonal antibodies fused with a fluorescent
compound, are designed to study antigen-antibody interaction in a given cell population and
specific tissue localization of those antigens. Significance of this method lies in the fact that it
helps in detection of haptens or causative antigens that invade the cells of the tissue and
triggers immune response (Zarkovic, Jakovcevic and Zarkovic 2017). Visualization of the
cells is achieved by fluorescence microscopy.
Epstein Barr virus (EBV) is one of the most pathogenic viruses in biology. It is a
subtype of human herpes virus which is connected to various lymphoproliferative disorders
along with Hodgkin’s lymphoma, HIV-induced malignancies and acute cerebellar ataxia.
Epidemiological evidence suggests, EBV infection elicit wide range of autoimmune diseases
and cancers. Most common type of EBV infection in human, exhibits infectious
mononucleosis (IM). Although, specific mechanism of pathogenesis is still to be identified.
Detailed research elaborated, a special type of short non-coding RNAs known as Bam H1-a
rightward transcript 7 micro-RNAs (ebv-miRBART7) are encoded by Epstein Barr virus,
which has been identified as a potential biomarker for nasopharyngeal carcinoma (Chan et al.
2013). The virus usually persist in a quiescent state, which resides in memory B lymphocytes
for lifelong among various individuals. It plays an important role in transformation of the
virus into B lymphocytes in-vitro, which is often associated with various disease like
Burkett’s lymphoma, Hodgkin’s diseases along with immunoblastic lymphoma. The virus
usually passes through a cycle of infected stages which involves transcription of a discrete
viral gene (Cohen 2015).

In this essay, the main research question that will be addressed revolves around the
development of an appropriate immune-histological method for identification of Epstein Barr
Virus, collected from tonsil gland of a patient.
Method of Fixation
Researchers should extract Epstein Barr Virus (EBV) sample from the human tonsil
tissue sample, followed by fixation method. For the fixation process, Formalin-Fixed
Paraffin Embedded (FFPE) technique can be used. This technique is mostly used in
immunohistochemistry (IHC). In this method, tissue sections are mounted on a slide for
examination. Firstly, the collected tissue will be fixed by keeping in a formaldehyde solution
or formalin, in order to preserve the protein and other important structures that are present in
the sample tissues (Hassani and Khan 2015). According to the study of Yo et al. (2015),
fixation of tissue using formalin, will cause conversion of haemoglobin to haematin. The
application of formaldehyde to the fresh tissues during the fixation will help in formation of
crosslinks between the proteins and would provide a mechanical strength to the tissues.
Initially, the cross linking reaction takes place among protein molecules and then the reaction
is carried out between basic amino acid, lysine. Therefore, it is reported that the reaction
between formaldehyde and protein is highly reversible, only if water is applied within the
first 24 hours of reaction time of both agents. Then, the formaldehyde fixed tissues are
embedded in a paraffin wax block during microtome mediated tissue cutting procedure to
make the process easier (Zimmerman, Aligo and Weinstock 2017). During the process of
formaldehyde fixation, the tissue becomes hard in nature, so that the tissues can survive the
other process of PPFE. For paraffin fixation, the tissue is dehydrated and cleaned by using a

