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ELISA
ELISA
Immunochemistry ELISA
Department of Clinical Biochemistry
Created by:
June 21, 2024 A.N. Emami
Total Razavi
slides : 115 1
Enzyme Linked Immunosurbent
Assay
ELISA
ELISA
Immunochemistry ELISA
Outlines
What is ELISA
History
Application
Mechanism & Reagents
Types of ELISA
Methods
Quality control
HIV test
ELISA
ELISA
Immunochemistry ELISA
ELISA
ELISA
Immunochemistry ELISA
The American
The American physicist
physicist
Rosalyn S.Yalow
Rosalyn S.Yalow (born(born
1921) made
1921) made her her mostmost
outstanding contribution
outstanding contribution toto
modern
modern medicine
medicine inin
developing
developing
radioimmunoassay(RIA),
radioimmunoassay (RIA),for
for
which she
which she received
received aa Nobel
Nobel
Prizeininphysiology/medicine
Prize physiology/medicine
(1977).
(1977).
Dr.Solomon
Dr. SolomonA.A.Berson,
Berson,M.D.
M.D.
'45 (1919-1972)
'45 (1919-1972) and
and Dr.
Dr.
Rosalyn Sussman
Rosalyn Sussman Yalow
Yalow
(1921- )) co-developed
(1921- co-developed the
the
radioimmunoassay (RIA)
radioimmunoassay (RIA) inin
1959.
1959.
EvaEngvall
Eva Engvallisisone
oneof
ofthe
the
scientistswho
scientists whoinvented
inventedthethe
ELISAtest
ELISA testinin1971.
1971.She
Sheisis
shownatather
shown herhome
homenear
near
Buellton,Calif.
Buellton, Calif.Engvall
Engvallisis
currentlyaaprofessor
currently professoratatthe
the
BurnhamInstitute
Burnham InstituteininLa
La
Jolla,Calif.
Jolla, Calif.
ELISA
ELISA
Immunochemistry ELISA
Advantages of ELISA
Sensitive: nanogram levels or lower
Reproducible
Minimal reagents
Qualitative & Quantitative
Qualitative Eg. HIV testing
quantitative assays Eg. Drug Monitoring
Sensitivity
Relative sensitivities of tests (approx)
precipitation
immunoelectrophoresis 10 g/ml - 1 mg/ml
double/radial diffusion
Limitations
Materials needed
Testing sample
Antibody (1st, 2nd) / Antigen
Polystyrene microtiter plate
Blocking buffer
Washing buffer
Substrate
Enzyme
ELISA
ELISA
Immunochemistry ELISA
Mechanism
The basic mechanism involved in these test
utilizes absorption of antigen to a solid surface
which is placed in contact with a dilution of
serum. The reaction which detects and
quantifies the binding of antibody uses an
antibody labeled with an enzyme followed by
the addition of an appropriate substrate on
which the enzyme can act to produce a colour
reaction. Two distinct test mechanisms are
“noncompetitive " and "competitive".
Reagents
Antigens may be produced using any of the standard
techniques. They may be purified by precipitation and ultra-
centrifugation or by column chromatography before being
absorbed onto the surface. An alternative method which
allows the use of relatively impure antigens is to bind specific
antibody to the surface and allow it to absorb the antigen from
the preparation. Since the latter approach usually adds an extra
stage to the test this is not the preferred approach for
commercial test kits. In most cases the antigen is delivered
pre-absorbed onto plates, though they need to be carefully
dried and packed to maintain their stability at refrigerator
temperature for a reasonable period.
Reagents
Reagents
Finally, and most importantly, standard negative sera
and positive sera of known potency are required.
These are even more important than those used in
other serological tests since it is common practice to
express the results of the test sera by comparing them
with those of the control sera (serum-to-positive or
serum-to-negative ratios). The objective of this is to
remove some of the variation due to operator,
environment and plate effects.
