ELISA
razavinader
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135 slides
Jun 26, 2012
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About This Presentation
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. I...
Slide Content
Total slides : 115 1June 26, 2012
ELISAImmunochemistry
Isfahan University of Medical Science, School of Pharmacy
Department of Clinical Biochemistry
Created by:
A.N. Emami Razavi
ELISAImmunochemistry
Isfahan University of Medical Science, School of Pharmacy
Department of Clinical Biochemistry
Created by:
A.N. Emami Razavi
Enzyme Linked
Immunosurbent Assay
ELISA
Immunosurbent Assay
ELISA
Total slides : 115 3June 26, 2012
ELISAImmunochemistry
Outlines
What is ELISA
History
Application
Mechanism & Reagents
Types of ELISA
Methods
Quality control
HIV test
ELISAImmunochemistry
Outlines
What is ELISA
History
Application
Mechanism & Reagents
Types of ELISA
Methods
Quality control
HIV test
What is ELISA?
ELISA
ELISA
Total slides : 115 5June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 6June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 7June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
History
ELISA
ELISA
Total slides : 115 9June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 10June 26, 2012
ELISAImmunochemistry
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).
ELISAImmunochemistry
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).
Total slides : 115 11June 26, 2012
ELISAImmunochemistry
Dr. Solomon A. Berson, M.D.
'45 (1919-1972) and Dr.
Rosalyn Sussman Yalow
(1921- ) co-developed the
radioimmunoassay (RIA) in
1959.
ELISAImmunochemistry
Dr. Solomon A. Berson, M.D.
'45 (1919-1972) and Dr.
Rosalyn Sussman Yalow
(1921- ) co-developed the
radioimmunoassay (RIA) in
1959.
Total slides : 115 12June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 13June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 14June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Applications
ELISA
ELISA
Total slides : 115 16June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 17June 26, 2012
ELISAImmunochemistry
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)
ELISAImmunochemistry
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)
Total slides : 115 18June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 19June 26, 2012
ELISAImmunochemistry
Sensitivity
Relative sensitivities of tests (approx)
Usual operating range
[Ab] or [Ag]
precipitation
immunoelectrophoresis
double/radial diffusion
10 mg/ml - 1 mg/ml
immunofluorescence 0.1 - 10 mg/ml
ELISA (colour) 0.1 - 10 ng/ml
(chemiluminescence) 0.01 - 10 ng/ml
radioimmunoassay 0.01 - 10 ng/ml
ELISAImmunochemistry
Sensitivity
Relative sensitivities of tests (approx)
Usual operating range
[Ab] or [Ag]
precipitation
immunoelectrophoresis
double/radial diffusion
10 mg/ml - 1 mg/ml
immunofluorescence 0.1 - 10 mg/ml
ELISA (colour) 0.1 - 10 ng/ml
(chemiluminescence) 0.01 - 10 ng/ml
radioimmunoassay 0.01 - 10 ng/ml
Total slides : 115 20June 26, 2012
ELISAImmunochemistry
Enzymes with Chromogenic Substrates
High molar extinction coefficient (i.e., strong
color change)
Strong binding between enzyme and substrate
(low K
M
)
Linear relationship between color intensity and
[enzyme]
ELISAImmunochemistry
Enzymes with Chromogenic Substrates
High molar extinction coefficient (i.e., strong
color change)
Strong binding between enzyme and substrate
(low K
M
)
Linear relationship between color intensity and
[enzyme]
Total slides : 115 21June 26, 2012
ELISAImmunochemistry
Limitations
Results may not be absolute
False positive possible
False negative possible
Antibody must be available
ELISAImmunochemistry
Limitations
Results may not be absolute
False positive possible
False negative possible
Antibody must be available
Total slides : 115 22June 26, 2012
ELISAImmunochemistry
Materials needed
Testing sample
Antibody (1
st
, 2
nd
) / Antigen
Polystyrene microtiter plate
Blocking buffer
Washing buffer
Substrate
Enzyme
ELISAImmunochemistry
Materials needed
Testing sample
Antibody (1
st
, 2
nd
) / Antigen
Polystyrene microtiter plate
Blocking buffer
Washing buffer
Substrate
Enzyme
Mechanism &
Reagents
ELISA
Reagents
ELISA
Total slides : 115 24June 26, 2012
ELISAImmunochemistry
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".
ELISAImmunochemistry
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".
Total slides : 115 25June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 26June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 27June 26, 2012
ELISAImmunochemistry
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.
