IMMUNO-CHROMATOGRAPHIC ANTIGEN-

DETECTION AND ELISA TESTS

PROF. T. B. KWOFIE

UNIVERSITY OF HEALTH AND ALLIED SCIENCES

SCHOOL OF MEDICINE
DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY
 Medical Laboratory Tests

 A medical laboratory test is a procedure in which a sample

of blood, urine, other bodily fluid, or tissue is examined to get
information about a person’s health.

 Some laboratory tests provide precise and reliable information

about specific health problems.

 Other tests provide more general information that helps doctors

identify or rule out possible health problems.

 Doctors may also use other types of tests, such as imaging tests, in
addition to laboratory tests to learn more about a person’s health.
Diagnostic Methods in Medical Laboratory

1. Direct Examination
2. Indirect Examination (Virus Isolation)
3. Serology
 Serology Laboratory

• Antigen and Antibody Reaction test is the test of serology

blood test which is performed to detect and measure the
presence and levels of antibodies as a result of exposure
to a particular bacteria or virus (Antigen).

• When we are exposed to bacteria or viruses (antigens),

the body’s immune system produces specific antibodies
against the organism.

• Antibody levels (antibody titer) help physicians to

determine whether an infection had taken place and
whether the infection occurred recently, or occurred
years ago.
 Rational
• An antibody (Ab), also known as an immunoglobulin (Ig), is a large Y-
shaped protein produced by B cells that is used by the immune system to
identify and neutralize foreign objects such as bacteria and viruses (Ag).

• The antibody recognizes a unique part of the foreign antigen, called an

epitope.

• Each tip of the "Y" of an antibody contains a paratope (a structure

analogous to a lock) that is specific for one particular epitope (similarly
analogous to a key) on an antigen, allowing these two structures to bind
together with precision.

• Using this binding mechanism, an antibody can tag a microbe or an infected

cell for attack by other parts of the immune system, or can neutralize its
target directly (for example, by blocking a part of a microbe that is essential
for its invasion and survival).

• This essentially is the Antigen and Antibody Reaction

• In practice, the term, Antigen and Antibody Reaction, usually refers
to the diagnostic identification of antibodies in the serum.

• Antibodies can accurately find and detect almost any molecule.

• They are therefore ideal "search engines" for detecting minuscule

amounts of a particular target molecule, and are used daily as tools,
or reagents, in research laboratories worldwide.

• Antibodies also play a crucial role in diagnosing various conditions,

such as testing blood serum for the presence of various pathogens.

• Arguably their most important application is as drug treatments

• Anibody molecules are multivalent and antigens are
also often multivalent.

• This multivalency tends to increase the strength of

the interaction, and this really represents the true
state of affairs.

• This overall binding energy that results in the

binding of a multivalent antibody with a multivalent
antigen is called the functional affinity or the
avidity.
 Affinity and Avidity of Antibodies

• Antigen-antibody interactions are non-covalent and reversible, formed by a

combination of hydrogen bonds, hydrophobic interactions, electrostatic and
van der Waals forces.

• When describing the strength of the antigen-antibody complex, affinity and

avidity are always mentioned.

• Affinity measures the strength of interaction between an epitope and an

antibody’s antigen binding site.

• Avidity gives a measure of the overall strength of an antibody-antigen complex.

• Avidity is dependent on three major parameters:

 Affinity of the antibody for the epitope

 Valency of both the antibody and antigen
 Structural arrangement of the parts that interact
 MALARIA RAPID DIAGNOSTIC TESTS (RDTS)
 RDTs are lateral flow immuno-chromatographic antigen-
detection tests, which rely on the capture of dye-labeled
antibodies to produce a visible band on a strip of nitro-
cellulose, often encased in plastic housing, referred to as
cassettes.

 Malaria rapid diagnostic tests (RDTs) assist in the

diagnosis of malaria by providing evidence of the
presence of malaria parasites in human blood.

 RDTs are an alternative to diagnosis based on clinical

grounds or microscopy, particularly where good quality
microscopy services cannot be readily provided.

 Malaria RDTs detect specific antigens (proteins) produced

by malaria parasites in the blood of infected individuals.

 Some RDTs can detect only one species (Plasmodium

falciparum) while others detect multiple species
(P. vivax, P. malariae and P. ovale).
 Blood for the test is commonly obtained from a finger-
prick.
 RDT cassette
 With malaria RDTs, the
dye-labeled antibody first
binds to a parasite antigen,
and the resultant complex is
captured on the strip by a
band of bound antibody,
forming a visible line (T -
test line) in the results
window.  Inside the cassette is a strip made of
filter paper and nitrocellulose.

