ELISA

Isfahan University of Medical Science, School of Pharmacy
Immunochemistry ELISA
Department of Clinical Biochemistry

Created by:
June 26, 2012 A.N. Emami115
Total slides : Razavi 1

Enzyme Linked
Immunosurbent Assay

ELISA


Outlines

 What is ELISA
 History
 Application
 Mechanism & Reagents
 Types of ELISA
 Methods
 Quality control
 HIV test

June 26, 2012 Total slides : 115 3


 Enzyme-Linked ImmunoSorbent Assay, or ELISA, is
a biochemical technique used mainly in immunology
to detect the presence of an antibody or an antigen in
a sample. The ELISA has been used as a diagnostic
tool in medicine and plant pathology, as well as a
quality control check in various industries. In simple
terms, in ELISA an unknown amount of antigen is
affixed to a surface, and then a specific antibody is
washed over the surface so that it can bind the
antigen. This antibody is linked to an enzyme, and in
the final step a substance is added that the enzyme
can convert to some detectable signal.



 Between each step the plate is typically washed with
a mild detergent solution to remove any proteins or
antibodies that are not specifically bound. Older
ELISAs utilize chromogenic substrates, though newer
assays employ fluorogenic substrates with much
higher sensitivity.



Enzyme-linked immunosorbent assay
Name suggests three components
 Antibody
 Allows for specific detection of analyte of interest
 Solid phase (sorbent)
 Allows one to wash away all the material that is not
specifically captured
 Enzymatic amplification
 Allows you to turn a little capture into a visible color
change that can be quantified using an absorbance plate
reader



 Prior to the development of the EIA/ELISA, the only
option for conducting an immunoassay was
radioimmunoassay, a technique using radioactively-
labeled antigens or antibodies. In radioimmunoassay,
the radioactivity provides the signal which indicates
whether a specific antigen or antibody is present in
the sample. Radioimmunoassay was first described in
a paper by Rosalyn Sussman Yalow and Solomon
Berson published in 1960.



 The American physicist
Rosalyn S.Yalow (born
1921) made her most
outstanding contribution to
modern medicine in
developing
radioimmunoassay (RIA), for
which she received a Nobel
Prize in physiology/medicine
(1977).



Dr. Solomon A. Berson, M.D.
'45 (1919-1972) and Dr.
Rosalyn Sussman Yalow
(1921- ) co-developed the
radioimmunoassay (RIA) in
1959.



 Because radioactivity poses a health threat, a safer
alternative was sought. A suitable alternative to
radioimmunoassay would substitute a non-radioactive
signal in place of the radioactive signal. When certain
enzymes (such as peroxidase) react with appropriate
substrates (such as ABTS or 3,3’,5,5’-
Tetramethylbenzidine), they can result in changes in
color, which can be used as a signal. However, the
signal has to be associated with the presence of
antibody or antigen, which is why the enzyme has to
be linked to an appropriate antibody. This linking
process was independently developed by Stratis
Avrameas and G.B. Pierce.



 Since it is necessary to remove any unbound antibody
or antigen by washing, the antibody or antigen has to
be fixed to the surface of the container, i.e. the
immunosorbent has to be prepared. A technique to
accomplish this was published by Wide and Porath in
1966
 In 1971, Peter Perlmann and Eva Engvall at
Stockholm University in Sweden, as well as Anton
Schuurs and Bauke van Weemen in The Netherlands,
independently published papers which synthesized
this knowledge into methods to perform EIA/ELISA



 Eva Engvall is one of the
scientists who invented the
ELISA test in 1971. She is
shown at her home near
Buellton, Calif. Engvall is
currently a professor at the
Burnham Institute in La
Jolla, Calif.



 Because the ELISA can be performed to
evaluate either the presence of antigen or the
presence of antibody in a sample, it is a useful
tool both for determining serum antibody
concentrations (such as with the HIV test or
West Nile Virus) and also for detecting the
presence of antigen. It has also found
applications in the food industry in detecting
potential food allergens such as milk, peanuts,
walnuts, almonds, and eggs.



What is it used for?

