Technologies for Viral Detection: Pros and Cons of ELISA, Viral Flow Cytometry, and Transmission Electron Microscope

This study discusses the pros and cons of ELISA, viral flow cytometry, and transmission electron microscope for viral detection. It also explores the suitability of these technologies for low and high resource areas and as per the genotype of the virus. The study concludes that ELISA is the most suitable technology for detecting the virus that appeared to be spreading in a specific area due to its flexibility, high sensitivity, and specificity.

Molecular Diagnostics

Table of Contents
INTRODUCTION...........................................................................................................................3
MAIN BODY..................................................................................................................................3
Technologies for viral detection along with pros and cons.........................................................3
Disadvantages.............................................................................................................................5
Application of suitable technology that serve for low resource and high resource area (950)....6
Suitability of technology as per the genotype of virus................................................................7
Protocol process for verification and validation of the test.........................................................9
CONCLUSION................................................................................................................................9
REFERENCES................................................................................................................................1

INTRODUCTION
Molecular diagnostic is a collection of techniques that is being used to analyse biological
markers in the genome and proteome by detecting bacterial genes. This study is going to discuss
some different techniques that can help out laboratories in analysing and detecting the type of
virus. Some effective techniques that will be discussed in this study include: viral flow
cytometry, ELISA and transmission electron microscope. It will further discuss effectiveness of
ELISA in detecting the virus that appeared to be spreading in a certain area and has high
mutation rate. It will further discuss process or protocol of using or applying ELISA technology
to detect virus and accordingly taking actions.
MAIN BODY
Technologies for viral detection along with pros and cons
As it is found that virus that appeared to be spreading in a certain area. Some other
information that have been found regarding this virus is: it is spreading to the great extent and
affecting people to the great extent. Laboratories researched on this virus in order to know its
type, nature and structure and on the basis of result, it is found that this virus has high mutation
rate which means it has different variants and changes in a DNA sequence Zamani & et.al.,
(2021). Some patients have no symptoms but still tested positive and other infected person
presented with severe symptoms that may lead to severe complications.
In addition, it is found that people with specific genotype, presented with severe
complications and symptoms. For preventing patients from death and getting affected with this
virus to the great extent, it is important to detect this virus at early stage. Some technologies that
may help out laboratories in detecting this virus and identifying its nature as well as structure.
ELISA: ELISA stands for enzyme linked immunoassay that is an effective laboratory test
that is being used commonly for detecting antibodies in the blood. In regard to antibody, it can
be said that it is a protein that is produced by the body’s immune system when it detects harmful
substances, called antigens. For performing this test, there is need to take blood sample and sent
it to a laboratory where the target antibody is linked to a specific enzyme MacMullan & et.al.,
(2020). Of laboratories found that targeted antibody in blood then this test or blood solution turns
a different colour and on this basis, it can be identified as whether individual is affected with this
specific type of virus. This test is being performed to diagnose: rota virus, lime disease, HIV,
pernicious anaemia, toxoplasmosis, Zika virus and others.

Advantages
 Easily availability of commercial ELISA kits.
 Protocol is easy to follow.
 It can help out in determining the concentration of antigens in a sample.
Disadvantages
 Limitation to the amount of presence of the antigens in sample.
 There is temporary readout Spinu & et.al., (2018).
Viral flow cytometry: It is a laser based cell biology technique that is being used to analyse,
count and sort cells of interest from a mixed population. Fluorescence activated cell sorting is an
effective method in this virus detection technology that is being used to detect and discriminate
cells by its properties of light scattering and fluorescence. This test is being used for monitoring
cells expressing fluorescence proteins. It is found that this virus that appeared to be spreading in
a specific area can cause a life threatening respiratory illness as like Covid-19. In covid19 or
coronavirus, some people has symptoms and others do not present with specific symptoms
Niewold & et.al., (2020). Also, this corona virus had high mutation rate which means there is
changes in sequence of DNA. This test plays a key role in both viral research and diagnostic
laboratories. It is not easier to detect virus because they are very small in size but this technique
helps out in identifying and detecting virus because of its ability to separate and sort
heterogeneous mixture of biological particles. This technique can help out in identifying cell
type, bacteria and cyanobacteria. So, on the basis of ability of this type of technology to analyse
and sort large numbers of cells, it can be said that it is an effective one and can help out
laboratories in detecting the type of virus that outbreak suddenly and has high mutation rate.
Advantages
 This test sort and purifies small as well as complex subpopulations.
 By sorting virus particles, it can easily detect viruses Vázquez & et.al., (2018).
Disadvantages
 Machineries, used for detecting virus by this technique is costly
 Intense training is required to use this technique, otherwise, it may affect results.
Transmission electron microscope: It is a type of microscope that uses a particle beam of
electrons to visualise specimen and generate a highly magnified image. It is being used to view
tissue section and small molecules. In this technique, electrons can pass generating a projection

