1. ELISA is an immunoassay technique used to detect the presence of antigens or antibodies in samples. It relies on antibodies and enzyme-linked detection to provide sensitive, quantitative results.
2. There are several types of ELISA including direct, indirect, sandwich, and competitive, which differ in their use of capture and detection antibodies.
3. The ELISA procedure involves immobilizing an antibody on a plate, adding samples and detection reagents, washing away unbound material, and measuring enzyme activity to determine antigen or antibody levels.
1. ALISA Technique - Principles
-Types -How Can Make Kit to
Determine Any Biomarker
Assist. Prof. Dr. Shakir Faris Tuleab
Ph. D. Biochemistry
University of Anbar
College of Education For Pure Sciences
Chemistry Department
2. Enzyme Linked Immunosorbent Assay (ELISA)
Term Was Coined By Engvall and Pearlmann in 1971
Different Types
Sandwich, Indirect, Competitive----
Similar To RIA, Except No Radiolabel
Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal or polyclonal Abs
• 2 Antibodies Required
• Must Recognize different epitopes
• 1st Antibody is referred to as capture Ab
• 2nd Antibody detection Ab
• 2nd Antibody is biotinylated
• Enzymes Commonly Used: HRP (Horse Radish Peroxidase) And ALP
(Alkaline Phosphatase)
• Substrate is TMB (Chromogen)
3. The principle and method of ELISA
Enzyme-linked immunosorbent assay (ELISA) is a method of target antigen (or
antibody) capture in samples using a specific antibody (or antigen), and of target
molecule detection/quantitation using an enzyme reaction with its substrate.
The principle
In ELISA, various antigen-antibody combinations are used, always including an
enzyme-labeled antigen or antibody, and enzyme activity is measured colorimetrically.
The enzyme activity is measured using a substrate that changes color when modified by
the enzyme. Light absorption of the product formed after substrate addition is measured
and converted to numeric values.
Depending on the antigen-antibody combination, the assay is called a direct ELISA,
indirect ELISA, sandwich ELISA, competitive ELISA etc.
4. Definitions
Antibodies (also known as immunoglobulins
abbreviated Ig) are gamma globulin proteins that are
found in blood and are used by the immune system to
identify and neutralize foreign objects, such as
bacteria and viruses.
Antigens A substance that when introduced into
the body stimulates the production of an antibody.
Immunoassay A laboratory technique that
makes use of the binding between an antigen and its
homologous antibody in order to identify and quantify
the specific antigen or antibody in a sample
5.
6.
7. 1- Direct ELISA
A target protein (or a target antibody) is immobilized on the surface of
microplate wells and incubated with an enzyme-labeled antibody to the
target protein (or a specific antigen to the target antibody). After
washing, the activity of the microplate well-bound enzyme is
measured.
ELISA Types
8. 2- Indirect ELISA
A target protein is immobilized on the surface of microplate wells and
incubated with an antibody to the target protein (the primary antibody),
followed by a secondary antibody against the primary antibody. After
washing, the activity of the microplate well-bound enzyme is measured.
Although indirect ELISA requires more steps than direct ELISA, labeled
secondary antibodies are commercially available, eliminating the need to
label the primary antibody.
9. 3- Sandwich ELISA
An antibody to a target protein is immobilized on the surface of microplate wells
and incubated first with the target protein and then with another target protein-
specific antibody, which is labeled with an enzyme. After washing, the activity of
the microplate well-bound enzyme is measured. The immobilized antibody (orange)
and the enzyme-labeled antibody (green) must recognize different epitopes of the
target protein.
Compared to direct ELISA, the sandwich ELISA (combining antibodies to two
different epitopes on the target protein) has a higher specificity.
Sandwich ELISA is useful for applications that require a high accuracy.
10. 4- Competitive ELISA
An antibody specific for a target protein is immobilized on the surface of microplate
wells and incubated with samples containing the target protein and a known amount
of enzyme-labeled target protein. After the reaction, the activity of the microplate
well-bound enzyme is measured.
When the antigen level in the sample is high, the level of antibody-bound enzyme-
labeled antigen is lower and the color is lighter. Conversely, when it is low, the level
of antibody-bound enzyme-labeled antigen is higher and the color, darker. The graph
above and to the right illustrates the correlation between absorption and antigen
levels in samples.
11.
12. When a target antigen is a small molecule, such as histamine,
pesticide, and dioxin, two antibodies cannot simultaneously bind to
the antigen in sandwich ELISA. Competitive ELISA is useful for the
measurement of low molecular weight targets.
• Immobilization of antibody/antigen on microplate wells
• How are proteins bound (adsorbed) to the wells?
• Immobilization is commonly mediated by hydrophobic interaction
or covalent bonds.
Microplates that are pre-activated for different immobilization
methods are commercially available. The choice of method of
immobilization depends on the type of protein to be immobilized.
In general, covalent bonding is preferred for smaller molecules,
such as peptides, and hydrophobic interaction (physical adsorption)
is preferred for larger molecules, such as proteins. An appropriate
method should be determined for each assay system.