 Measure antibody levels (allergies, vaccines)
 Detect viruses (hepatitis, HIV, venereal
diseases)
 Detect hormonal changes (pregnancy)
 Detect circulatory inflammatory markers
(cytokines)



Advantages of ELISA
 Sensitive: nanogram levels or lower
 Reproducible
 Minimal reagents
 Qualitative & Quantitative
 Qualitative  Eg. HIV testing
 quantitative assays  Eg. Drug Monitoring

 Wells can be coated with Antigens OR Antibodies
 Suitable for automation high speed
 NO radiation hazards



Sensitivity
Relative sensitivities of tests (approx)

Usual operating range
[Ab] or [Ag]

precipitation
immunoelectrophoresis 10 µg/ml - 1 mg/ml
double/radial diffusion

immunofluorescence 0.1 - 10 µg/ml

ELISA (colour) 0.1 - 10 ng/ml
(chemiluminescence) 0.01 - 10 ng/ml

radioimmunoassay 0.01 - 10 ng/ml



Enzymes with Chromogenic Substrates

 High molar extinction coefficient (i.e., strong
color change)
 Strong binding between enzyme and substrate
(low KM)
 Linear relationship between color intensity and
[enzyme]



Limitations

 Results may not be absolute
 False positive possible
 Falsenegative possible
 Antibody must be available



Materials needed
 Testing sample
 Antibody (1st, 2nd) / Antigen
 Polystyrene microtiter plate
 Blocking buffer
 Washing buffer
 Substrate
 Enzyme


Mechanism &
Reagents

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

 The labeled anti-globulin used in the standard test is
included in the test kits as is the appropriate
substrates and stop-solutions for use with it. All of
these reagents may be purchased separately from a
number of sources however their titration to a
standard level of activity for a specific purpose is
rarely worthwhile for a commercial laboratory
carrying out routine serology.



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.



 Noncompetitive binding assay or
Sandwich method
 Antigen measuring system [Titrewells coated
with antibodies ; Enzyme labelled antibodies]
 Antibody measuring system [Titrewells coated
with antigens ; Enzyme labelled antiantibodies]

 Competitive binding assay
 Titrewells coated with antibodies ; Enzyme labelled
antigens



Noncompetitive or Sandwich Assay
 Antigen measuring system
 Titre wells coated with suitable antibody
 Add patient sample containing the antigen
 Incubate: till antigen antibody reaction is complete
 Wash remove unbound antigen
 Add Antibody labelled with Enzyme
 Incubate till antigen binds labelled antibody
 Wash  remove unbound labelled antibody
 Add substrate ; incubate
 Enzyme + Substrate  Product  measure colour
 Colour proportional to antigen in patient sample



Noncompetitive or Sandwich Assay
 Antibody measuring system
 Titre wells coated with suitable antigen
 Add patient sample containing the antibody
 Wash remove unbound antibody
 Add Antiantibody labelled with Enzyme
 Incubate till labelled antiantibodies binds antigen-
antibody complex
 Wash  remove unbound labelled antiantibody
 Colour proportional to antibody in patient sample



Competitive binding assay
 Titrewells coated with antibodies
 Known quantities of patient sample containing
antigen + antigen labelled with enzyme
 Wash remove unbound antigens
 Colour inversely related to antigen in patient sample



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

Substrate Abbrev. Color
Diaminobenzidine DAB Brown

Enzyme Alkaline Phosphatase (AP)
Substrate Abbrev. Color
BromochloroindolylphosphateNitr BCIP (AP substrate) Purple
o Blue Tetrazolium NBT (Enhance color)



BASIC FORMAT

Solid phase = 96 / 384-well microplate



96 Well 350µL Polypropylene Plates



96 Well 1mL Polypropylene Plates



96 Well 2mL Polypropylene Plates



384 Well Polystyrene Assay Plates



384 Well Polypropylene Microtiter Plates



1. Coat solid phase with
antigen when analysing antibody
antibody when analysing antigen

Analyte = antibody Analyte = antigen

Incubate, wash



2. Block free binding sites. Incubate. Wash.




3. Add sample. Incubate. Wash




4. Add conjugate. Incubate. Wash.

E E E E




5. Add substrate
6. Incubate, stop, measure colour change

ENZYME

Colourless

OD

CONCENTRATION


COATING THE PLATE
 Dilute the Ag in PBS containing 0.02% NaN3 (5-10
ug/ml) and despense 50 ul into each well of 96-well
microtiter plate using a multipipette/dispenser. (Micro
plates vary in their ability to bind Ag. So each
resercher should test for the plates of choice for their
specific Ag). Seal plates with adhesive plate sealer
and incubate overnight at 4° C or 2 to 3 h at 37° C
(coated plates can be prepared and stored in the
refrigerator for several weeks).



WASHING THE PLATE
Wash the plate three time with PBS using the Nunc-
Immuno wash device (this device simultaneously
delivers and aspirates fluid)



BLOCKING THE PLATE

block any residual binding capacity by adding 50 ul of a
blocking buffer (PBS containing 5% BSA, 0.02%
NaN3) to each well. Incubate the plate for 1 h at room
temperature.