image. One of the main characteristic of this technique is: magnifying objects up to 2 million
times. It requires high voltage beam in order to create an image. It observes particles at a much
higher magnification and resolution and provides higher resolution images than a scanning
electron microscope. This microscope makes laboratories able in scanning and viewing the
surface of a sample Hage & et.al., (2020). It requires very thin specimen that is semi-transparent
to electrons. It has attached electron gun that fires a beam of electrons. This technique has
number of operating modes that all together makes able to laboratories to detect virus and its
nature such as: diffraction, scanning imaging TEM, conventional imaging and spectroscopy. The
very first TEM was demonstrated by Max knoll and Ernst Ruska in the year of 1931. On
samples, it provides morphologic and crystallographic information by making use of energetic
electrons. Images that this test produce are two dimensional and high resolutions in nature that
allow for a wide range of science and industry application. Some major components that this
technique consists of include: thermionic gun, electron beam, an electron source, vacuum
chamber, electromagnetic lenses, 2 condensers, sample stage, computer and fluorescent screen.
Advantages
 It provides the most powerful magnification.
 Produced images are high quality and give detailed information to laboratories.
 TEMs have an ability to yield information of surface features, size, shape and structure
Hachtel, Idrobo & Chi, (2018).
 With proper training, people can easily operate.
Disadvantages
 As compared to other, TEMS application is large and costly.
 For analysis and operations, there is requirement training that may increase overall cost.
 TEMs require special housing and maintenance.
 Images are represented and produced in only white and black.
So, on the basis of above discussed technologies, it can be said that all these can help out
laboratories in knowing the type of virus that appeared to be spreading in a specific area and on
the basis of nature and structure of this virus, appropriate actions can be taken in a timely
manner.

Application of suitable technology that serve for low resource and high resource area
On the basis of above discussed all three types of technologies, it can be said that ELISA
would be the best and suitable in identifying or detecting the virus that appeared to be spreading
in a specific area. As it is found that this virus has high mutation rate and has a single autosomal
variant that is dominant. There are 4 types of ELISA technology such as: direct Elisa, indirect
Elisa, sandwich and competitive. In direct ELISA, laboratory uses only an enzyme labelled
primary anti body and secondary antibodies are not needed. Whereas, within indirect type of
ELISA, both primary as well as secondary antibody are used but primary antibody is not labelled
with an enzyme and only secondary antibody is labelled with enzyme Zhang & et.al., (2020).
In sandwich type of ELISA, it is an antigen that is mobilized to the plate and this antibody
is called capture antibody. In this type, primary antibody is not labelled and enzyme labelled
secondary detection antibody. As compared to other discussed all three types pf ELISA, it can be
said that competitive ELISA is complex because in this type, laboratory needs to use inhibitor
antigen. As per the usage of inhibitor antigen, thus type of ELISA technology is known as
inhibitor antigen. The process of using and implementing is: interested antigen and inhibitor
antigen compete for binding to the primary antibody.
For this virus detection, sandwich WLISA will be used. It is important to apply this technique in
an effective manner so that it can be easily used for detecting the nature of virus that appeared to
be spreading Li & et.al., (2019). Some common steps that need to be followed for applying this
technique include:
 Preparing a surface to which a known quantity of capture antibody is bound.
 It is important to block any nonspecific binding sites on the surface.
 Antigen containing sample need to be added to the plate.
 For removing unbound antigen, it is important to wash the plate.
 As detection antibodies, laboratory needs to add enzyme linked secondary antibodies.
 For removing unbound antibody enzyme conjugates, there is need to wash the plate.
 Converted substrate by the enzyme into a colour also need to be added.
 It is the last stage in which laboratories need to measure fluorescence signal of the plate
wells for determining the presence, nature as well as quantity of antigen.