13. Pre-treatment of microplates
• Many types of ELISA microplates are commercially
available, including the hydrophobic type, hydrophilic
type as well as types surface-activated with amino or
carboxyl groups. Surface-activated microplates are
useful for covalent bonding.
It is important to select the correct type of ELISA
microplate for the intended use.
14. ELISA procedure
• Sandwich ELISA
Step-by-step procedure
Preparation of reagents and equipment
Immobilization of antibody
Add diluted antibody to each well
of a 96-well ELISA plate. Seal the
plate to prevent evaporation, and
allow it to incubate at 4°C for 15-
18 hours to immobilize the
antibody
16. • Blocking
Add blocking buffer to each well, and allow it to
incubate at 37°C for 1 hour to reduce non-specific
binding of the target protein to the well.
17. Washing
Remove the blocking buffer, and wash 3 times with washing solution.
•Addition of samples
Dilute the samples with sample dilution buffer, and
add 100 µL of each sample to each well. For the
calibration curve, prepare a dilution series of the
standard on the same plate. Allow it to incubate at
37°C for 1 hour.
•Washing
Remove the samples, and wash 5 times with washing
solution.
18. Addition of the detection antibody
Dilute the detection antibody in sample dilution buffer, and add 100
µL to each well. Allow it to incubate at 37°C for 1 hour.
Washing
After reaction, remove the detection antibody, and wash 5 times
with washing solution.
19. Addition of enzyme-labeled secondary antibody
Dilute an enzyme-labeled secondary antibody with sample dilution
buffer, and add 100 µL to each well. Allow it to incubate at 37°C for 1
hour.
Washing
After reaction, remove the secondary antibody, and wash 5 times
with washing solution.
20. Add a substrate solution.
Allow it to incubate as the color develops.
21. Add a stop solution to stop the reaction
when the color is sufficiently developed.
23. • Antibodies suitable for ELISA
• List of primary antibodies
• IgG, IgG2b, IgG1 κ, IgG1, IgG2bκ, IgG2aκ, ---etc
• List of secondary antibodies
• Fab'(aff.)
24. What are antibodies?
•Antibodies are proteins produced and secreted by B cells. They bind to
foreign substances that invade the body, such as pathogens.
The term “antibody” refers to its function, which is to bind to an antigen.
Another name for this protein molecule is immunoglobulin (abbreviated
Ig).
25. Antibodies are Y-shaped molecules, consisting of two heavy chains (H
chains) and two light chains (L chains) arranged as shown in the diagram
on the right. An antigen binds to the antigen-binding site at the tip of the
“Y.” An important feature is that each antibody recognizes a specific
antigen as illustrated in the diagram. This is called "antibody specificity"
• Domain names
26. How to generate polyclonal antibodies
• Overview of the procedure
An antigen (immunogen) injected into animals induces them to produce
and secrete high levels of antibodies into the blood.
Several months after repeated immunization, the blood (plasma, serum)
is collected, and antibodies are purified.The antibodies generated by this
method are called polyclonal antibodies because they are derived from
different B cell clones and the resulting antiserum contains numerous
different antibodies that react to the injected immunogen.
27. Animals used for immunization (immunized animals)
In addition to mice and rabbits, various mammals and birds are used for
immunization, including rats, hamsters, guinea pigs, chickens, goats,
sheep, and donkeys. Immunoglobulin isotypes, organization of
immunoglobulin genes, mechanism of diversification, and the organ
sites of antibody diversification differ between vertebrate animals.
29. Difference between polyclonal and
monoclonal antibodies
Antigen binding Monoclonal antibody reacts to a single epitope.
Therefore, only one antibody molecule can bind to an antigen
molecule. In contrast, polyclonal antibody is a collection of
immunoglobulin molecules that react against a specific antigen, each
recognizes a different epitope. Therefore, multiple antibody
molecules bind to an antigen molecule
30. ELISA Plate
•96 well plate
•Made of plastic on which protein can be
adsorbed (bind) easily
•Usually done overnight @ 4C
•Special buffer used that will not denature Ab
and maximize binding
•Blocking step ensures no empty spaces are
left
•Blocking reagent is often 10% FBS (Fetal
Bovine Serum)
31. Blocking
• Blocking Reagent 10% FBS ((Fetal bovine serum is
the most widely used serum-supplement for the in
vitro cell culture of eukaryotic cells. This is due to it
having a very low level of antibodies and containing
more growth factors, allowing for versatility in many
different cell culture applications)). in PBS
• Alternatively 1% BSA (Immunoassay Grade) Bovine
serum albumin (BSA)
• Filter To Remove Particulates
• Plate Is Brought To R.T
• Add 200 L per well Blocking Buffer
• Wait For 2 Hours At R.T
32. After Blocking
•Wash x3 with PBS/tween (detergent)
•Add standards + samples
•Samples are typically supernatants from cultures or
patient serum/plasma
•Use 100 L
•Often dilution is required if signal is too strong
•standards?
33. Standard Curve
•Serial dilutions of the cytokine being measured
•Exact concentration is needed
•A plot of concentration (pg/mL or ng/mL) is plotted
against OD (optical density)