Reagents
ELISAImmunochemistry
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.
Reagents
Types of ELISA
ELISA
ELISA
Total slides : 115 29June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 30June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 31June 26, 2012
ELISAImmunochemistry
Noncompetitive or Sandwich Assay
Antibody measuring system
Titre wells coated with suitable antigen
Add patient sample containing the antibody
Incubate: till antigen antibody reaction is complete
Wash remove unbound antibody
Add Antiantibody labelled with Enzyme
Incubate till labelled antiantibodies binds antigen-
antibody complex
Wash remove unbound labelled antiantibody
Add substrate ; incubate
Enzyme + Substrate Product measure colour
Colour proportional to antibody in patient sample
ELISAImmunochemistry
Noncompetitive or Sandwich Assay
Antibody measuring system
Titre wells coated with suitable antigen
Add patient sample containing the antibody
Incubate: till antigen antibody reaction is complete
Wash remove unbound antibody
Add Antiantibody labelled with Enzyme
Incubate till labelled antiantibodies binds antigen-
antibody complex
Wash remove unbound labelled antiantibody
Add substrate ; incubate
Enzyme + Substrate Product measure colour
Colour proportional to antibody in patient sample
Total slides : 115 32June 26, 2012
ELISAImmunochemistry
Competitive binding assay
Titrewells coated with antibodies
Known quantities of patient sample containing
antigen + antigen labelled with enzyme
Incubate: till antigen antibody reaction is complete
Wash remove unbound antigens
Add substrate ; incubate
Enzyme + Substrate Product measure colour
Colour inversely related to antigen in patient sample
ELISAImmunochemistry
Competitive binding assay
Titrewells coated with antibodies
Known quantities of patient sample containing
antigen + antigen labelled with enzyme
Incubate: till antigen antibody reaction is complete
Wash remove unbound antigens
Add substrate ; incubate
Enzyme + Substrate Product measure colour
Colour inversely related to antigen in patient sample
Total slides : 115 33June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 34June 26, 2012
ELISAImmunochemistry
Enzyme-Mediated Detection
PurpleBCIP (AP substrate)
NBT (Enhance color)
BromochloroindolylphosphateNitr
o Blue Tetrazolium
ColorAbbrev.Substrate
Alkaline Phosphatase (AP)Enzyme
BrownDABDiaminobenzidine
ColorAbbrev.Substrate
Horseradish PeroxidaseEnzyme
ELISAImmunochemistry
Enzyme-Mediated Detection
PurpleBCIP (AP substrate)
NBT (Enhance color)