There are some specific kits that can also be used for knowing the nature and type of virus
that outbreaks in society on the basis of their spread rate and their genotype. Specific procedure
of using or applying this technique for detecting virus include:
 Attachment of an antibody to a polystyrene plate which is a solid surface and has an
affinity towards bacteria, other hormones and other antibodies.
 An attached microtiter coated with antigen is filled with this antigen and antibody
mixture in order to remove free antibodies by washing.
 A second antibody, associated to primary is added that is conjugated with an enzyme.
 Free enzyme is being removed that are linked with secondary antibodies and for this
laboratories need to wash the plate Shao & et.al., (2019).
 At the last stage, substrate is added that is converted by the enzyme to form a coloured
product.
For detecting viral in an effective manner, a capture antibody that is directed against the protein
is linked to a solid support like plastic 96 well microtiter plate. Clinical specimen needs to be
added and if there are viral antigens in human body or blood sample then they will automatically
be captured by the bound antibody. Overall, it can be said that this effective technique can be
used for number of reasons as for detecting cancer, HIV and others. For detecting viral, the
antigen is immobilized to a solid surface. It can be done in 2 manner or two types such as:
directly or via the use of a capture antibody that itself immobilised to surface.
Suitability of technology as per the genotype of virus
There are number of advantages and reasons of using sandwich antigen such as:
 It has high flexibility which means an ability to adapt changes and it is found that this
virus that appeared has high mutation rate. This virus has changes in DNA sequence and
has variances so, as per the characteristic of this sandwich type, it can be said that, it can
help out in detecting virus type, nature and structure as well.
 Other advantage of this technology is high sensitivity.
 It is high specificity as different antibodies blind to the same antigen for detection. So, on
the basis of this, it can be said that it can help out in knowing as which type of virus
occur with single autosomal variant that is dominant.
Along with advantages, there are some limitation or factors that need to be considered for
detecting the nature of virus such as: interest of antigen must be large enough so that it can easily

detect the nature as well as structure. It is important to bind two different antibodies so, target
antigen needs to be large Li & et.al., (2020). Along with this, it is found that sometimes, it
becomes difficult for laboratories to find two different antibodies that recognise different
epitopes on the antigen of interest and cooperate in an effective manner and in a sandwich
format.
If there is an antibodies present against the virus, then this technique binds to the
immobilized antigen. Bound antibodies can be detected easily by using a second antibody. There
is a feature of using this type of technology as it can be used in both diagnostic and in an
experimental manner. Sandwich ELISA is being used to measure antigen between 2 layers of
antibodies. It is important to contain at least 2 antigen sites in target antigen so that it can give
appropriate outcomes by binding them. Some other reasons of using this technique that makes it
suitable in detecting this virus include: needs to follow simple procedure and easy to perform for
everyone, high sensitivity and flexibility as well. ELISA is based on antigen antibody reaction. It
is also found that false positive results are rare in this technique Wang & et.al., (2021). False
outcomes can be occurred if patients have not developed antibodies to virus or if mistake has
made at the end of laboratory. On the basis of survey and results of this technique, it is found that
ELISA test is 99.9% accurate and reliable.
Genotype of this virus is single stranded RNA associated with nucleoprotein within a capsid
comprised of matrix protein. On the basis of its genotype, it can be said that it is like corona
virus. Coronavirus also has high mutation rate and has variation. Some of positive or affected
people of Coronavirus has severe symptoms and some presented with no specific symptoms. It
happens in this virus that is unknown in nature and structure. So, on the basis of genotype of this
virus, it can be said that sandwich ELISA will be suitable technique. Antigen is not required to
get purified and that is the reason as it can be used for complex samples. As compared to other
techniques and assays, it is easier to process and it requires the presence of radioactive materials.
Antibodies have 2 regions such as: variable and constant. In the variable region and at each tip of
Y shaped of this technique, it is highly specific and bind to only one specific antigen. Whereas,
constant region is the same for every antibody for the same type. If individual is infected with
virus, then antibody concentration increases and antibodies of human body binds to virus and
target then for ruin by the immune system Sas & et.al. (2018). Finally, and after binding, the
viral antigen concentration drops. Infection is now cleared and seen to laboratories and

