‫جامعة دارة السالم الدولية للعلوم والتكنلوجيا‬

‫قسم الهندسة وتكنولوجيا المعلومات‬

‫تخصص هندسة أجهزة ومعدات طبية‬

‫‪ELISA‬‬

‫إعـــداد‪/‬‬
‫رضوان محمد أحمد قايد‬
‫جبران عبدالواحد األديب‬
‫غانم أنور محمد العلفي‬

‫إشــــراف‬
‫د‪ .‬محمد العلفي‬

‫‪2023‬‬
 Dedication

I dedicate this modest effort to my father and my

mother who have long supported me and gave me

the strength to reach this level and also to my batch

who has long been an example of hard work and

role model.

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 Acknowledge
All praise is to Allah for what He has bestowed upon us
of great graces,
Thank you for your help and for doing your utmost to get to
where we are today.
We also extend our sincere thanks and praise
to the supervisor

Dr. Mohammad Al-Alfi

May God protect her and take care of her

For the scientific and ethical guidance she has provided us.

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 Introduction:
Enzyme immunoassays (EIAs) use the catalytic properties of enzymes to
detect and quantify immunologic reactions. Enzyme-linked immunosorbent
assay (ELISA) is a heterogeneous EIA technique used in clinical analyses. In
this type of assay, one of the reaction components is nonspecifically
adsorbed or covalently bound to the surface of a solid phase, such as a
microtiter well, a magnetic particle, or a plastic bead. This attachment
facilitates the separation of bound and free-labeled reactants.
In the most common approach to using the ELISA technique, an aliquot of
sample or calibrator containing the antigen (Ag) to be quantified is added to
and allowed to bind with a solid-phase antibody (Ab). After washing, an
enzyme-labeled antibody is added and forms a “sandwich complex” of solid-
phase Ab-Ag-Ab enzyme. Unbound antibody is then washed away, and
enzyme substrate is added. The amount of product generated is proportional
to the quantity of antigen in the sample.
Specific antibodies in a sample can also be quantified using an ELISA
procedure in which antigen instead of antibody is bound to a solid phase. The
second reagent is an enzyme-labeled antibody specific to the analyte
antibody. In addition, ELISA assays have been used extensively to detect
antibodies to viruses and autoantigens in serum or whole blood. In addition,
enzyme conjugates coupled with substrates that produce visible products
have been used to develop ELISA-type assays with results that can be
interpreted visually. Such assays are very useful in screening, point-of-care,
and home testing applications.

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dentification

ELISA is the basic assay technique, known as enzyme-linked

immunosorbent assay (also referred to as EIA: Enzyme
Immunoassay) that is carried out to detect and measure
antibodies, hormones, peptides and proteins in the blood.

Antibodies are blood proteins produced in response to a specific

antigen. It helps to examine the presence of antibodies in the
body, in case of certain infectious diseases.

ELISA is a distinguished analysis compared to other antibody-

assays as it yields quantitative results and separation of non-
specific and specific interactions that take place through serial
binding to solid surfaces, which is normally a polystyrene
multiwell plate.

Types Of ELISA

ELISA tests can be classified into three types depending upon

the different methods used for binding between antigen and
antibodies, namely:

 Indirect ELISA – Antigen is coated to the microtiter well

 Sandwich ELISA – Antibody is coated on the microtiter
well
 Competitive ELISA – Microtiter well which is antigen-
coated is filled with the antigen-antibody mixture.

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Indirect ELISA

 Indirect ELISA detects the presence of an antibody in a

sample.
 The antigen is attached to the wells of the microtitre plate.
 A sample containing the antibodies is added to the antigen-
coated wells for binding with the antigen.
 The free primary antibodies are washed away and the
antigen-antibody complex is detected by adding a
secondary antibody conjugated with an enzyme that can
bind with the primary antibody.
 All the free secondary antibodies are washed away. A
specific substrate is added which gives a coloured product.
 The absorbance of the coloured product is measured by
spectrophotometry.

Sandwich ELISA

 Sandwich ELISA helps to detect the presence of antigen in

a sample.
 The microtitre well is coated by the antibody.
 The sample containing the antigen is added to the well and
washed to remove free antigens.
 Then an enzyme-linked secondary antibody, which binds
to another epitope on the antigen is added. The well is
washed to remove any free secondary antibodies.