ELISA
ELISA
Immunochemistry ELISA
Enzyme labels
Enzyme labels should have high specific reactivity
Should be easily coupled to ligands & the labelled
complex must be stable
The reactivity should be retained after linking of the
enzyme to the antigen/antibody
The chosen enzymes should not be normally present
in the patient samples
Examples of enzyme labels
Horse radish peroxidase, Alkaline phosphatase, Glucose
oxidase
Enzyme-Mediated Detection
Enzyme Horseradish Peroxidase
ELISA
ELISA
Immunochemistry ELISA
BASIC FORMAT
Incubate, wash
E E E E
5. Add substrate
6. Incubate, stop, measure colour change
ENZYME
Colourless
OD
CONCENTRATION
June 21, 2024 Total slides : 115 48
Immunochemistry ELISA
SAMPLE
Dilute in buffer-Tween 20
Include known positive and negative samples
Standards……. recombinant protein
International standard antibody
Double-dilute from 10 pg/ml - 10 ng/ml
100 l/well, duplicates
2 - 4 hours 20/37oC or overnight 4oC
3 - 6 washes with buffer-Tween 20
CONJUGATE
AMPLIFICATION
amplify with
E
E
or
E
S
E-S B S-E
Biotin-labelled anti-Ig
followed by
streptavidin-enzyme
SUBSTRATES
See Sigma catalogue for list of conjugates and substrates
Spectrophotometer
Alkaline phosphatase
365 nm 445 nm
Fluorimeter
Micro-titre plate with a standard Elisa test seen from below. The first 2
wells at top right are the negative controls. The following 2 are positive
controls. The remaining sera are field test sera. Usually at least 2 wells
with a known laboratory positive control are included on each plate.
1. Antigen
1. Antigen
E E E
3. Anti-(human) Ig-enzyme
1. Antigen
4. Substrate
E E E
3. Anti-(human) Ig-enzyme
1. Antigen
1. Specific antibody
2. Impure antigen
eg tissue homogenate
1. Specific antibody
2. Impure antigen
1. Specific antibody
4. Sample (human
antibody)
2. Impure antigen
1. Specific antibody
June 21, 2024 Total slides : 115 71
Immunochemistry ELISA
5. Anti-human Ig-enzyme
E E
4. Sample (human antibody)
2. Impure antigen
1. Specific antibody
6. Substrate
5. Anti-human Ig-enzyme
4. Sample (human
antibody)
2. Impure antigen
1. Specific antibody
June 21, 2024 Total slides : 115 73
Immunochemistry ELISA
eg. hormones
drugs
tumour antigens
cytokines
1. Anti-analyte
2. Sample
1. Anti-analyte
E E
3. Anti-analyte-enzyme
2. Sample
1. Anti-analyte
3. Or: E E
anti-analyte-biotin S S
followed by E-S B S-E E-S B S-E
streptavidin-enzyme
2. Sample
1. Anti-analyte
4. Substrate
3. Or:
anti-analyte-biotin
followed by
streptavidin-
enzyme
2. Sample
1. Anti-analyte
1. Analyte
E E E
E
E
E
E E E 2. Anti-analyte-E
E
+ sample
1. Analyte
3. Wash
E E E 2. Anti-analyte-E E
+ sample
1. Analyte
4. Substrate
3. Wash
2. Anti-analyte-E
+ sample
1. Analyte
1. Anti-analyte
2. Analyte-E
+ sample
1. Anti-analyte
3. Wash
2. Analyte-E
E E E E E + sample E E
1. Anti-analyte
4. Substrate
3. Wash
2. Analyte-E
+ sample
1. Analyte
CYTOKINES
Type 1 Type 2
IL2 HEALTH IL4
IL12 IL5
IFN IL6
TNF IL10
4o overnight
RT 60 minutes
Add streptavidin-peroxidase
RT 30 min
Read A490 0
[rCK]
ELISA
ELISA
Immunochemistry ELISA
HIV
HIV
An HIV ELISA, sometimes called an HIV
enzyme immunoassay (EIA) is the first and
most basic test to determine if an individual is
positive for a selected pathogen, such as HIV.