healthcare professional and person can be recovered by providing them with effective treatment.
So, on the basis of its effectiveness, characteristics and benefits, it can be said that ELISA is the
most used and effective one and can help out in knowing all about this virus. On the basis of the
nature and structure of virus, people can be provided with appropriate treatment.
Protocol process for verification and validation of the test
As it is known that ELISA is an abbreviation for enzyme linked immunosorbent assay. This
test is versatile and medical professional finds easier to perform this test as compared to other
complicated tests. It is also known that this test is based on microtiter plate that has a solid phase
substrate. It is mainly used for detecting proteins as well as ions like: potassium and glucose.
Medical professional often uses this test in order to detect antigens and it helps them out in
identifying the type of virus or infectious disease. In regard to Sandwich ELISA, it can be said
that it is based on the detection and quantification of target protein Hougs & et.al., (2019). The
reason of calling or naming it sandwich is because target antigen is sandwiched between primary
and secondary antibodies.
Some materials that are required in ELISA sandwich protocol include: clear 96 well plate,
coating buffer, blocking buffer, wash buffer, plate sealers, reagent reservoirs, detection antibody,
capture, TMB substrate solution, streptavidin and stop solution. Some examples of protocol
include:
Run time: 4 hours and 30 minutes’ hands on time.
It is important to allow all reagent to reach room temperature before using.
In regard to biochemical markers, it can be said that it plays a vital role in improving decision in
clinical medical. For making sure that this test gives accurate outcomes, it is important healthcare
professionals to use validation and verification of test. Validation is conformation by
examination and provision of objective evidence. It makes sure that specific requirement for
intended use are fulfilled. Intended use of method needs to be verified. There is a standard
procedure that needs to be performed in order to verify and validate this test and getting positive
outcomes (Ling & et.al., (2018). some other detailed steps that need to be followed for getting
positive as well as validate outcomes such as:
1. Preparing coating solution by diluting the capture antibody in coating buffer.
2. After this step, it is important to coat plates with 100 μL per well of coating solution.
Covering plates for around 12-18 hours at 2-8 °C.

