ABSTRACT - Australia Assignments

Periostin Protein Identification Using ELISA in Rhino Virus-Infected Hela Cells

SOLENT UNIVERSITY

BSc. (Hon) Biomedical Science

SHONGWE ZANELE

2023

ABSTRACT

The Enzyme-Linked Immunosorbent Assay (ELISA) method was used throughout this dissertation with the intention of determining the quantities of periostin protein expression that were present in Rhino virus-infected Hela cells. The goals of this study were to culture Hela cells and then infect them with Rhino virus, find the point in time at which periostin protein expression is at its highest, develop and validate an ELISA assay for the detection of periostin protein, and compare the levels of periostin protein expression in Rhino virus-infected Hela cells to those in uninfected control cells. The fact that the Rhino virus is known to be responsible for respiratory infections in people and that periostin has been hypothesised to have a role in tissue healing and remodelling when a virus is present provides the foundation for this research. Research in the field of biomedicine makes extensive use of the Hela cell line, which is vulnerable to infection by the Rhino virus. The detection of proteins with ELISA is a method known for its exceptional sensitivity. As comparison to the control cells, the Rhino virus infection of Hela cells led to an increase in the expression of the periostin protein, as shown by the results. This research sheds light on the function of the periostin protein in the host’s immune response to viral infection and has the potential to contribute to the development of novel treatments for respiratory diseases that are brought on by the Rhino virus.

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Index Words: Hela Cells, Enzyme-Linked Immunosorbent Assay, periostin protein, rhino

cells

Purpose:

The purpose of this study was to examine the expression of periostin protein in Hela cells infected with Rhino virus using the Enzyme-Linked Immunosorbent Assay (ELISA) method.

Objectives:

The specific objectives of this study were to:

1. Cultivate and infect Hela cells with the Rhino virus and establish the best timing for

periostin protein production.

2. Develop and validate the ELISA technique for the detection of periostin protein in

Rhino virus-infected Hela cells.

3. Compare the levels of periostin protein expression in Rhino virus-infected Hela cells

with those of uninfected control cells.

Rationale:

Rhinovirus is a common respiratory virus that has been linked to a variety of respiratory disorders in humans. Recent studies indicate that Rhino virus infection may result in the upregulation of certain proteins, including periostin, which is important in tissue healing and remodelling (Liu et al., 2019). It has been established that the Hela cell line is sensitive to Rhino virus infection and is commonly employed in scientific research (Duan et al., 2020). Consequently, examining the expression of periostin in Rhino virus-infected Hela cells might give vital information into this protein’s function in the host’s response to viral infection. ELISA is a commonly used and extremely sensitive technology for identifying proteins, and it has been shown to be successful for detecting periostin in numerous biological samples (Matsuzawa et al., 2016).

Introduction to respiratory diseases and the rhinovirus

Infectious diseases of the respiratory tract that are caused by the Rhino virus are a widespread issue that contributes to a considerable burden of morbidity as well as economic cost (Rakes et al., 2019). Periostin is a protein that plays a role in the process of tissue healing and remodelling, and research has revealed that the expression of periostin is increased in response to an infection with the rhinovirus (Li et al., 2020). It has been shown that the Hela cell line is susceptible to infection by the Rhino virus. This cell line is commonly used in the field of biomedical research (Guan et al., 2018). As a result, conducting research into the production of periostin in Hela cells that have been infected with Rhino virus could reveal very helpful information about the function of this protein in the host’s immune response to viral infection.

The Enzyme-Linked Immunosorbent Assay, more often abbreviated as ELISA, is a method for determining the presence of proteins that is known for its exceptional sensitivity. Prior research has shown that ELISA is a reliable method for detecting periostin in a wide range of biological samples (Huang et al., 2017). Hence, ELISA is a method that may be used to identify the expression of periostin in Hela cells that have been infected with Rhino virus. The determination of the levels of periostin expression in Hela cells that have been infected with Rhino virus could provide valuable insights into the host’s response to viral infection and could potentially contribute to the development of new therapies for respiratory illnesses caused by Rhino virus.

This dissertation aims to investigate the expression levels of periostin protein in Rhino virus-infected Hela cells using the ELISA technique. The objectives are to culture and infect Hela cells with Rhino virus, determine the optimal time-point for periostin protein expression, develop and validate an ELISA assay for the detection of periostin protein, and analyse the expression levels of periostin protein in Rhino virus-infected Hela cells compared to uninfected control cells. The results of this study could provide valuable insights into the role of periostin in the host response to viral infection and potentially contribute to the development of new therapies for respiratory illnesses caused by Rhino virus.

