NNMD 5272 Nanomedicine General Seminar

The aim of the report was to detect the proteins that are related to the infection caused by the hepatitis C virus. The different methods that can be applied for estimation of the proteins related to diseases was emphasized in the discussion.

The application of the immunofluorescence method is elaborated in the discussion. Alternative methods like enzyme linked immune sorbent assay are also briefly highlighted.

Introduction:

Hepatitis C is an infection caused by the RNA containing hepatitis C virus. The infection is detrimental as it can lead to liver and other cancers (Gan & Patel, 2013). The virus is blood borne and it comes in contact with the human body mostly by infected blood transfusion (Gower et al., 2014). The number of people infected by the virus is potentially increasing with time. The detection of the virus should be done in the earliest stage to avoid inimical consequences. There are several structural protein of the virus those are responsible for the occurrence of the infection (Romero-Brey et al., 2012). In the initial stage of the infection process, the envelop proteins of the virus like (E1 and E2) can be selected as markers to detect the viral infection in the people suffering from the symptoms associated with the disease (Luo & Davis, 2013). The other structural proteins can also be detected in later stage of the infection.

Method for detection of the protein responsible for the disease:

There are several methods available for the detection of the proteins responsible for the infection. Immunofluorescence is one of the most widely used detection methods for detecting the hepatitis C virus (Odell & Cook, 2013). This method is based on the detection of the antibodies specific to the antigens responsible for the infection. The antibodies are released only in the presence of the specific antigens and hence can only be detected when the virus is in the body.

Requirement table:

 The steps involved in the detection of the hepatitis viral protein by direct immunofluorescence:

• The process begins with the fixation of the samples of tissues or cells on the slide.
• The cells are then totally covered with the specific antibody solution. The antibody solution that is used in the initial step is known as the primary antibody.
• The antibody is labeled for identification after it binds to the specific antigens. Fluorescent dyes are generally used for labelling.
• After the time required for the reaction to take place, the binding is observed by fluorescent microscope.

Alternative of the same method:

The primary antibody labelling is expensive and hence most laboratories use another labeled antibody to bind with the primary antibody. The secondary antibody has to have a precursor association with primary antibody for them to bind. The process in which a secondary antibody is used is known as indirect immunofluorescence.

DIRECT IMMUNOFLOURESCENCE METHOD

Source: www.sinobiological.com

INDIRECT FLOURESCENCE METHOD

Source: www.sinobiological.com

Drawbacks of the immunofluorescence method:

There is a possibility of cross reactivity in case of the indirect immunofluorescence method. The chances of false positives are minimal but the potential cannot be ruled out.

The process is highly sensitive as it deals with the specific reactions between the antigen and antibody.

Alternative methods used for the detection of the proteins:

There are several other methods that are in use for the protein detection, some of them are enzymatic assays, enzyme linked immunosorbent assay (ELISA), mass spectroscopy and western blot (Tozzoli et al., 2012).

The enzyme linked immune sorbent assay is one of the most extensively used method for the detection of certain proteins related to specific diseases. The method is commercially known as ELISA. In this type of assay, the specific protein is detected in solutions. The method like the immunofluorescence assay, requires two different types of antibodies. In the beginning a monoclonal antibody is used, in a solid surface, it is adsorbed which are known as well plates. The solid surface is then totally covered with the sample under inspection. The monoclonal antibody captures the antigen. A polyclonal antibody is then used.  the captured antigen gets bound to this antibody. In the final step, another labeled antibody is used, this antibody is called the secondary antibody which is required for the detection of the antigen.

Conclusion:

Therefore, from the report it can be inferred that the detection of the proteins related to detrimental diseases is highly significant. It helps to analyze the reason of infection in minimal time, unlike the other detection procedures available. Hepatitis C infection is on the rise across the globe, diagnosis is significant in the initial period of infection. The detection of disease based on the proteins of the agent is used extensively as it gives minimal false positive results. The processes discussed in the report can be developed further to have zero false results.

References:

Gan, S. D., & Patel, K. R. (2013). Enzyme immunoassay and enzyme-linked immunosorbent assay. J. Invest. Dermatol, 133(9), e12.

Gower, E., Estes, C., Blach, S., Razavi-Shearer, K., & Razavi, H. (2014). Global epidemiology and genotype distribution of the hepatitis C virus infection. Journal of hepatology, 61(1), S45-S57.

Luo, X., & Davis, J. J. (2013). Electrical biosensors and the label free detection of protein disease biomarkers. Chemical Society Reviews, 42(13), 5944-5962.

Odell, I. D., & Cook, D. (2013). Immunofluorescence techniques. The Journal of investigative dermatology, 133(1), e4.

Romero-Brey, I., Merz, A., Chiramel, A., Lee, J. Y., Chlanda, P., Haselman, U., ... & Walther, P. (2012). Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS pathogens, 8(12), e1003056.

Tozzoli, R., Antico, A., Porcelli, B., & Bassetti, D. (2012). Automation in indirect immunofluorescence testing: a new step in the evolution of the autoimmunology laboratory. Autoimmunity Highlights, 3(2), 59-65.