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 µl/well, duplicates
 2 - 4 hours 20/37oC or overnight 4oC
 3 - 6 washes with buffer-Tween 20



CONJUGATE

 For assays of (human) antibodies use anti-
(human) Ig-enzyme

 For assays of antigens use enzyme-conjugated
antibody



Conjugating antibodies to Alkaline
Phosphatase
 Centrifuge 5 mg of alkaline phosphatase suspension (supplied
as a suspension in 65% (NH4)SO4) in a microfuge for 5 to 10
min. resuspend the enzyme pellet in a total volume of 1 mg of
the antibody to be labeled.
 Dialyzed overnight against 0.1 M sodium phosphate buffer,
pH 6.8 to remove any contaminating free amino groups.
 In a fume hood, slowly add 50 ug 1% glutaraldehyde solution
while stirring for 5 min. Incubate at room temperatyre for 2 to
3 h, add 100 ul of 1 M ethanolamine, pH 7, and then incubate
for additional 2 h at room temperature.
 Dialyze overnight against PBS and remove any insoluble
materials by centrifugation at 40,000 x g for 30 min.
 Store the conjugate (supernatant) in the presence of 50%
glycerole,1mM MgCl2 1mM ZnCl2, and 0.02% NaN3 at 4ºC


AMPLIFICATION

E

Directly conjugated developing
antibody may give weak signal



amplify with

E
E

unlabelled (rabbit) anti-(human) Ig
followed by
anti-(rabbit) Ig-enzyme



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

Orthophenylene diamine Tetramethyl
hydrochloride (OPD) benzidine (TMP)

Horse radish peroxidase (HRP)

Orange, 490 nm Yellow, 450 nm

Spectrophotometer



Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate

Alkaline phosphatase

Yellow, 405 nm Methyl umbelliferone
Spectrophotometer

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.



INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES

• screening hybridoma supernatants
• detecting clinically important antibodies
- autoantibodies
- anti-pathogens
- anti-allergens

1. Antigen




2. Sample (human) antibody

1. Antigen




E E E
3. Anti-(human) Ig-enzyme


1. Antigen




4. Substrate
E E E
3. Anti-(human) Ig-enzyme


1. Antigen



ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

Useful when pure antigen not available
or antigen coats poorly

1. Specific antibody




2. Impure antigen
eg tissue homogenate





3. Wash → pure antigen

2. Impure antigen





4. Sample (human
antibody)


2. Impure antigen




5. Anti-human Ig-enzyme
E E
4. Sample (human antibody)


2. Impure antigen





6. Substrate

5. Anti-human Ig-enzyme

4. Sample (human
antibody)


2. Impure antigen



ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

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



COMPETITION ELISA TO DETECT ANTIGENS
(antigen-coated plate)

1. Analyte




Low [analyte] High [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



(antibody-coated plate)

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
IFNγ IL6
TNFα IL10

RHEUMATOID ARTHRITIS CANCER
MULTIPLE SCLEROSIS VIRUSES
DIABETES MYCOBACTERIA
2 1
ASTHMA, ALLERGY
LUPUS
1 2


Detection of cytokines by ELISA

 Plasma or supernatant of cultured mononuclear cells

 Coat plate with anti-CK (Pharmingen) 0.5 µg/ml in
bicarbonate buffer, 4o overnight

 Wash x 2 with PBS-T

 Block with PBS + 10% FCS, 2 hours RT

 Wash x2 with PBS-T



 Add standards (recombinant CK 10 pg-10 ng /ml), controls
and samples

 4o overnight


 Add biotinylated anti-CK (Pharmingen) 0.5 µg/ml

 RT 60 minutes


 Add streptavidin-peroxidase

 RT 30 min



 Wash x8 with PBS-T 2

 Add OPD substrate

 15 min RT, dark 1

 Stop with N H2SO4

 Read A490 0
[rCK]



HIV

 The ELISA test, or the enzyme immunoassay
(EIA), was the first screening test commonly
employed for HIV. It has a high sensitivity.



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



The ELISA Method
 Partially purified, inactivated HIV antigens pre-
coated onto an ELISA plate
 Patient serum which contains antibodies. If the
patient is HIV+, then this serum will contain
antibodies to HIV, and those antibodies will bind to
the HIV antigens on the plate.
 Anti-human immunoglobulin coupled to an enzyme.
This is the second antibody, and it binds to human
antibodies.
 Chromogen or substrate which changes color when
cleaved by the enzyme attached to the second
antibody.



 Negative ELISA  PositiveELISA
Test Test



ELISA data from three patients

Positive Control Negative Control Patient A Patient B Patient C Assay Control

1.689 0.153 O.055 0.412 1.999 0.123

 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.