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  The enzyme-specific substrate is added to the plate to form
a coloured product, which can be measured.

Competitive ELISA

 Competitive ELISA helps to detect antigen concentration

in a sample.
 The microtitre wells are coated with the antigen.
 Antibodies are incubated in a solution having the antigen.
 The solution of the antigen-antibody complex is added to
the microtitre wells. The well is then washed to remove
any unbound antibodies.
 More the concentration of antigen in the sample, lesser the
free antibodies available to interact with the antigen, which
is coated in the well.
 The enzyme-linked secondary antibody is added to detect
the number of primary antibodies present in the well.
 The concentration is then determined by
spectrophotometry.

Also Read: Antigens and Immunology

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Principle of ELISA

ELISA works on the principle that specific antibodies bind the

target antigen and detect the presence and quantity of antigens
binding. In order to increase the sensitivity and precision of the
assay, the plate must be coated with antibodies with high
affinity. ELISA can provide a useful measurement of antigen-
antibody concentration.

ELISA Procedure

ELISA is one of the easiest blood tests that can be carried out. It
is rapid, quick and requires a blood sample of the patient. The
entire procedure of ELISA is mentioned below.

 An antibody is attached to a polystyrene plate which is a

solid surface and is attracted or has an affinity towards
bacteria, other antibodies and hormones.
 A microtiter coated with antigen is filled with this antigen-
antibody mixture after which free antibodies are removed
by washing.
 A second antibody specific to primary antibody is added
which is usually conjugated with an enzyme.
 Free enzyme-linked secondary antibodies are removed by
washing the plate.
 Finally, the substrate is added. The substrate is converted
by the enzyme to form a coloured product, which can be
measured by spectrophotometry.
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HCG protein which indicates pregnancy is detected by ELISA.
A combination of blood or urine sample and purified HCG
linked to an enzyme is added to the system. If HCG is absent in
the test sample, then only the linked enzyme binds to the solid
surface.

The more the substance of interest is present, the more reaction

takes place and less of linked enzyme binds to the solid surface.
These reactions are indicated usually with a change in the colour
of the solution.

Diseases That Can Be Diagnosed Using ELISA

ELISA can be used to detect some of these conditions:

 Ebola
 Pernicious anaemia
 AIDS
 Rotavirus
 Lyme disease
 Syphilis
 Toxoplasmosis
 Zika virus
 Carcinoma of the epithelial cells

Further Reading: Blood Groups

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Advantages Of ELISA

Following are some of the advantages of the ELISA technique:

 Results fetched from ELISA gives an accurate diagnosis of

a particular disease since two antibodies are used.
 Can be carried out for complex samples as the antigen is
not required to get purified to detect.
 It is highly responsive since direct and indirect analysis
methods can be carried out.
 It is a rapid test, yields results quickly.
 Possible detection for ELISA ranges from the quantitative,
semi-quantitative, standard curve, qualitative, calibration
curve models etc.
 Easier to perform and uncomplicated process as compared
to other assays which require the presence of radioactive
materials.

Applications of ELISA

The applications of ELISA are discussed below:

1. The presence of antibodies and antigens in a sample can be

determined.
2. It is used in the food industry to detect any food allergens
present.
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3. To determine the concentration of serum antibody in a
virus test.
4. During a disease outbreak, to evaluate the spread of the
disease, e.g. during recent COVID-19 outbreak, rapid
testing kits are being used to determine presence of
antibodies in the blood sample

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Quality Control and Lab Safety

As with quantitative procedures, it is important to verify that the

results of qualitative and semi-quantitative examinations are
correct before reporting them to the requesting healthcare
provider. The laboratory must establish a quality control
program for all of its qualitative and semi-quantitative
tests. When establishing this program, set policies, train staff,
assign responsibilities, and ensure all necessary resources are
available. Ensure that the recording of all quality control data is
complete and that the quality manager and the laboratory
director conduct an appropriate review of the information

Positive and negative controls are recommended for many

qualitative and semi-quantitative tests, including some
procedures that use special stains or reagents and tests with
endpoints such as agglutination or color change. These controls
should generally be used with each test run. The use of controls
will also help validate a new lot number of test kits or reagents,
check on temperatures of storage and testing areas, and evaluate
the process when new testing personnel is carrying out the
testing.