BromochloroindolylphosphateNitr
o Blue Tetrazolium
ColorAbbrev.Substrate
Alkaline Phosphatase (AP)Enzyme
BrownDABDiaminobenzidine
ColorAbbrev.Substrate
Horseradish PeroxidaseEnzyme
Methods
ELISA
ELISA
Total slides : 115 36June 26, 2012
ELISAImmunochemistry
BASIC FORMAT
Solid phase = 96 / 384-well microplate
ELISAImmunochemistry
BASIC FORMAT
Solid phase = 96 / 384-well microplate
Total slides : 115 37June 26, 2012
ELISAImmunochemistry
96 Well 350µL Polypropylene Plates
ELISAImmunochemistry
96 Well 350µL Polypropylene Plates
Total slides : 115 38June 26, 2012
ELISAImmunochemistry
96 Well 500µL Polypropylene Plates
ELISAImmunochemistry
96 Well 500µL Polypropylene Plates
Total slides : 115 39June 26, 2012
ELISAImmunochemistry
96 Well 600µL Polypropylene Plates
ELISAImmunochemistry
96 Well 600µL Polypropylene Plates
Total slides : 115 40June 26, 2012
ELISAImmunochemistry
96 Well 1mL Polypropylene Plates
ELISAImmunochemistry
96 Well 1mL Polypropylene Plates
Total slides : 115 41June 26, 2012
ELISAImmunochemistry
96 Well 2mL Polypropylene Plates
ELISAImmunochemistry
96 Well 2mL Polypropylene Plates
Total slides : 115 42June 26, 2012
ELISAImmunochemistry
384 Well Polystyrene Assay Plates
ELISAImmunochemistry
384 Well Polystyrene Assay Plates
Total slides : 115 43June 26, 2012
ELISAImmunochemistry
384 Well Polypropylene Microtiter Plates
ELISAImmunochemistry
384 Well Polypropylene Microtiter Plates
Total slides : 115 44June 26, 2012
ELISAImmunochemistry
Analyte = antibody Analyte = antigen
Incubate, wash
1. Coat solid phase with
antigen when analysing antibody
antibody when analysing antigen
ELISAImmunochemistry
Analyte = antibody Analyte = antigen
Incubate, wash
1. Coat solid phase with
antigen when analysing antibody
antibody when analysing antigen
Total slides : 115 45June 26, 2012
ELISAImmunochemistry
2. Block free binding sites. Incubate. Wash.
Analyte = antibody Analyte = antigen
ELISAImmunochemistry
2. Block free binding sites. Incubate. Wash.
Analyte = antibody Analyte = antigen
Total slides : 115 46June 26, 2012
ELISAImmunochemistry
3. Add sample. Incubate. Wash
Analyte = antibody Analyte = antigen
ELISAImmunochemistry
3. Add sample. Incubate. Wash
Analyte = antibody Analyte = antigen
Total slides : 115 47June 26, 2012
ELISAImmunochemistry
4. Add conjugate. Incubate. Wash.
Analyte = antibody Analyte = antigen
E E E E
ELISAImmunochemistry
4. Add conjugate. Incubate. Wash.
Analyte = antibody Analyte = antigen
E E E E
Total slides : 115 48June 26, 2012
ELISAImmunochemistry
5. Add substrate
6. Incubate, stop, measure colour change
Colourless
ENZYME
OD
CONCENTRATION
ELISAImmunochemistry
5. Add substrate
6. Incubate, stop, measure colour change
Colourless
ENZYME
OD
CONCENTRATION
Total slides : 115 49June 26, 2012
ELISAImmunochemistry
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).
ELISAImmunochemistry
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).
Total slides : 115 50June 26, 2012
ELISAImmunochemistry
WASHING THE PLATE
Wash the plate three time with PBS using the Nunc-
Immuno wash device (this device simultaneously
delivers and aspirates fluid)
ELISAImmunochemistry
WASHING THE PLATE
Wash the plate three time with PBS using the Nunc-
Immuno wash device (this device simultaneously
delivers and aspirates fluid)
Total slides : 115 51June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 52June 26, 2012
ELISAImmunochemistry
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 ml/well, duplicates
2 - 4 hours 20/37
o
C or overnight 4
o
C
3 - 6 washes with buffer-Tween 20
ELISAImmunochemistry
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 ml/well, duplicates
2 - 4 hours 20/37
o
C or overnight 4
o
C
3 - 6 washes with buffer-Tween 20
Total slides : 115 53June 26, 2012
ELISAImmunochemistry
CONJUGATE
For assays of (human) antibodies use anti-
(human) Ig-enzyme
For assays of antigens use enzyme-conjugated
antibody
ELISAImmunochemistry
CONJUGATE
For assays of (human) antibodies use anti-
(human) Ig-enzyme
For assays of antigens use enzyme-conjugated
antibody
Total slides : 115 54June 26, 2012
ELISAImmunochemistry
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 MgCl
2
1mM ZnCl
2
, and 0.02% NaN3 at 4ºC
ELISAImmunochemistry
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 MgCl
2
1mM ZnCl
2
, and 0.02% NaN3 at 4ºC
Total slides : 115 55June 26, 2012
ELISAImmunochemistry
AMPLIFICATION
E
Directly conjugated developing
antibody may give weak signal
ELISAImmunochemistry
AMPLIFICATION
E
Directly conjugated developing
antibody may give weak signal
Total slides : 115 56June 26, 2012
ELISAImmunochemistry
amplify with
unlabelled (rabbit) anti-(human) Ig
followed by
anti-(rabbit) Ig-enzyme
E
E
ELISAImmunochemistry
amplify with
unlabelled (rabbit) anti-(human) Ig
followed by
anti-(rabbit) Ig-enzyme
E
E
Total slides : 115 57June 26, 2012