gradual dehydration method. During this process, different concentration of ethanol is used in
a gradual manner. This paraffin embedding of tissue further helps in marinating the tissue
structure for a long period of time and removal of water from the tissues will prevent the
alteration of tissue structure as well (Zimmerman, Aligo and Weinstock 2017).
Antigen Retrieval
After the formaldehyde-paraffin fixation method antigen retrieval process is required,
before initiation of actual immunohistochemical process (IHC). It is reported that, during the
formaldehyde fixation process, formation of methylene bridges promotes masking of antigen
sites due to the cross linking of the proteins. In case of antigen retrieval process, these
methylene bridges are broken and the antigenic sites are exposed, thereby causing the
antibodies to bind with the antigens. Generally, there are two different methods of antigen
isolation, namely, proteolytic-induced epitope retrieval which is enzyme mediated retrieval
and heat-induced epitope retrieval (HIER) (Vinod, Jones and Udupa 2016). The former
process refers to the antigen retrieval method using proteolytic enzymes like trypsin,
proteinase K, or pepsin to isolate antigen of interest. On the other hand, heat-induced epitope
retrieval (HIER) uses heat to enhance epitope availability. However it is reported that using
enzymatic retrieval of epitope may cause damage of the tissue morphology and so this
enzymatic retrieval process should be avoided for this kinds of experiment, as it is completely
based on examination of Epstein Barr Virus sample extracted from the tissue sample of tonsil
(Kim, Roh and Park 2016).
In case of heat-induced epitope retrieval (HIER), a pretreatment with acidic, basic or
universal antigen retrieval reagents is required, and it is reported that these multistep
treatment of the tissue sample can greatly enhance immunoreactivity of these tissues. During
this process, preparation of working solution is needed. In order to do so, 10X antigen

retrieval concentrate and deionized water are mixed together, in 1:9 ratio. Then, the retrieval
solution is preheated at a temperature of 92-95 degree centigrade. Nextly, the slides are
immersed in the retrieval solution for 2-10 minutes, based on the optimal conditions of the
tissue as antigen retrieval of tissues is dependent on their incubation time and temperature
(Kabiraj et al. 2015). After incubation, slides are removed from the water bath placed in the
retrieval solution and they are allowed to cool until reached the room temperature. When the
slide reaches to room temperature, deionized water is applied to rinse through the slides in a
very slow and gentle manner and it is followed by washing with PBS. However, it is
recommended that vigorous rinsing should not be used as after the retrieval process of tissue,
it becomes loose and may cause detachment of sample from the slides. After this process,
tissues are ready to incubate with primary antibody (Kabiraj et al. 2015).
Choice of Primary Antibody
Antigen detection is one of the most crucial steps of immunohistochemical process.
At first, the primary antibody of the antigen should be identified according to its specific
epitope. After labelling of the antigen with the primary antibody, a secondary antibody is
added, so that it can bind with the primary antibody. An enzyme is then added, so that it can
bind with the secondary antibody, thereby easing the process of detection by the colorimetric
method. The study of Tabll et al. (2015) also supported the use of monoclonal antibody in
the identification of EBV, as monoclonal antibodies can bind with a specific epitope of an
antigen that needs to be identified. So, it can be said that a monoclonal antibody is more
specific in nature. On the other hand, use of polyclonal antibody produces higher chances of
cross reactivity and there is a possibility of obtaining wrong results during result
interpretation and this is due to production of huge amount of non-specific antibodies against
a specific antigen as well. So, in this experiment using monoclonal antibody will be fruitful.

Detection System
Detection of EBV in human tissue can be achieved by Lawrence Livermore
Microbial Detection Array (LLMDA) method. According to the study of Tellez et al. (2014),
this method is one of the most effective and inclusive assay, which utilizes probes for all
kinds of bacteria and viruses. In this study, this detection system was successfully used for
detection of EBV in human lymphoma sample.
Controls
During these experiments, all materials should be handled carefully in order to avoid
any kind of contamination of the reagents that may hamper the actual study results as well.
As a part of this process, it is recommended that the researchers should not pipette the
reagents through oral suction and should avoid any kind of contact with the reagents and
specimen (Quentmeier et al. 2016).
Result Interpretation
Presence of VCA IgG, Epstein-Barr nuclear antigen (EBNA) and VCA IgM indicates
the presence of EBV in the tissue (Sickinger et al. 2014).
Conclusion
The selected use of FFPE technique is effective as it helps in preventing deformities
of the tissue structure and thereby causing proper fixation. However, it is highly unsafe to
consume formaldehyde vapor during the process as it is harmful for health. On the other
hand, use of heat-induced antigen retrieval method will cause fixation of tissues and it will
give advantage to the researchers over the enzymatic method as enzymatic method may cause
alteration of tissue structures. Additionally, use of monoclonal antibodies as primary antibody
will allow greater specificity of the antigen- antibody reaction in the experiments.

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