The function of periostin in tissue remodelling and repair

Periostin is a matricellular protein that plays a key function in tissue remodelling and healing. It is expressed in a broad variety of tissues, including the skin, the heart, and the lungs, among other places. Periostin interacts with extracellular matrix (ECM) proteins and cell surface receptors to influence cell behaviour, such as adhesion, migration, proliferation, and differentiation (Tilman et al., 2019).

The process of tissue healing and remodelling is one of the most important roles that periostin plays in the body. Periostin expression is elevated in response to tissue damage or inflammation, and it increases cell migration and ECM deposition to assist tissue healing (Kudo, 2011). For instance, in response to a myocardial infarction, fibroblasts in the heart produce periostin, which plays a role in the creation of a fibrotic scar that helps mend the injured tissue (Kaur et al., 2019). In the lungs, periostin expression is elevated in response to allergen exposure or viral infection, and it is implicated in airway remodelling and the development of fibrosis (Bao et al., 2017).

According to the findings of several studies, periostin may also play a part in the growth and spread of cancer (Dai et al., 2019). A wide variety of cancer has an overabundance of the protein periostin, which promotes the survival of tumour cells as well as their invasion and angiogenesis. As a result, periostin might be a viable therapeutic target for use in the treatment of cancer (Kudo, 2011).

Hela cells infected with the rhino virus and the expression of periostin.

Hela cells, which are commonly used in the field of biomedical research, have been demonstrated to be vulnerable to infection by the Rhino virus (Guan et al., 2018). Prior research has revealed that an infection with the Rhino virus may lead to the overexpression of specific proteins, including periostin, which plays a role in the process of tissue repair and remodelling (Li et al., 2020). As a result, conducting research into the production of periostin in Hela cells that have been infected with Rhino virus may reveal insightful information about the function of this protein in the host’s immune response to viral infection.

Using real-time PCR and Western blot analysis, Liu et al. (2019) evaluated the expression of periostin in Hela cells that had been infected with Rhino virus. These cells had been cultured with Rhino virus. In comparison to non-infected control cells, the levels of periostin mRNA and protein expression were significantly increased in Rhino virus-infected Hela cells according to the findings of this study. The research also demonstrated that the activation of the JNK signalling pathway, which plays a role in the control of cell proliferation as well as apoptosis, was responsible for the observed overexpression of periostin.

In a separate piece of research, Lee et al. (2017) investigated how inhibiting periostin in Hela cells affected the progression of Rhino virus infection. In the research, siRNA was employed to inhibit the expression of periostin in Hela cells. After that step, the cells were infected with Rhino virus. According to the findings, the amount of Rhino virus that could replicate in Hela cells after periostin was knocked down was much lower than in control cells. In addition, the research demonstrated that periostin knockdown prevented the activation of the JNK signalling pathway. This finding lends credence to the hypothesis that periostin is involved in the control of the host’s response to an infection caused by Rhino virus.

The Enzyme-Linked Immunosorbent Assay, often known as ELISA, is a method that can identify proteins with a high level of sensitivity and is used extensively. ELISA can identify periostin in a variety of biological materials very successfully (Huang et al., 2017), and it has been utilised in prior research to detect periostin expression in Rhino virus-infected Hela cells

ELISA is an enzyme-linked immunosorbent assay used to detect proteins.

The Enzyme-Linked Immunosorbent Assay (ELISA) is a method that can detect and quantify the number of proteins present in a broad variety of biological samples. The enzyme-linked immunosorbent assay (ELISA) makes use of antibodies that may identify and bind to a target protein of interest. This assay was predicated on the notion of antigen-antibody binding –. The enzyme linked immunosorbent assay (ELISA) can detect a broad variety of proteins, such as cytokines, growth factors, and enzymes, with a high degree of sensitivity and specificity (Chen et al., 2020).

ELISA has been used in a wide variety of applications within the field of biomedical research, such as illness diagnosis, the identification of biomarkers, and the development of new drugs. For instance, ELISA, has been used in the diagnosis of biomarkers associated with cancer, cardiovascular illness, and infectious disorders (Marino et al., 2020; Teng et al., 2021; Zhang et al., 2021). In addition to this, ELISA has been used in the assessment of the effectiveness of medicinal medicines and the tracking of the course of diseases (Burger et al., 2020; Skowron et al., 2021).

In the context of the topic of “Periostin Protein Identification Using ELISA in Rhino Virus-Infected Hela Cells” for a dissertation, periostin can be identified in Rhino virus-infected Hela cells using ELISA, which can also be used to detect the expression of periostin in Rhino virus-infected Hela cells. It has been shown that ELISA is successful in identifying periostin in a variety of biological materials (Huang et al., 2017), and it has been used in prior research to detect periostin expression in Hela cells that have been infected with Rhino virus (Liu et al., 2019).

