Immunodiagnostics

Definition of terms
• Antigen = foreign substance that, when introduced into the
body, is capable of stimulating an immune reaction e.g.
foreign molecules in bacteria, viruses, protozoa, serum
components, etc.

• Antibody also known as immunoglobulin = large protein

produced by plasma B cells which identifies antigens

• Immune reaction = reversible binding of antigen to

antibody.
Immune reaction: Antigen-Antibody
reaction (Ag-Ab)
High specificity:
Antigen-binding site of the
antibody molecule perfectly
matches the antigen
(”key in hole”)
 Immunodiagnostics
In immunodiagnostics, the same basic principle
is applied: specific detection and binding of
antigen by antibody → Ag-Ab complex
 Types of Immunoassays
• Agglutination

• Enzyme-linked Immunosorbent Assay (ELISA)

• Immunofluorescence

• Lateral Flow Immunoassays

• Immunoblotting, Western blot

 Agglutination

•Agglutination is the process whereby specific antigens

aggregate to form larger visible clumps when the
corresponding specific antibody is present in the serum.

•The antibodies or the antigens are conjugated to a carrier.

•Carriers used in agglutination methods could be artificial

(e.g. latex or charcoal) or biological (e.g. erythrocytes ).
A. Latex Agglutination
• In latex agglutination procedures,
antibody molecules are bound to the
surface of latex beads.
• Many antibody molecules are bound to
each latex particle, increasing the
potential number of exposed antigen-
binding sites.
• If an antigen is present in a test specimen
the antigen will bind to the combining
sites of the antibody, forming visible
cross-linked aggregates of latex beads and
antigen.
• In some procedures, latex particles can be
coated with antigen. In the presence of
serum antibodies, these particles
agglutinate into large visible clumps.
 Examples…..

1. Pregnancy Testing
• Pregnancy tests are designed to detect minute amounts of human
chorionic gonadotropin (hCG), a hormone secreted by the
developing embryo that rapidly increases in the urine or serum
during the early stages of pregnancy.
• The hormone consists of two alpha (α) and beta (β) subunits.
• Many pregnancy test kits contain antibody directed against the β
subunit of the hormone.
• The latex particles are coated with the anti-hCG antibodies that will
agglutinate with the hCG, if it is present in the sample.
1. Pregnancy Testing

Results:
• Agglutination
within 2 minutes
represents a positive
reaction.

• No agglutination
represents a
Antigen e.g. b-subunit
hCG
negative reaction.
 2. Test for rheumatoid factor (RF)

• Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting

primarily the joints.
• It is diagnosed by the presence of the Rheumatoid Factor (RF),
which are autoantibodies that are generated by the affected
individual.
• The RF is actually an IgM class of antibody that reacts with the
affected individual’s IgG, leading to activation of the immune system
and destructive inflammation, causing rheumatoid arthritis.
 2. Test for rheumatoid factor (RF)
Principle of RF test
• A number of methods are available for testing of RF. The most
commonly used serological method is based on latex
agglutination test.
• If RF is present in the blood of the patient, it will agglutinate
the latex particles coated with IgG molecule.
 Test for rheumatoid factor (RF)

Interpretation:
Agglutination of latex particles is considered a positive reaction, indicating
the presence of rheumatoid factor at a significant and detectable level.
• Positive result: An agglutination of the latex particles suspension will
occur within two minutes.
• Negative result: No agglutination of the latex particles suspension
within two minutes.
 ELISA

• ELISA (enzyme-linked immunosorbent assay) is a plate-

based assay technique designed for detecting and
quantifying soluble substances such as proteins,
antibodies, and hormones in samples such as serum,
plasma, saliva, tissue lysates, and urine.
 ELISA
• In an ELISA, the antigen is immobilized on a solid surface
(microplate) and then complexed with an antibody that is linked
to a reporter enzyme.
• A colorless substrate (chromogen) is added. The reporter enzyme
converts the substrate into a measurable colored product.
• The most crucial element of an ELISA is a highly specific antibody-
antigen interaction.
There are different formats of ELISA:

• Direct

• Indirect

• Sandwich
 Direct ELISA

• In a direct ELISA, an antigen or sample is immobilized

directly on the plate and a conjugated detection antibody
binds to the target protein.