ELISAImmunochemistry
or
Biotin-labelled anti-Ig
followed by
streptavidin-enzyme
E-S B S-E
E
S
ELISAImmunochemistry
or
Biotin-labelled anti-Ig
followed by
streptavidin-enzyme
E-S B S-E
E
S
Total slides : 115 58June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 59June 26, 2012
ELISAImmunochemistry
Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate
Alkaline phosphatase
Yellow, 405 nm Methyl umbelliferone
Spectrophotometer
365 nm 445 nm
Fluorimeter
ELISAImmunochemistry
Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate
Alkaline phosphatase
Yellow, 405 nm Methyl umbelliferone
Spectrophotometer
365 nm 445 nm
Fluorimeter
Total slides : 115 60June 26, 2012
ELISAImmunochemistry
ELISAImmunochemistry
Total slides : 115 61June 26, 2012
ELISAImmunochemistry
ELISAImmunochemistry
Total slides : 115 62June 26, 2012
ELISAImmunochemistry
ELISAImmunochemistry
Total slides : 115 63June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 64June 26, 2012
ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
• screening hybridoma supernatants
• detecting clinically important antibodies
- autoantibodies
- anti-pathogens
- anti-allergens
1. Antigen
ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
• screening hybridoma supernatants
• detecting clinically important antibodies
- autoantibodies
- anti-pathogens
- anti-allergens
1. Antigen
Total slides : 115 65June 26, 2012
ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
2. Sample (human) antibody
1. Antigen
ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
2. Sample (human) antibody
1. Antigen
Total slides : 115 66June 26, 2012
ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
Total slides : 115 67June 26, 2012
ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E
4. Substrate
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
ELISAImmunochemistry
INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES
E E E
4. Substrate
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
Total slides : 115 68June 26, 2012
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
Useful when pure antigen not available
or antigen coats poorly
1. Specific antibody
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
Useful when pure antigen not available
or antigen coats poorly
1. Specific antibody
Total slides : 115 69June 26, 2012
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
2. Impure antigen
eg tissue homogenate
1. Specific antibody
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
2. Impure antigen
eg tissue homogenate
1. Specific antibody
Total slides : 115 70June 26, 2012
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
3. Wash ® pure antigen
2. Impure antigen
1. Specific antibody
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
3. Wash ® pure antigen
2. Impure antigen
1. Specific antibody
Total slides : 115 71June 26, 2012
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
4. Sample (human
antibody)
3. Wash ® pure antigen
2. Impure antigen
1. Specific antibody
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
4. Sample (human
antibody)
3. Wash ® pure antigen
2. Impure antigen
1. Specific antibody
Total slides : 115 72June 26, 2012
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
E E
5. Anti-human Ig-enzyme
4. Sample (human antibody)
3. Wash ® pure antigen
2. Impure antigen
1. Specific antibody
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
E E
5. Anti-human Ig-enzyme
4. Sample (human antibody)
3. Wash ® pure antigen
2. Impure antigen
1. Specific antibody
Total slides : 115 73June 26, 2012
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
6. Substrate
5. Anti-human Ig-enzyme
4. Sample (human
antibody)
3. Wash ® pure antigen
2. Impure antigen
1. Specific antibody
ELISAImmunochemistry
ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES
6. Substrate
5. Anti-human Ig-enzyme
4. Sample (human
antibody)
3. Wash ® pure antigen
2. Impure antigen
1. Specific antibody
Total slides : 115 74June 26, 2012
ELISAImmunochemistry
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
eg. hormones
drugs
tumour antigens
cytokines
1. Anti-analyte
ELISAImmunochemistry
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
eg. hormones
drugs
tumour antigens
cytokines
1. Anti-analyte
Total slides : 115 75June 26, 2012
ELISAImmunochemistry
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
ELISAImmunochemistry
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
1. Anti-analyte
2. Sample
Total slides : 115 76June 26, 2012
ELISAImmunochemistry
1. Anti-analyte
2. Sample
3. Anti-analyte-enzyme
E E
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
1. Anti-analyte
2. Sample
3. Anti-analyte-enzyme
E E
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
Total slides : 115 77June 26, 2012
ELISAImmunochemistry
1. Anti-analyte
2. Sample
3. Or:
anti-analyte-biotin
followed by