Keep the following in mind when using traditional controls for

qualitative or semi-quantitative tests: test control materials in the
same manner as testing patient samples; use a positive and
negative control, preferably once each day of testing, or at least
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as often as recommended by the manufacturer; choose positive
controls that are close to the cut-off value of the test, to be sure
the test can detect weak positive reactions; for agglutination
procedures, include a weak positive control as well as a negative
control and stronger positive control.

Basic safety rules for laboratory conduct should be observed

whenever working in a laboratory. Consider all specimens,
control materials, and calibrator materials as potentially
infectious. Exercise the usual precautions required for handling
all laboratory reagents. Disposal of all waste material should be
in accordance with local guidelines. Wear gloves, a lab coat, and
safety glasses when handling human blood specimens. Place all
plastic tips, sample cups, and gloves that come into contact with
blood in a biohazard waste container. Discard all disposable
glassware into sharps waste containers.

Protect all work surfaces with disposable absorbent bench top

paper, discarded into biohazard waste containers weekly or
whenever blood contamination occurs. Wipe all work surfaces
weekly. All equipment should be regularly inspected for wear or
deterioration. Equipment should be maintained according to the
manufacturer’s requirements, and records of certification,
maintenance, or repairs should be maintained for the life of the
equipment. Computers and instrumentation should be labeled to
indicate whether gloves should be worn. Inconsistent glove use
around keyboards/keypads is a source of potential
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contamination. Avoid wearing jewelry in the lab, as this can
pose multiple safety hazards. Safety rules for laboratory-specific
operations will be provided in appropriate laboratory SOPs.

Enhancing Healthcare Team Outcomes

ELISA testing is an important part of medical care and scientific

research. Collaboration between scientists, laboratory
technicians, phlebotomists, clinicians, nurses, and other medical
professionals is necessary for appropriate specimen collection,
testing, interpretation, diagnosis, and effective patient education
and treatment planning. ELISA technologies continue to grow
and play a major role in clinical research allowing for
the development of more diagnostic and screening tests. The
continued evolution of ELISA testing is promising for the future
of medicine and has improved early diagnosis of HIV and
pregnancy detection.

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 References:

1. Aydin S. A short history, principles, and types of ELISA, and our laboratory
experience with peptide/protein analyses using ELISA. Peptides. 2015
2. Engvall E. The ELISA, enzyme-linked immunosorbent assay. Clin
Chem. 2010
3. Shah K, Maghsoudlou P. Enzyme-linked immunosorbent assay (ELISA): the
basics. Br J Hosp Med (Lond). 2016
4. Konstantinou GN. Enzyme-Linked Immunosorbent Assay (ELISA). Methods
Mol Biol. 2017;
5. Leng SX, McElhaney JE, Walston JD, Xie D, Fedarko NS, Kuchel GA.
ELISA and multiplex technologies for cytokine measurement in
inflammation and aging research. J Gerontol A Biol Sci Med Sci. 2008
6. Gelkop S, Sobarzo A, Brangel P, Vincke C, Romão E, Fedida-Metula S,
Strom N, Ataliba I, Mwiine FN, Ochwo S, Velazquez-Salinas L, McKendry
RA, Muyldermans S, Lutwama JJ, Rieder E, Yavelsky V, Lobel L. The
Development and Validation of a Novel Nanobody-Based Competitive
ELISA for the Detection of Foot and Mouth Disease 3ABC Antibodies in
Cattle. Front Vet Sci. 2018.
7. de la Rica R, Stevens MM. Plasmonic ELISA for the ultrasensitive detection
of disease biomarkers with the naked eye. Nat Nanotechnol. 2012 .
8. Tabatabaei MS, Ahmed M. Enzyme-Linked Immunosorbent Assay
(ELISA). Methods Mol Biol. 2022.
9. Lin AV. Direct ELISA. Methods Mol Biol. 2015.

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