• Substrate is then added, producing a signal that is

proportional to the amount of analyte in the sample.
 Direct ELISA
Application: 

Mainly for research purposes such as:

• Assessing antibody affinity and specificity.

Advantages:
•Fast and simple protocol

Disadvantages:
•Less specific since using only 1 antibody.
•Potential for high background if all proteins from a sample are
immobilized in well.
 Indirect ELISA

• An antigen is immobilized on a plate.

• First, an unconjugated primary detection antibody is added
and binds to the specific antigen.
• A conjugated secondary antibody directed against the primary
antibody is then added.
• Substrate then produces a signal proportional to the amount of
antigen bound in the well.
 Indirect ELISA

Application: 
 

• Measuring endogenous antibodies.

Advantages:

•Amplification using a secondary antibody

Disadvantages:

•Potential for cross-reactivity caused by secondary antibody

 Sandwich ELISA
• Sandwich ELISAs are the most common type of ELISA.
• Two specific antibodies are used to sandwich the antigen,
commonly referred to as matched antibody pairs.
• Capture antibody is coated on a microplate, sample is added, and
the protein of interest binds and is immobilized on the plate.
• A conjugated-detection antibody is then added and binds to an
additional epitope on the target protein. Substrate is added and
produces a signal that is proportional to the amount of analyte
present in the sample.
• Sandwich ELISAs are highly specific, since two antibodies are
required to bind to the protein of interest.
 Sandwich ELISA

Applications: 

Determining antigen presence in a biological sample.

Advantages:

•Highest specificity and sensitivity

Disadvantages:

•Longer protocol
•Challenging to develop
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
 SARS-CoV-2 (COVID-19) ELISA Kit

• SARS-CoV-2 (COVID-19) ELISA kit is intended for the qualitative

determination of IgG class antibodies against SARS-CoV-2 in
human serum to support the diagnosis of COVID-19 disease and
constitutes a supplement to direct pathogen detection.

• By testing antibodies among the general public through random

sampling (i.e., serosurvey), public health agencies can estimate the
true size of infection (prevalence) and its fatality rate.
SARS-CoV-2 (COVID-19) ELISA Kit
 SARS-CoV-2 (COVID-19) ELISA Kit
• Microtiter plates are coated with specific antigens to bind
corresponding antibodies of the sample.
• The patient sample is added to the microtiter plate and incubated,
so that the patient Ab can bind to the specific antigens on the
plate.
• The wells are washed to remove all unbound sample material.
• Then horseradish peroxidase (HRP) labelled conjugate is added
which binds to the captured antibodies.
• In a second washing step unbound conjugate is removed.
• The immune complex is visualized by adding
Tetramethylbenzidine (TMB) substrate which gives a blue reaction
product. The intensity of this product is proportional to the amount
of specific antibodies in the sample. Absorbance at 450/620 nm is
read using an ELISA Microtiter plate reader.
 Dengue NS1 Antigen ELISA kit
• Dengue is an acute viral disease which is transmitted by Aedes
mosquitoes. Dengue is characterized clinically by biphasic fever,
rash and hematopoietic depression etc.
• Dengue virus (DENV) is a single-stranded RNA virus. The DENV
genome encodes 3 structural (capsid C, membrane M, and
envelope E) and 7 nonstructural (NS1, NS2A, NS2B, NS3, NS4A,
NS4B, and NS5) proteins. 
• Dengue NS1 protein is a hexametric secreted protein.
 Dengue NS1 Antigen ELISA Test Principle

• This Dengue NS1 ELISA kit uses sandwich ELISA to detect

low levels of NS1 antigen in circulating blood during acute
Dengue infection.
• In the Dengue NS1 ELISA kit the wells are coated with a
highly effective NS1 capture antibody.
• The patient serum is added to the microtiter plate and
incubated.
• Wash to remove unbound materials.
• Then the HRP-conjugated secondary antibody is added.
• Wash to remove unbound conjugate.
• Add TMB substrate.
• Color change indicates positive result.
Dengue NS1 Antigen ELISA Test Principle
ELISA can detect:
•Antibodies to HIV that the immune system developed in response to HIV infection
•HIV proteins, such as p24, that were developed by HIV.