streptavidin-enzyme
E-S B S-E
E
S
E
S
E-S B S-E
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
1. Anti-analyte
2. Sample
3. Or:
anti-analyte-biotin
followed by
streptavidin-enzyme
E-S B S-E
E
S
E
S
E-S B S-E
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
Total slides : 115 78June 26, 2012
ELISAImmunochemistry
1. Anti-analyte
2. Sample
4. Substrate
3. Or:
anti-analyte-biotin
followed by
streptavidin-
enzyme
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
1. Anti-analyte
2. Sample
4. Substrate
3. Or:
anti-analyte-biotin
followed by
streptavidin-
enzyme
ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
Total slides : 115 79June 26, 2012
ELISAImmunochemistry
COMPETITION ELISA TO DETECT ANTIGENS
1. Analyte
(antigen-coated plate)
ELISAImmunochemistry
COMPETITION ELISA TO DETECT ANTIGENS
1. Analyte
(antigen-coated plate)
Total slides : 115 80June 26, 2012
ELISAImmunochemistry
2. Anti-analyte-E
+ sample
1. Analyte
Low [analyte] High [analyte]
E E E
E E
E
E
E
E
E
COMPETITION ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
2. Anti-analyte-E
+ sample
1. Analyte
Low [analyte] High [analyte]
E E E
E E
E
E
E
E
E
COMPETITION ELISA TO DETECT ANTIGENS
Total slides : 115 81June 26, 2012
ELISAImmunochemistry
3. Wash
2. Anti-analyte-E
+ sample
1. Analyte
Low [analyte] High [analyte]
E E E
E
COMPETITION ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
3. Wash
2. Anti-analyte-E
+ sample
1. Analyte
Low [analyte] High [analyte]
E E E
E
COMPETITION ELISA TO DETECT ANTIGENS
Total slides : 115 82June 26, 2012
ELISAImmunochemistry
3. Wash
2. Anti-analyte-E
+ sample
1. Analyte
4. Substrate
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
3. Wash
2. Anti-analyte-E
+ sample
1. Analyte
4. Substrate
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
Total slides : 115 83June 26, 2012
ELISAImmunochemistry
COMPETITION ELISA TO DETECT ANTIGENS
1. Anti-analyte
(antibody-coated plate)
ELISAImmunochemistry
COMPETITION ELISA TO DETECT ANTIGENS
1. Anti-analyte
(antibody-coated plate)
Total slides : 115 84June 26, 2012
ELISAImmunochemistry
2. Analyte-E
+ sample
1. Anti-analyte
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
2. Analyte-E
+ sample
1. Anti-analyte
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
Total slides : 115 85June 26, 2012
ELISAImmunochemistry
3. Wash
2. Analyte-E
+ sample
1. Anti-analyte
E E E E E E E
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
3. Wash
2. Analyte-E
+ sample
1. Anti-analyte
E E E E E E E
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
Total slides : 115 86June 26, 2012
ELISAImmunochemistry
3. Wash
2. Analyte-E
+ sample
1. Analyte
4. Substrate
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
ELISAImmunochemistry
3. Wash
2. Analyte-E
+ sample
1. Analyte
4. Substrate
Low [analyte] High [analyte]
COMPETITION ELISA TO DETECT ANTIGENS
Total slides : 115 87June 26, 2012
ELISAImmunochemistry
CYTOKINES
1
2 1
2
HEALTH
CANCER
VIRUSES
MYCOBACTERIA
ASTHMA, ALLERGY
LUPUS
RHEUMATOID ARTHRITIS
MULTIPLE SCLEROSIS
DIABETES
IL2
IL12
IFNg
TNFa
IL4
IL5
IL6
IL10
Type 1 Type 2
ELISAImmunochemistry
CYTOKINES
1
2 1
2
HEALTH
CANCER
VIRUSES
MYCOBACTERIA
ASTHMA, ALLERGY
LUPUS
RHEUMATOID ARTHRITIS
MULTIPLE SCLEROSIS
DIABETES
IL2
IL12
IFNg
TNFa
IL4
IL5
IL6
IL10
Type 1 Type 2
Total slides : 115 88June 26, 2012
ELISAImmunochemistry
Detection of cytokines by ELISA
Plasma or supernatant of cultured mononuclear cells
Coat plate with anti-CK (Pharmingen) 0.5 mg/ml in
bicarbonate buffer, 4
o
overnight
Wash x 2 with PBS-T
Block with PBS + 10% FCS, 2 hours RT
Wash x2 with PBS-T
ELISAImmunochemistry
Detection of cytokines by ELISA
Plasma or supernatant of cultured mononuclear cells
Coat plate with anti-CK (Pharmingen) 0.5 mg/ml in
bicarbonate buffer, 4
o
overnight
Wash x 2 with PBS-T
Block with PBS + 10% FCS, 2 hours RT
Wash x2 with PBS-T
Total slides : 115 89June 26, 2012
ELISAImmunochemistry
Add standards (recombinant CK 10 pg-10 ng /ml), controls
and samples
4
o
overnight
Wash x3 with PBS-T
Add biotinylated anti-CK (Pharmingen) 0.5 mg/ml
RT 60 minutes
Wash x6 with PBS-T
Add streptavidin-peroxidase
RT 30 min
ELISAImmunochemistry
Add standards (recombinant CK 10 pg-10 ng /ml), controls
and samples
4
o
overnight
Wash x3 with PBS-T
Add biotinylated anti-CK (Pharmingen) 0.5 mg/ml
RT 60 minutes
Wash x6 with PBS-T
Add streptavidin-peroxidase
RT 30 min
Total slides : 115 90June 26, 2012
ELISAImmunochemistry
Wash x8 with PBS-T
Add OPD substrate
15 min RT, dark
Stop with N H
2
SO
4
Read A
490
2
1
0
[rCK]
ELISAImmunochemistry
Wash x8 with PBS-T
Add OPD substrate
15 min RT, dark
Stop with N H
2
SO
4
Read A
490
2
1
0
[rCK]
HIV Test
ELISA
ELISA
Total slides : 115 92June 26, 2012
ELISAImmunochemistry
HIV
The ELISA test, or the enzyme immunoassay
(EIA), was the first screening test commonly
employed for HIV. It has a high sensitivity.