 A control line (C- control  Typically, a drop of blood is added to

line) gives information on the RDT through one hole (A; sample
well), and then a number of drops of
the integrity of the
buffer usually through another hole (B;
antibody-dye conjugate, but
buffer well).
does not confirm the ability
 Buffer carries the blood along the
to detect parasite antigen.
length of the RDT.
Mode of action of common malaria RDT format
1. The first step of the test procedure involves mixing the patient’s
blood with a lysing agent in a test strip or well. This ruptures the
red blood cells, releasing more parasite protein.
2. Dye-labeled antibody, specific for target antigen, is present on
the lower end of nitrocellulose strip or in a plastic well provided
with the strip. Antibody, also specific for the target antigen, is
bound to the strip in a thin (test) line, and either antibody
specific for the labeled antibody, or antigen, is bound at the
control line.
3. Blood and buffer, which have been placed on strip or in the well,
are mixed with labeled antibody and are drawn up the strip
across the lines of bound antibody.
4. If antigen is present, some labeled antibody-antigen complex will
be trapped and accumulate on the test line.
 Excess-labeled antibody is trapped and accumulates on the
control line.
 A visible control line indicates that labeled antibody has
traversed the full length of the strip, past the test line, and that
at least some free antibody remains conjugated to the dye and
that some of the capturing properties of the antibodies remain
intact.
5. The intensity of the test band will vary with the amount of
antigen present, at least at low parasite densities (antigen
concentration), as this will determine the amount of dye particles
which will accumulate on the line.

The control band intensity may decrease at higher parasite

densities, as much of the labeled antibody will have been captured
by the test band before reaching the control.
Specific Viral Diagnosis:
Rapid and ELISA Tests
 Rationale for Specific Viral Diagnosis

• About 70% of all human diseases are caused by viruses yet historically,
diagnostic virology has had to justify its use

• The reasons have been that traditional viral diagnostic techniques, especially
culture, are slow, expensive, and often peripheral to clinical decision-making,
particularly when no therapeutic agents are available.

• The availability of antiviral therapeutic agents such as acyclovir, ganciclovir,

foscarnet, cidofovir, antiretroviral drugs, neuraminidase inhibitors, and IFN-α
that are effective for specific viral infections but expensive (and in some cases
potentially toxic) has created an obvious need for specific viral diagnosis.

• In some cases, establishing a specific viral diagnosis limits other diagnostic

procedures and may allow discontinuation of antibiotic therapy

• Likewise, in some cases, confirmation of a specific viral diagnosis helps in

determining prognosis
• Diagnostic virology is rapidly moving into the mainstream of clinical medicine
as a result of the convergence of several independent developments, namely:

1. Progress in antiviral therapeutics has increased the need for specific viral
diagnoses.

2. Technological developments, particularly in the area of nucleic acid

chemistry, have provided important new tools for viral diagnosis.

3. The number of patients at risk for opportunistic viral infections has

expanded greatly as a result of the HIV/AIDS epidemic.

4. Modern management of HIV infection and hepatitis C is providing a new

paradigm for the integration of molecular techniques into management of
chronic viral infections.

• These developments are not only increasing the use of diagnostic virology but
are reshaping the field.
 Specimen choice and collection
• Specimen quality limits test quality

• Pathogen detection depends on:

– Appropriate collection site.
– Proper timing of specimen collection.
– Effective and timely processing of sufficient
specimen.
 Specimen storage and transport
• Keep specimens other than blood at 4ºC
• If delay >24hrs, freeze at -70ºC or below.
• Avoid any storage at -20ºC: greater loss in
infectivity
• Non-enveloped viruses (adenovirus,
enteroviruses) more stable than enveloped
(e.g. RSV, VZV, CMV).
 Viral Transport Medium
• Salt solution – ensures proper ionic
concentrations

• Buffer - maintains pH

• Protein - for virus stability

• Antibiotics or antifungals – to prevent

contamination
 Diagnostic Methods in Virology

1. Direct Examination
2. Indirect Examination (Virus Isolation)
3. Serology
 The role of Serology in Virus Diagnosis

• Serology remains the mainstay for the diagnosis of virus infections in a routine
diagnostic laboratory, especially for the diagnosis of virus infections.

• However, it plays a much lesser or absent role in other areas of virological testing, such
as epidemiological research, monitoring the response to anti-viral therapy, and
antiviral resistance testing.

• Serology remains the bulk of the work carried out by a routine diagnostic virus
laboratory.

• A large variety of serological tests are available eg. complement-fixation (CFT) ,

haeagglutination-inhibition (HAI), enzyme-linked immunoassay (EIA),
radioimmunoassay (RIA), particle agglutination, immunofluorescence, single radial
haemolysis, western blot etc.

• The sensitivity and specificity varies greatly between different techniques.