The test is performed in a 8 cm x 12 cm plastic
plate which contains an 8 x 12 matrix of 96
wells, each of which are about 1 cm high and
0.7 cm in diameter.
An ELISA plate
Above is ELISA data from three patients. Numbers are expressed as optical density
at 450 nm. The cutoff value indicating a positive result is 0.500. Optical densities
of 0.300 to 0.499 are indeterminate and need to be retested. Values below 0.300 are
considered to be negative. In most cases, a patient will be retested if the serum
gives a positive result. If the ELISA retests are positive, the patient will then be
retested by western blotting analysis.
Early Seroconverters
the window between infection and an antibody response to the virus
Operator Error
Fail to add serum or reagent to the correct well
Reagent diluted in wrong diluvent or in wrong dilution
Equipment Error
MULTIPLE TRANSFUSION
CHRONIC HEPATITIS,
CHRONIC ALCOHOLIC
HBV VACCINATION
ANTIBODY TO POLYSTERENE
Equipment
Cross contamination
Sample quality
Personnel training
Kit integrity
Controls
In-house controls
Kit controls are designed to be quite robust and do not reflect subtle
changes in testing.
In-house controls should be calibrated to test as a low positive (above
cut-off, below maximum)
Equipment : Pipettes
Set the washer to wash the recommended number of times (with correct
volume)
Check for accurate dispensing and complete aspiration in each plate well,
if not clean the washer head
Listen for changes in the sound the washer makes, this can indicate a
vacuum leak
Monthly
Run a 10% solution of ethanol through the washer to disinfect.
This can also be done if the washer exhibits signs of contamination
(high background).
Thoroughly rinse the washer after alcohol is used.
Weekly
Run a control plate weekly. Variations in positive or negative specimens
could be a sign of a bad diode or a spill on a diode .
Cross contamination
Sample Quality
Transport conditions
Storage conditions
Age of sample
Problem 1
What is the ELISA test intended to measure?
Antibody
A
to HIV only
Antigen
B
to HIV only
Presence
C
of free, circulating virus in the patient
Antibodies
D
directed against HLA molecules
Problem 2
What would happen if serum were omitted from the
ELISA, but all other steps remained the same and
were performed properly?
A
Anti-human Ig-conjugate would not bind and be washed
away.
B
Anti-human Ig-conjugate would bind non-specifically to
the ELISA plate.
C The O.D. values would be nearly the same as the assay
control.
D
Both A and C.
Problem 3
What would happen if the anti-human Ig-conjugate
were not washed free of the well before the substrate
was added?
A The ELISA would not develop when the substrate was
added.
B The ELISA would develop normally.
C All wells would show uniform over development due to
unbound and excess anti-human Ig enzyme conjugate.
D
Both A and B.
Problem 4
From the ELISA data, which patient is
seropositive for HIV?
Positive Control Negative Control Patient A Patient B Patient C Assay Control
A
Patient A C
Patients A and B
B
Patient B D
Patient C
Questions
Questions
Immunochemistry ELISA
A
Antibody to HIV only
Correct!
Incorrect!
C
Presence of free, circulating virus in the
patient
Incorrect!
D
Antibodies directed against HLA
molecules
Incorrect!
A
Anti-human Ig-conjugate would not bind and
be washed away.
Correct!
B
Anti-human Ig-conjugate would bind non-
specifically to the ELISA plate.
Incorrect!
C
The O.D. values would be nearly the same as
the assay control.
Correct!
D
Both A and C.
Correct!
A
The ELISA would not develop when the
substrate was added.
Incorrect!
B
The ELISA would develop normally.
Incorrect!
C
All wells would show uniform over
development due to unbound and excess anti-
human Ig enzyme conjugate.
Correct!
Since the enzyme which acts on the
substrate is in excess, it would turn all the
wells a uniform color whether they were truly
positive or not.
D
Both A and B.
Incorrect!
A
Patient A
Incorrect!
B
Patient B
Incorrect!
C
Patients B and C
Incorrect!
D
Patient C
Correct!
Extinction coefficient