3. Aspiration of wells and washing 1 time with > 200 μL of wash buffer per well is specific
protocol.
4. Block plate with 200 μL per well with blocking buffer for at least 1 hour at room
temperature.
5. Aspirate, invent as well as tap on a paper that have absorption ability in order to remove
excess liquid.
6. It is the most important step in which, medical professionals need to prepare standard and
sample dilution in blocking buffer.
7. Pipette 100 μL of standards and samples into designated wells.
8. Washing and aspiration 5 times with > 200 μL of wash buffer per well.
9. Preparation of detection antibody solution by diluting the detection antibody in blocking
buffer Nicolella & et.al., (2018).
10. Again it is need to add around 100 μL of detection antibody solution into each well.
11. Again there is need to wash 5 times with > 200 μL of wash buffer per well.
12. Make working solution of streptavidin HRP with blocking buffer by diluting 1:5,000.
13. After making this solution, it is important to add that solution of > 100 μL into each well.
14. After adding every time, it is essential to wash them 5 times with > 200 μL of wash
buffer per well.
15. After adding > 100 μL of streptavidin HRP solution into each well, it is important to add
around > 100 μL of TMB substrate solution and incubating plate for 30 minutes at room
temperature.
16. It is again an important step of ELISA implementation for getting validate result in which
100 μL of stop solution needs to be added.
17. Measuring absorbance at 450 nm within 25-30 minutes after adding stop solution.
18. It is the last stage in which, medical professionals and laboratories need to calculate
results by making an effective use of 4 parameter curve fit.
There are some detailed steps that can also be followed for getting positive and validate results
such as:
Antibody coating: As the main aim of applying this test is to identify antigen in human body
and to know as whether individual is affected with virus or infectious disease or not. For this
purpose, it is important in this first step to immobilise captured antibody on high protein binding

plates by incubation it overnight. Plates are then blocked with irrelevant protein and it is called
albumin. Laboratories can omit this step when they use Mabtech’s pre coated plates.
Protein capture: Taken samples as well as standard dilution are being added to the wells and
will be then captured by the bound antibodies Nicolella & et.al. (2018).
Detection antibody: After capturing, specific biotinylated detection antibody is added to the
bottom well in order to make detection of captured protein able to act accordingly.
Streptavidin enzyme conjugate: Streptavidin conjugated with alkaline phosphatase is added to
the wells. This step needs to be performed just after adding biotinylated detection antibody
because it is being bind with Streptavidin conjugated.
Addition of substrate: As this result can be detected when antibody changes its colour and for
that laboratories need to add colorimetric substrate. It forms a coloured solution when it is
catalysed by the enzyme.
Analysis: It is the last stage in which absorbance is measured in this ELISA reader. It helps out
laboratories in determining the amount of protein in collected samples Yeong & et.al. (2018).
For measuring validity and verifying results, there is requirement to use different types of
validation tests such as:
Intra assay precision: This validation shows reproducibility between wells within used
assay plate. Data and results, gathered from this intra assay precision can make laboratories
know that taken samples run in a different wells of the plate and it gives comparable results. In
addition, it can also be said that this precision is typically <10%. It also makes sure that results
that obtained will be consistent over time. On the basis of above discussed process, types and
protocols, it can be said that there are variations in this test but the most used type of this test
consists of an antibody, attached to a solid surface Ford & et.al. (2020). A mixture of purified
HCG is attached to an enzyme and then the test sample is being added to the test system. On the
basis of above discussed steps for knowing validation, it can clearly be said that knowing
accuracy of test is important as it can make able to people to believe on it. It can help out
laboratories in taking appropriate decisions and protecting people against getting affected with
virus and improving their overall health.
So, on the basis of above evaluation and analysis, it can be said that ELISA is one of the
most used and effective viral detection test that can help out in knowing the nature, type and

structure of virus that appeared to be spreading in a specific area. It has high mutation rate which
means it varies in DNA structure. It is also found that people affected with this have severe
symptoms and some presented with no symptoms. In addition, it is also stated that this virus has
specific genotype or single autosomal variant that is dominant. So, on the basis of this, it is found
that it belongs to genera beta virus. It has similarities with beta coronavirus but also have
differences in their genomic as well as phenotypic structure. It has an ability to influence their
pathogenesis Ji & et.al. (2019).
CONCLUSION
It has been summarised from the above study that ELISA, transmission electron microscope
and viral flow cytometry are effective techniques of detecting viral and helping out healthcare
professionals in developing strategies accordingly. By identifying the nature and structures of
viral, it becomes easier to protect people from getting affected with virus and it can be done by
implementing appropriate viral detection technique. This study has discussed effectiveness of
sandwich ERICA viral detection technique and steps that need to be followed for getting positive
outcomes. It has shown protocol process for validation of ERICA viral detection test for
improving overall health of people.

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