Understanding ELISA
Tests
Lab: 4
Pharmacy/2nd stage
Medical Microbiology
2025
Asst.Proff. Dr. Lina A. Hassan
A.L. Hassan Al Ardawi
A.L. Kawther H.Sabti
What is an ELISA test ?
ELISA, or Enzyme-Linked ImmunoSorbent
Assay, detects proteins, hormones and
antibodies or antigens (microbes) in biological
samples. It's a versatile technique with
numerous applications in diagnostics and
research.
Diagnosing with ELISA
1 Antibody Detection 4 Past Exposures
Detects autoantibodies in Detects past exposures to diseases
autoimmun disease as ANA like Hepatitis (IgG).
(Antinuclear Antibodies) and
antibodies against infectious diseases 5 Drug Abuse
(Varicella-Zoster virus,
Screens for drug abuse,
Toxoplasmosis ,Syphilis...etc).
2 Tumor Markers including amphetamine and
Estimates levels of tumor markers like cocaine.
.prostate-specific antigen (PSA) 6 Infectious Diseases (Antigen)
Detects on fungi, viruses, bacteria, and
3 Hormone Levels parasites.
Detects and estimates hormone levels
such as Thyroid , prolactin and
.testosterone
Sample Preparation
Serum
Collect whole blood in a test tube then leave it at
room temperature for 20 minutes and centrifuge it.
Immediately aliquot supernatant (serum) and store
samples at -80°C.
Plasma
Collect whole blood into the anticoagulant-containing
tube, then centrifuge it. Immediately withdraw the
supernatant (plasma) and store samples at -80°C.
.
Urine and Saliva
Collect samples and centrifuge it. withdraw the
supernatant and store samples at -80°C.
Basic Components of ELISA:
• 96 or 384-well plates
• Primary antibodies
• Secondary antibodies
• Enzymes (peroxidase/alkaline
phosphatase)
• Substrate (chromagen)
• Plate reader for measurement
ELISA Principles
In an ELISA assay, the antigen is immobilized to a solid surface
(wells). This is done either directly or via the use of a capture
antibody itself immobilized on the surface. The antigen is then
complexed with a detection antibody conjugated with an enzyme
(peroxidase ) a molecule responsive for detection.
ELISA Results
The enzyme converted a colorless substrate into a
colored product. This indicates the presence of the
target antibody. The amount of colored product
indicates the quantity of the antibody. Higher color
intensity means more antibody present. After an
incubation period, the secondary antibody solution is
Determined The Optical
removed, and loosely adherent Densi
ones are washed off as
before.
The plate is placed in a plate reader and the optical densi
amount of colored product) is determined for each well.
Types of ELISA
There are four main types of ELISA: Direct, Indirect, Sandwich and Competitive ELISA.
Each has unique advantages, disadvantages and suitability.
1-Direct ELISA
How it Works
In a direct ELISA, the antigen is immobilized directly onto
the surface of a multi-well plate. An enzyme-labeled
primary antibody, specific to the antigen, is then added.
This antibody binds to the antigen, and when a substrate
is introduced, the enzyme catalyzes a visible color
change.
Detection
The color change is measured using a spectrophotometer
or absorbance microplate reader. Direct ELISAs are
suitable for both qualitative and quantitative detection
of antigens in various samples.
2-Indirect ELISA
The plate is first coated with a specific capture antigen. The target antibody (primary antibody) attaches
to this antigen, forming an antigen-antibody complex. A secondary antibody is used to detect the primary
antibody. This secondary antibody is typically enzyme-linked, allowing for detection via a substrate
reaction.
Applications
Indirect ELISA is frequently used to determine the outcome of an immunological response, such as
measuring the concentration of an antibody in a sample.
3-Sandwich ELISA
The plate is coated with a capture antibody. The sample is added, and any antigen present binds to
the capture antibody. A primary antibody is added, which binds to the antigen. An enzyme-linked
secondary antibody is then added, binding to the detecting antibody. Finally, a substrate is added,
which is converted by the enzyme into a detectable form, allowing for quantification of the antigen.
It is termed a “sandwich” because the antigens are sandwiched between two layers of antibodies
(capture and detection antibodies).*The sandwich ELISA has the highest sensitivity and specificy
among all the ELISA types. The major disadvantages of this type are the time consumption and
.
expense.
.
4-Competitive ELISA
1 Incubation 2 Washing 3 Detection
Unlabeled antibody is incubated The plate is washed to remove A secondary antibody, specific to
with its antigen (sample). These unbound antibodies. The more the primary antibody and coupled
bound antibody/antigen antigen in the sample, the fewer to an enzyme, is added. A substrate
complexes are then added to an unbound antibodies are available is then added, producing a
antigen-coated well. to bind to the antigen in the well. chromogenic or fluorescent signal.
hence "competition“.
Applications of Competitive ELISA
Advantage
Despite its limitations, competitive ELISA is
particularly useful for measuring low
molecular weight targets, such as
histamine, where other ELISA methods may
be less effective.
Disadvantage
The competitive ELISA has a lower
specificity compared to other ELISA types,
which can limit its use in complex samples.
Choosing the Right ELISA
Assay
Selecting the appropriate ELISA assay depends
on the specific application and the
characteristics of the target antigen or antibody.
Direct ELISAs are simple and quick, while indirect
ELISAs offer increased sensitivity and measuring
antibody concentrations. Sandwich ELISAs
provide high specificity and sensitivity, and
competitive ELISAs are suitable for small
molecules.