ELISAImmunochemistry
HIV
The ELISA test, or the enzyme immunoassay
(EIA), was the first screening test commonly
employed for HIV. It has a high sensitivity.
Total slides : 115 93June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 94June 26, 2012
ELISAImmunochemistry
An ELISA plate
ELISAImmunochemistry
An ELISA plate
Total slides : 115 95June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 96June 26, 2012
ELISAImmunochemistry
Negative ELISA
Test
Positive ELISA
Test
ELISAImmunochemistry
Negative ELISA
Test
Positive ELISA
Test
Total slides : 115 97June 26, 2012
ELISAImmunochemistry
ELISA data from three patients
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.
Positive ControlNegative Control Patient A Patient B Patient C Assay Control
1.689 0.153 O.055 0.412 1.999 0.123
ELISAImmunochemistry
ELISA data from three patients
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.
Positive ControlNegative Control Patient A Patient B Patient C Assay Control
1.689 0.153 O.055 0.412 1.999 0.123
Total slides : 115 98June 26, 2012
ELISAImmunochemistry
Sources of Error for HIV EIA Tests
ELISAImmunochemistry
Sources of Error for HIV EIA Tests
Total slides : 115 99June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 100June 26, 2012
ELISAImmunochemistry
Source of False Positive Results
MULTIPLE PREGNANCY
MULTIPLE TRANSFUSION
AUTO IMMUNE DISORDER
CHRONIC HEPATITIS,
CHRONIC ALCOHOLIC
HBV VACCINATION
ANTIBODY TO POLYSTERENE
ELISAImmunochemistry
Source of False Positive Results
MULTIPLE PREGNANCY
MULTIPLE TRANSFUSION
AUTO IMMUNE DISORDER
CHRONIC HEPATITIS,
CHRONIC ALCOHOLIC
HBV VACCINATION
ANTIBODY TO POLYSTERENE
Total slides : 115 101June 26, 2012
ELISAImmunochemistry
Trouble shooting EIA
Kit integrity
Controls (kit and in-house)
Equipment
Cross contamination
Sample quality
Personnel training
Correct validation and interpretation of results
ELISAImmunochemistry
Trouble shooting EIA
Kit integrity
Controls (kit and in-house)
Equipment
Cross contamination
Sample quality
Personnel training
Correct validation and interpretation of results
Total slides : 115 102June 26, 2012
ELISAImmunochemistry
Kit integrity
Was cold chain maintained
during kit transport?
Has the kit expired?
Has the kit been stored
properly in your lab?
ELISAImmunochemistry
Kit integrity
Was cold chain maintained
during kit transport?
Has the kit expired?
Has the kit been stored
properly in your lab?
Total slides : 115 103June 26, 2012
ELISAImmunochemistry
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)
ELISAImmunochemistry
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)
Total slides : 115 104June 26, 2012
ELISAImmunochemistry
Equipment : Pipettes
Single and Multi channel Pipettes should be
calibrated on a monthly basis.
ELISAImmunochemistry
Equipment : Pipettes
Single and Multi channel Pipettes should be
calibrated on a monthly basis.