• Most techniques will detect all classes of antibody whereas some assays eg. RIA, EIA
and IF can be made to detect one specific class only ie. IgM, IgG or IgA.
 Serology
Detection of rising titres of antibody between acute and convalescent
stages of infection, or the detection of IgM in primary infection.

Classical Techniques Newer Techniques

1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)

2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)
3. Immunofluorescence techniques (IF) 3. Particle agglutination
4. Neutralization tests 4. Western Blot (WB)
5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay
 What types of specimens are collected to diagnose?

Respiratory tract infections Nasal and bronchial washings, throat and

nasal swabs, sputum
Eye infections throat and conjunctival swab/scraping
Gastrointestinal tract stool and rectal swabs
infections
Vesicular rash vesicle fluid, skin scrapings
Maculopapular rash throat, stool, and rectal swabs
CNS (encephalitis and stool, tissue, saliva, brain biopsy,
meningitis cases) cerebrospinal fluid
Genital infections vesicle fluid or swab
Urinary tract infections urine
Bloodborne infections blood
ELISA test
 Principle of ELISA Test

 ELISA tests are the standard test for

finding out if someone has antibodies to
a particular antigen.

 ELISA stands for Enzyme-Linked

Immunosorbent Assay.

 Surface of solid phase (microtitre plate)

coated with antibody

 Antigen of interest binds if present.

 Second enzyme-conjugated antibody

added

 Substrate added and colour

generated/read by spectrophotometer.
 ELISA Serological Test
I. The ELISA test is one of the main laboratory testing methods of viral
infections.
II. ELISA is a common laboratory testing technique that detects and counts
certain antibodies, antigens, proteins and hormones in bodily fluid
samples such as blood, plasma, urine, saliva and cerebrospinal fluid
(CSF).
[Link] stands for “ENZYME-LINKED IMMUNOSORBENT ASSAY.” Another
name for it is an EIA test.
[Link] consider ELISA to be the gold standard of immunoassays.
V. Tests that use ELISA can help diagnose a wide range of conditions, from
bacterial and viral infections (like Lyme disease and HIV)
to endocrine conditions, like thyroid disease to even pregnancy tests (in
pregnancy tests they detect the presence of a hormone called human
chorionic gonadotrophin (HCG)
What then is ELISA test? Antigens are usually proteins
ELISA test are immunoassays or sugars found on the
I. Immunoassays are tests that rely on the surfaces of cells or viruses.
interaction between antigens and antibodies in Antigens exist on several
a laboratory setting
types of cells, including:
II. In this ELISA test, enzymes such as alkaline
 Viruses. ???
phosphatase, horseradish peroxidase or beta-
galactosidase are conjugated to form secondary  Bacteria.
antibodies, also called detection antibodies,  Allergens, like food
which bind to the primary viral antigen-
allergens or airborne
antibody complexes to form a coloured end-
product that can be visualized and quantified allergens.
when the appropriate substrate is added and  Parasites.
the sample incubated.
 Proteins.
[Link] intensity of the color change is proportional
to the amount of the antibody. So ELISA can
 Tumor cells.
determine both the presence of the antibody  Normal cells in your body.
and how much of it there is.
TYPES OF ELISA
 Optical Density (OD) value

 What does the number after the HIV ELISA

test result mean?

 Some people are given test results which say

something like ‘non-reactive (OD: 0.219)’.
This number at the end is called the Optical
Density (OD) value.

 This is the measure of how much colour

there is in the dish at the end of the ELISA.

 As the colour in the dish is an indicator of

whether the result is negative or positive
these numbers give the result more precisely
than just a simple ‘positive’ or ‘negative’
answer.
ELISA Microplate for HIV antibody Test:
coloured wells indicate reactivity
 The cut-off values for different tests vary. In
general, any numbers below 1.0 mean it’s a
negative result. Any numbers above 1.0
mean it’s a positive result.
 Rapid Tests
 Rapid tests are a simplified  The buffer causes the antibodies in
version of antibody ELISA tests. the blood to flow along the test
stick.
 They look for HIV antibodies in
the blood.  When they pass over the section
with the antigens, if there are any
 The antigens for HIV are fixed on antibodies for HIV present then
one particular strip along the they will stick to these antigens and
rapid test stick. change colour.

 Towards the end of the testing  Once the test is complete, if there is
stick are control antigens to show one stripe it means it is a negative
that the test worked. result. If there are two stripes then
it means it’s a positive result. If
 A sample is placed at the end of there are no stripes it means the
the testing stick. Then a buffer, to test did not work properly.
facilitate the testing process is
added.
 Summary
• Viral diagnostics is a dynamic field

• New applications of molecular technology being

introduced continuously

• Increased therapeutic options for viral

infections have increased the clinical relevance
of making a viral diagnosis.