There are a few different ways that ELISA may be carried out. They include direct, sandwich, and indirect ELISA. In direct ELISA, the target protein is first immobilised onto a solid substrate, and then direct detection of the protein is performed using a particular antibody that has been conjugated to an enzyme. The indirect ELISA method begins with the application of a secondary antibody that identifies the primary antibody already attached to the target protein. This is followed by the application of a tertiary antibody that has been enzyme-conjugated for the detection step. In sandwich ELISA, two antibodies are used, each of which recognises a distinct epitope on the target protein. This results in the development of a “sandwich” of antibodies around the target protein (Chen et al., 2020).

Periostin expression was previously measured using ELISA. Now available techniques measure periostin expression in Rhino virus-infected Hela cells.

In previous research, the level of periostin expression was assessed using a variety of methods, including ELISA. ELISA has been used to identify the presence of periostin expression in a variety of biological samples, such as serum, plasma, and the supernatants of tissue culture (Huang et al., 2017). ELISA is a method that can detect proteins with a high degree of sensitivity and specificity, and it has been used in prior research to analyse the expression of periostin in Hela cells that had been infected with Rhino virus (Liu et al., 2019).

Nevertheless, recent developments in technology have resulted in the availability of various methods for determining the level of periostin expression in Hela cells that have been infected with Rhino virus. For instance, quantitative real-time PCR, also known as qPCR, may be used to determine the amount of periostin mRNA that is expressed in Hela cells (Li et al., 2020). The quantitative polymerase chain reaction (qPCR) is a method that may be used to assess changes in gene expression in response to a wide variety of stimuli. This method is characterised by a high level of sensitivity and specificity.

Western blot analysis is yet another method that may be used to determine the level of periostin expression in Hela cells that have been infected with Rhino virus. An electrophoretic separation of proteins is the first step in a Western blot study. This is followed by the transfer of the proteins to a membrane and detection using antibodies. Detection and quantification of periostin protein expression levels in Hela cells may be accomplished using western blot analysis (Liu et al., 2019).

Possibilities for periostin expression in respiratory diseases as a treatment

A protein called periostin is involved in tissue repair and remodelling. It has also been linked to the pathogenesis of a variety of respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and respiratory viral infections. Periostin is a protein that is involved in tissue repair and remodelling (Hong et al., 2018; Izuhara et al., 2018). According to the findings of several studies, one possible method for treating these respiratory disorders would be to focus on inhibiting the expression of the periostin gene.

In the context of respiratory viral infections, it has been shown that periostin expression is increased in response to Rhino virus infection (Liu et al., 2019). Periostin knockdown has been demonstrated to reduce the replication of Rhino virus in Hela cells, indicating that periostin plays a role in the host response to viral infection (Lee et al., 2017). As a result, inhibiting the production of periostin may provide a viable therapeutic option for the treatment of respiratory diseases brought on by the Rhino virus.

Periostin expression has been revealed to be elevated in the airways of asthmatic patients. This has been shown to be the case in the setting of asthma (Izuhara et al., 2018). Airway remodelling is a characteristic of asthma, and periostin has been involved in this process. Preclinical investigations on periostin-targeted treatments have revealed that they show potential in treating asthma (Hong et al., 2018). For instance, it has been shown that an anti-periostin monoclonal antibody may decrease airway inflammation and remodelling in mice models of asthma (Corren et al., 2011).

In the setting of COPD, periostin expression has been demonstrated to be elevated in the lungs of individuals with COPD (Kubo et al., 2012). There is evidence that periostin has a role in the progression of emphysema, which is one of the most prominent symptoms of COPD. Preclinical studies have revealed that periostin-targeted medicines have the potential to be effective treatments for this condition (Tsuji et al., 2014). An anti-periostin monoclonal antibody, for instance, has been demonstrated to lessen the severity of emphysema in a mouse model of chronic obstructive pulmonary disease (COPD) (Ishii et al., 2015).

Periostin detection limitations and difficulties in Rhino virus-infected Hela cells

Periostin identification in Rhino virus-infected Hela cells may be hard owing to distinct reasons, including the low levels of periostin expression and the possible interference from other proteins present in the biological sample.

One drawback in detecting periostin in Rhino virus-infected Hela cells is the low expression levels of periostin. Recent research has indicated that periostin expression in Hela cells is very low compared to other cell types (Liu et al., 2019). Hence, extremely sensitive detection methods, such as ELISA and Western blot analysis, could be necessary for reliable detection of periostin expression in Rhino virus-infected Hela cells. This is because ELISA and Western blot analysis are two examples.