Total slides : 115 105June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 106June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 107June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 108June 26, 2012
ELISAImmunochemistry
Cross contamination
Can be caused by:
Reusing pipette tips (contaminated with + plasma)
Splashes from one well to another during removal of plate covers
ELISAImmunochemistry
Cross contamination
Can be caused by:
Reusing pipette tips (contaminated with + plasma)
Splashes from one well to another during removal of plate covers
Total slides : 115 109June 26, 2012
ELISAImmunochemistry
Sample Quality
Properly collected (no haemolysis)
Transport conditions
Storage conditions
Number of freeze/thaw cycles
Age of sample
ELISAImmunochemistry
Sample Quality
Properly collected (no haemolysis)
Transport conditions
Storage conditions
Number of freeze/thaw cycles
Age of sample
Total slides : 115 110June 26, 2012
ELISAImmunochemistry
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
ELISAImmunochemistry
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
Total slides : 115 111June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
Total slides : 115 112June 26, 2012
ELISAImmunochemistry
Problem 1
What is the ELISA test intended to measure?
Antibody to HIV only
Antigen to HIV only
Presence of free, circulating virus in the patient
Antibodies directed against HLA molecules
B
A
C
D
ELISAImmunochemistry
Problem 1
What is the ELISA test intended to measure?
Antibody to HIV only
Antigen to HIV only
Presence of free, circulating virus in the patient
Antibodies directed against HLA molecules
B
A
C
D
Total slides : 115 113June 26, 2012
ELISAImmunochemistry
Problem 2
What would happen if serum were omitted from the
ELISA, but all other steps remained the same and
were performed properly?
Anti-human Ig-conjugate would not bind and be washed
away.
Anti-human Ig-conjugate would bind non-specifically to
the ELISA plate.
The O.D. values would be nearly the same as the assay
control.
Both A and C.
B
A
C
D
ELISAImmunochemistry
Problem 2
What would happen if serum were omitted from the
ELISA, but all other steps remained the same and
were performed properly?
Anti-human Ig-conjugate would not bind and be washed
away.
Anti-human Ig-conjugate would bind non-specifically to
the ELISA plate.
The O.D. values would be nearly the same as the assay
control.
Both A and C.
B
A
C
D
Total slides : 115 114June 26, 2012
ELISAImmunochemistry
Problem 3
What would happen if the anti-human Ig-conjugate
were not washed free of the well before the substrate
was added?
The ELISA would not develop when the substrate was
added.
The ELISA would develop normally.
All wells would show uniform over development due to
unbound and excess anti-human Ig enzyme conjugate.
Both A and B.
B
A
C
D
ELISAImmunochemistry
Problem 3
What would happen if the anti-human Ig-conjugate
were not washed free of the well before the substrate
was added?
The ELISA would not develop when the substrate was
added.
The ELISA would develop normally.
All wells would show uniform over development due to
unbound and excess anti-human Ig enzyme conjugate.
Both A and B.
B
A
C
D
Total slides : 115 115June 26, 2012
ELISAImmunochemistry
Problem 4
From the ELISA data, which patient is
seropositive for HIV?
Patient A Patients A and B
Patient B Patient C
Positive ControlNegative Control Patient A Patient B Patient C Assay Control
1.689 0.153 O.055 0.412 1.999 0.123
B
A C
D
ELISAImmunochemistry
Problem 4
From the ELISA data, which patient is
seropositive for HIV?
Patient A Patients A and B
Patient B Patient C
Positive ControlNegative Control Patient A Patient B Patient C Assay Control
1.689 0.153 O.055 0.412 1.999 0.123
B
A C
D
Thank you
Questions
Questions
Total slides : 115 117June 26, 2012
ELISAImmunochemistry
Antibody to HIV only
Antibody from patient serum made in response
to various HIV proteins binds to antigen.
A
Correct!
ELISAImmunochemistry
Antibody to HIV only
Antibody from patient serum made in response
to various HIV proteins binds to antigen.
A
Correct!
Total slides : 115 118June 26, 2012
ELISAImmunochemistry
Antigen to HIV only
HIV antigens are already coated onto the plate.
Antibody from the patient binds to the HIV
antigens.
B
Incorrect!
ELISAImmunochemistry
Antigen to HIV only
HIV antigens are already coated onto the plate.
Antibody from the patient binds to the HIV
antigens.
B
Incorrect!
Total slides : 115 119June 26, 2012
ELISAImmunochemistry
Presence of free, circulating virus in the
patient
Serum antibody is detected, not circulating
virus in the serum.
C
Incorrect!
ELISAImmunochemistry
Presence of free, circulating virus in the
patient
Serum antibody is detected, not circulating
virus in the serum.
C
Incorrect!
Total slides : 115 120June 26, 2012
ELISAImmunochemistry
Antibodies directed against HLA
molecules
Although there may be anti-HLA
antibodies present in the serum, the test is
not designed to measure them.
D
Incorrect!
