2. Objectives
• To understand the steps involved in
performing an ELISA and how it is used as a
diagnostic tool
• To understand the disease-causing agent and
transmission patterns of certain infectious
diseases
3. Quick Recall:
1. The body possesses several lines of defense
against infection by pathogenic organisms.
2. The first two defense modes are nonspecific.
1. body’s physical barriers
2. nonspecific immune system
3. Specific immune responses are tailored to
the type of invading pathogen.
4. Specific Immune Response
• The specific immune system is complex and
involves several organs and tissues, including the
thymus, spleen, lymph nodes, bone marrow, and
white blood cells.
• Specific immune responses are triggered by
antigen molecules. Antigens include proteins and
other molecules produced by pathogens.
• The key players in the specific immune defense
are dendritic cells, macrophages, and small white
blood cells called B lymphocytes (B cells) and T
lymphocytes (T cells).
5. • Phagocytic macrophages and dendritic cells break
down pathogens and display antigenic fragments from
the pathogens on the surface of their cell membranes.
http://www.youtube.com/watch?v=BDr44vLNnPY
• B and T lymphocytes circulate through the body in the
blood and lymph.
• When T cells see displayed antigenic fragments, they
stimulate specific B cells to reproduce and generate
antibodies designed against the specific structure of
the antigen encountered. Thus, the word antigen is
derived from the term “antibody generator.”
6. • Antibodies are a group of serum proteins (also
referred to as immunoglobulins) that are found in
the bloodstream or bound to cell membranes.
• These proteins all have the same basic Y-shaped
structure, but have different antigen binding sites
at their ends.
• Antigen binding sites are designed to fit the
shape of specific antigens. Antibodies bind to
antigens like a lock and key, forming antigenantibody complexes
7.
8. • When an antibody forms an antigen-antibody
complex, generally it marks the invading
organism/antigen for destruction or for clearance
from the bloodstream by phagocytic cells.
• This removal is designed to prevent the
organism/antigen from infecting the cell. Antigenantibody complexes also stimulate additional
immune responses to aid the body in clearing an
infection.
10. Assay for detecting
specific organisms
infection
by
• ELISA is commonly used to test blood serum
for the presence of antibodies against
disease-causing pathogens such as viruses and
bacteria.
• In this way, the assay indirectly detects
infection by particular disease-causing agents.
11. • Based on the principle that antibodies
produced in response to pathogens attach to
their antigen targets with great specificity to
form antigen-antibody complexes.
• ELISAs can be used to test for infection by HIV,
influenza virus, the bacterium that causes
Lyme Disease, smallpox virus, SARS
coronavirus, West Nile virus, and other
disease agents.
12. Types of ELISA
• Indirect ELISA
• Direct ELISA
• Sandwhich ELISA
NON -COMPETETIVE
ELISA
• COMPETETIVE ELISA
13. MATERIALS NEEDED FOR ELISA KIT
ELISA Plate
Positive control
Negative control
Dilution Buffer
Conjugate
TMB Substrate
Stop Solution
14. Indirect ELISA
• Indirect ELISA is used to detect infection by
testing patients’ blood for the presence or
absence of ANTIBODIES against a particular
pathogen.
• The presence of such antibodies indicates that
the individual has been infected and that their
body has launched an immune response
against the disease-causing agent.
15. INDIRECT ELISA
Added Blocking buffer.
Suitable primary antibody is added.
Secondary antibody- HRPO is then
added which recognizes and binds to
primary antibody.
Antigen is added to plate.
TMB substrate is added, is converted
to detectable form.
Under standard condition ,the enzyme activity measured is proportional to
amount of specific antibody in the original serum.
17. ADVANTAGES OF INDIRECT DETECTION
Wide variety of labeled secondary antibodies are available commercially.
Versatile, since many primary antibodies can be made in one species and the
Same labeled secondary antibody can be used for detection.
Immunoreactivity of the primary antibody is not affected by labeling.
Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing for
signal amplification.