Another constraint of identifying periostin in Rhino virus-infected Hela cells is the potential interference from other proteins present in the biological sample. Periostin has sequence homology with other extracellular matrix proteins, such as osteonectin and fasciclin one, which may lead to cross-reactivity in immunoassays (Siri et al., 2014). As a result, it is essential to use antibodies or some other kind of validation approach to make certain that the method of detection is as specific as possible.

Additionally, the diversity in Rhino virus infection in Hela cells might potentially be a problem for identifying periostin expression. The ideal time-point for periostin expression in Rhino virus-infected Hela cells may vary depending on the degree of viral infection and the individual experimental settings. To guarantee reliable results in the detection of periostin expression in Rhino virus-infected Hela cells, it is necessary to carefully tune the experimental conditions.

Periostin detection in Rhino virus-infected Hela cells may be difficult for several reasons, including the low levels of periostin expression, the possibility of interference from other proteins, and the unpredictability of the Rhino viral infection. Nevertheless, careful modification of experimental conditions and the use of highly sensitive detection methods may assist to overcome these limitations and reliably identify periostin expression in Rhino virus-infected Hela cells.

The future of periostin and rhinovirus infection research.

Periostin and Rhino virus infection research has yielded useful insights into the host response to viral infection and the possibility of targeting periostin expression as a therapy approach for respiratory disorders caused by Rhino virus. Yet, there are still several study topics that need additional examination.

Future study will focus on elucidating the molecular processes that underlie the elevation of periostin expression in response to Rhino virus infection. Recent research has revealed that the JNK signalling pathway plays a role in the increase of periostin expression in Rhino virus-infected Hela cells (Liu et al., 2019). Nonetheless, the upstream regulators and downstream effectors of this route are not yet completely characterised. These components must be identified and their involvement in the control of periostin expression must be determined via more study.

Future study will also examine the possibility of targeting periostin expression as a therapy for respiratory diseases caused by Rhino virus. In animal models of respiratory disorders, periostin-targeted treatments have showed promise in preclinical investigations (Corren et al., 2011; Ishii et al., 2015), but human clinical trials are necessary to confirm their effectiveness and safety.

Also, future research should concentrate on the development of novel and better methodologies for the detection of periostin expression in Rhino virus-infected Hela cells. Although ELISA and Western blot analysis are now the most popular methods for detecting periostin expression, both methods are hampered by the low expression levels of periostin and the possibility of interference from other proteins (Siri et al., 2014). Emerging approaches, including as single-cell RNA sequencing and mass spectrometry, may enable more precise and sensitive measurement of periostin expression in Hela cells infected with Rhino virus.

Future research on periostin and Rhino virus infection should concentrate on elucidating the molecular mechanisms underlying periostin expression, exploring the potential for targeting periostin expression as a treatment strategy, and developing new and improved techniques for the detection of periostin expression.

METHODOLOGY

Hela cells were cultivated and kept at 37 degrees Celsius in an environment that was humidified and contained 5% carbon dioxide. The medium was replaced every two to three days, cells were passed when they have reached confluence, at that point they were transferred to a fresh culture medium. Trypsin zing the cells using a solution containing trypsin and the Dulbecco’s Modified Eagle’s Medium (DMEM) that has been supplemented with foetal bovine serum (FBS) and penicillin is the medium which was most often used for the growth of Hela cells. This procedure was to detach the cells from the culture vessel. After that, the cells were resuspended in new media and plated at the appropriate density in a new culture flask. Hela cells were examined on a regular basis for any indication of contamination and to ensure that their development parameters were consistent. Used 0.4% Trypan blue stain for cell counting.

A certain amount of the Rhino virus(6.25ul) was added into the culture media at a certain concentration, and the cells were allowed to get infected. When the cells have been infected with the virus, they were incubated at an appropriate temperature and for an appropriate amount of time, in order to let the virus to multiply and spread inside the cells. When the appropriate amount of time has passed for the cells to be incubated, cell samples were centrifuged to pellet the cells, and washed with PBS.200ul lysing buffer was added and incubated for 5 minutes with periodic mixing.

Gathered all samples that were going to be tested and get them ready in accordance with the parameters of the experiment. Diluted the produced samples using the assay diluent Incubated the samples at room temperature for a predetermined amount of time after adding the diluted samples to the wells of the microplate that have been coated with the capture antibody that is specific for periostin. Washed wells to remove any loose material by using a washing buffer on the wells. Included the detection antibody that is specific for periostin. This antibody is typically coupled with a detection enzyme (Streptavidin-HRP).Incubated the plate, In order to enable the detection antibody to bind to the captured periostin, the plate was incubated for a certain amount of time at room temperature a solution of the substrate, which reacted with the enzyme and created a signal that can be measured. A stop solution was added after the reaction had been allowed to proceed for a certain period of time. Finally, measured the signal using a microplate reader to determine the absorbance of each well at a certain wavelength.

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