ELISAImmunochemistry
Antibodies directed against HLA
molecules
Although there may be anti-HLA
antibodies present in the serum, the test is
not designed to measure them.
D
Incorrect!
Total slides : 115 121June 26, 2012
ELISAImmunochemistry
Anti-human Ig-conjugate would not bind and
be washed away.
but there is another correct answer
A
Correct!
ELISAImmunochemistry
Anti-human Ig-conjugate would not bind and
be washed away.
but there is another correct answer
A
Correct!
Total slides : 115 122June 26, 2012
ELISAImmunochemistry
Anti-human Ig-conjugate would bind non-
specifically to the ELISA plate.
The anti-human Ig conjugate is specific
for human Ig and has very little nonspecific
binding activity.
B
Incorrect!
ELISAImmunochemistry
Anti-human Ig-conjugate would bind non-
specifically to the ELISA plate.
The anti-human Ig conjugate is specific
for human Ig and has very little nonspecific
binding activity.
B
Incorrect!
Total slides : 115 123June 26, 2012
ELISAImmunochemistry
The O.D. values would be nearly the same as
the assay control.
but there is another correct answer.
C
Correct!
ELISAImmunochemistry
The O.D. values would be nearly the same as
the assay control.
but there is another correct answer.
C
Correct!
Total slides : 115 124June 26, 2012
ELISAImmunochemistry
Both A and C.
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.
D
Correct!
ELISAImmunochemistry
Both A and C.
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.
D
Correct!
Total slides : 115 125June 26, 2012
ELISAImmunochemistry
The ELISA would not develop when the
substrate was added.
The substrate would overdevelop because
an excess of enzyme coupled to the anti-
human Ig was present.
A
Incorrect!
ELISAImmunochemistry
The ELISA would not develop when the
substrate was added.
The substrate would overdevelop because
an excess of enzyme coupled to the anti-
human Ig was present.
A
Incorrect!
Total slides : 115 126June 26, 2012
ELISAImmunochemistry
The ELISA would develop normally.
The substrate would overdevelop because
an excess of enzyme coupled to the anti-
human Ig was present.
B
Incorrect!
ELISAImmunochemistry
The ELISA would develop normally.
The substrate would overdevelop because
an excess of enzyme coupled to the anti-
human Ig was present.
B
Incorrect!
Total slides : 115 127June 26, 2012
ELISAImmunochemistry
All wells would show uniform over
development due to unbound and excess anti-
human Ig enzyme conjugate.
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.
C
Correct!
ELISAImmunochemistry
All wells would show uniform over
development due to unbound and excess anti-
human Ig enzyme conjugate.
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.
C
Correct!
Total slides : 115 128June 26, 2012
ELISAImmunochemistry
Both A and B.
The substrate would overdevelop because
an excess of enzyme coupled to the anti-
human Ig was present.
D
Incorrect!
ELISAImmunochemistry
Both A and B.
The substrate would overdevelop because
an excess of enzyme coupled to the anti-
human Ig was present.
D
Incorrect!
Total slides : 115 129June 26, 2012
ELISAImmunochemistry
Patient A
Patient A has an O.D. of 0.055 and
would be considered negative.
A
Incorrect!
ELISAImmunochemistry
Patient A
Patient A has an O.D. of 0.055 and
would be considered negative.
A
Incorrect!
Total slides : 115 130June 26, 2012
ELISAImmunochemistry
Patient B
Patient B would be considered
indeterminate and would need to be retested.
B
Incorrect!
ELISAImmunochemistry
Patient B
Patient B would be considered
indeterminate and would need to be retested.
B
Incorrect!
Total slides : 115 131June 26, 2012
ELISAImmunochemistry
Patients B and C
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.
C
Incorrect!
ELISAImmunochemistry
Patients B and C
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.
C
Incorrect!
Total slides : 115 132June 26, 2012
ELISAImmunochemistry
Patient C
Serum from patient C showed an O.D. of
1.999 which is even higher than the positive
control O.D. for the assay.
D
Correct!
ELISAImmunochemistry
Patient C
Serum from patient C showed an O.D. of
1.999 which is even higher than the positive
control O.D. for the assay.
D
Correct!
Total slides : 115 133June 26, 2012
ELISAImmunochemistry
ELISAImmunochemistry
Total slides : 115 134June 26, 2012
ELISAImmunochemistry
ELISAImmunochemistry
Total slides : 115 135June 26, 2012
ELISAImmunochemistry
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.
ELISAImmunochemistry
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.
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