Sources of Error for HIV EIA Tests



Sources of False Negative Results

 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



Source of False Positive Results
 MULTIPLE PREGNANCY

 MULTIPLE TRANSFUSION

 AUTO IMMUNE DISORDER

 CHRONIC HEPATITIS,

 CHRONIC ALCOHOLIC

 HBV VACCINATION

 ANTIBODY TO POLYSTERENE



Trouble shooting EIA
 Kit integrity

 Controls (kit and in-house)

 Equipment

 Cross contamination

 Sample quality

 Personnel training

 Correct validation and interpretation of results



Kit integrity

 Was cold chain maintained
during kit transport?

 Has the kit expired?

 Has the kit been stored
properly in your lab?



Controls

 Kit Controls should be run on each EIA plate.
 Indicates whether the kit components are functioning.
 Used to validate EIA run, calculate cut off

 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

 Single and Multi channel Pipettes should be
calibrated on a monthly basis.



Equipment : Microplate Washers
 Daily
 Prime the washer with wash solution before running sample plates

 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

 At the end of the day prime the washer with DI water



Equipment : Microplate Washers
 Weekly
 If a washer is not used during the week rinse it out with DI water
to reduce microbial growth.

 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.



Equipment : Micro-plate Reader
 Daily
 Each time a reader is turned on it runs a self test, it will then report any
errors.

 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

 Can be caused by:

 Reusing pipette tips (contaminated with + plasma)

 Splashes from one well to another during removal of plate covers



Sample Quality

 Properly collected (no haemolysis)

 Transport conditions

 Storage conditions

 Number of freeze/thaw cycles

 Age of sample



Validation and Interpretation of Results

 Positive and Negative controls must fall within a certain range.

 Controls are used to calculate a cut-off.

 Samples below cut-off are negative, those above are positive



Test your self

1. What does ELISA measure?
2. What if serum were left out?
3. Omission of the wash step
4. Which patient is HIV positive?

Use these problems to test your understanding of
this topic.



Problem 1
What is the ELISA test intended to measure?
A Antibody to HIV only

B
 Antigen to HIV only

C
 Presence of free, circulating virus in the patient

D
 Antibodies 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

1.689 0.153 O.055 0.412 1.999 0.123

A
Patient A C
Patients A and B

B
Patient B D
Patient C



A
Antibody to HIV only

Correct!

Antibody from patient serum made in response
to various HIV proteins binds to antigen.



B Antigen to HIV only

Incorrect!

HIV antigens are already coated onto the plate.
Antibody from the patient binds to the HIV
antigens.



C
Presence of free, circulating virus in the
patient

Incorrect!

Serum antibody is detected, not circulating
virus in the serum.



D
Antibodies directed against HLA
molecules

Incorrect!

Although there may be anti-HLA
antibodies present in the serum, the test is
not designed to measure them.



A
Anti-human Ig-conjugate would not bind and
be washed away.

Correct!

 but there is another correct answer



B
Anti-human Ig-conjugate would bind non-
specifically to the ELISA plate.

Incorrect!

The anti-human Ig conjugate is specific
for human Ig and has very little nonspecific
binding activity.



C
The O.D. values would be nearly the same as
the assay control.

Correct!

but there is another correct answer.



D
Both A and C.

Correct!

Since no serum was added, the anti-human
Ig conjugate would not be able to bind to
human Ig and the O.D. values would be the
same as the assay control.



A
The ELISA would not develop when the
substrate was added.

Incorrect!

The substrate would overdevelop because
an excess of enzyme coupled to the anti-
human Ig was present.



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!

Patient A has an O.D. of 0.055 and
would be considered negative.



B
Patient B

Incorrect!

Patient B would be considered
indeterminate and would need to be retested.



C
Patients B and C

Incorrect!

Patient A has an O.D. of 0.055 and would
be considered negative and patient B would be
considered indeterminate and would need to be
retested.



D
Patient C

Correct!

Serum from patient C showed an O.D. of
1.999 which is even higher than the positive
control O.D. for the assay.



Extinction coefficient

 The extinction coefficient for a particular substance is a
measure of how well it scatters and absorbs electromagnetic
radiation (EM waves). If the EM wave can pass through very
easily, the material has a low extinction coefficient.
Conversely, if the radiation hardly penetrates the material, but
rather quickly becomes "extinct" within it, the extinction
coefficient is high.
 A material can behave differently for different wavelengths of
electromagnetic radiation. Glass is transparent to visible light,
but many types of glass are opaque to ultra-violet
wavelengths. In general, the extinction coefficient for any
material is a function of the incident wavelength. The
extinction coefficient is used widely in ultraviolet-visible
spectroscopy.

ELISA

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