DISADVANTAGES OF INDIRECT DETECTION
Cross-reactivity may occur with the secondary antibody, resulting in
nonspecific signal.
An extra incubation step is required in the procedure.
18. Direct ELISA
• assays for the presence or absence of certain
ANTIGENS in patients’ blood.
19. DIRECT ELISA
1. Apply a sample of known antigen to
a surface.
2. Enzyme linked primary antibody is
applied to the plate.
3. Washed, After this wash, only the
antibody-antigen complexes remain
attached.
4. Apply a substrate which is
converted by the enzyme to elicit a
chromogenic signal.
Under standard condition ,the enzyme
activity measured is proportional to
amount of specific antibody in the
original serum.
20. ADVANTAGES OF DIRECT DETECTION
Quick methodology since only one antibody is used.
Cross-reactivity of secondary antibody is eliminated.
DISADVANTAGES OF DIRECT DETECTION
Immunoreactivity of the primary antibody may be reduced as a result of
labeling.
Labeling of every primary antibody is time-consuming and expensive.
No flexibility in choice of primary antibody label from one experiment to
another.
Little signal amplification.
21. SANDWICH ELISA
1. a.
Plate is coated with suitable antibody.
b.
Blocking buffer is added.
2. Sample is added to plate so antigen is bounded by capture antibody.
3. A suitable biotin labeled detection antibody is added to plate.
4. Enzyme HRPO is added and binds the biotin labeled detection antibody.
5. TMB substrate is added and converted by HRPO to colored product.
Under standard condition ,the enzyme activity measured is proportional to the Amount of
specific antigen in the original serum.
22.
PROCEDURE OF ELISA
A
B
C
E
D
Wash
Wash
Wash
Wash
3X
4X
4X
4X
Wells are
coated with
0.2 μg
primary
antibody
Diluted
plasma
is added to
coated wells,
which bind
to antibodies
2h
Incubated
overnight at 4˚C
0.1 μg of
biotinylated
(biotin = – )
antihuman
secondary
antibody
2h
Add 1.2000
dilution of
streptavidin
conjugate to
alkaline
phosphatase
( E)
1h
Incubated at room temperature (24˚C)
Alkaline phosphatase
substrate is added
and developed colour
is read at 405 nm
wavelength to
measure plasma
cencentration
24. COMPETETIVE ELISA
COMPETETIVE ELISA
Solid phase coated with antibody
Add unknown amount of unlabeled
antigen and known amount of labeled
antigen
Free and labeled antigen are captured
Color formation by oxidation of substrate
into a colored compound
Under standard condition ,the enzyme activity measured is proportional to the
proportion of labeled antigen in the mixture of labeled and unlabled antigen.
25.
ADVANTAGES:
Suitable for complex (crude or impure) samples, since
the antigen does not require purification prior to
measurement.
DISADVANTAGES
Each antigen may require a different method to couple it to
the enzyme.
26. COMPARISON BETWEEN VARIOUS TYPES OF ELISA
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA
27. SENSITIVITY
ELISAs are one of the most sensitive immunoassays
available. The typical detection range for an ELISA is 0.1 to 1 fmole
or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular
characteristics of the antibody – antigen interaction.
In addition, some substrates such as those yielding
enhanced chemiluminescent or fluorescent signal, can be used to
improve results.
As mentioned earlier, indirect detection will produce
higher levels of signal and should therefore be more sensitive.
However, it can also cause higher background signal thus reducing
net specific signal levels.
28. APPLICATIONS
Screening donated blood for evidence of viral contamination by
HIV-1 and HIV-2 (presence of anti-HIV antibodies)
Hepatitis C (presence of antibodies)
Hepatitis B (testing for both antibodies and a viral antigen)
Measuring hormone levels
HCG (as a test for pregnancy)
LH (determining the time of ovulation)
TSH, T3 and T4 (for thyroid function)
Detecting infections
Sexually-transmitted agents like HIV, syphilis and chlamydia
Hepatitis B and C
Toxoplasma gondii
Detecting illicit drugs.
Detecting allergens in food and house dust
