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Biochemical instrumental analysis - 12
Dr. Maha Al-Sedik2015
CLS 332
ELISA Technique
Enzyme Linked Immuno Sorbent Assay
• Used to detect the presence of Ag, Ab from the Serum.
• Ag or Ab is fixed to the well.
• By measuring the enzymatic activity which interacts with
substrate to produce color.
• This Enzyme may be linked to Ag or Ab.
• The density of color is either directly or indirectly proportional
to target according to the type of ELISA.
ELISA Screening test
Positive ELISA Test
antigens coated the well
Patient serum Ab Anti-human immunoglobulin coupled to an enzyme.
Chromogen or substrate
Negative ELISA Test
1-Non Competitive ELISA to detect Ag
Ab labeled with enzyme
HBsAg absorbed from serum
Specific Ab coating the well
2-Non Competitive ELISA to detect Ab:
HCV AbHCV coating Ags
Enzyme labeled anti-Ab
3-Competitive ELISA with labeled Ag
Ag from the serum
Ag labeled (tracer)
Ab Coating the well
Ag from the serum
Ab Coating the well
Ag labeled (tracer)Washed
Ag from the serumCompete with tracer And binds to coating
well
4-Competitive ELISA with labeled Ab.
Ag coating well
Ab from the serum
Ab labeled with enzyme
Ab from the serumCompete with labled Ab
and bind to Ag coating well
Op
tic
al
De
ns
ity (
co
lor)
Concentration of Analyte
Op
tic
al
De
ns
ity (
co
lor)
Concentration of Analyte
Ab from serumbind with Ag and removedby washing
Ab labeled will notbind with Ag and
removed by washing
Op
tic
al
De
ns
ity
(co
lor)
Concentration of Analyte
Op
tic
al
De
ns
ity
(co
lor)
Concentration of Analyte
5- Competitive Neutralizing ELISA ( HBe Ab).
ELISA Technique
Sample 1 positive
Sample 2 negative
Antigen-specific antibody is attached to a solid-phase surface
Sample 1 Sample 2
Test specimen is added, which may or may not contain the antigen
Washing will remove excess or un bounded Ag
Sample 1 Sample 2
An enzyme-labeled antibody specific to the antigen is added (conjugate)
Washing will remove excess Conjugate
Sample 1 Sample 2Chromogenic substrate is added, which in the presence of the enzyme, changes its color.
Stopping solution is added to stop any reaction
ss
s
ss
s
ssss s s
The amount of color that develops is directly proportional to the amount of antigen in the test specimen.
Opti
cal D
ensi
ty (c
olor
)
Concentration of Analyte
Sample 1 Sample 2
Specific antigen is attached to a solid-phase surface
Sample 1 Sample 2
Test specimen is added, which may or may not contain the antibody
Washing will remove excess serum with un bounded Ab
Sample 1 Sample 2An enzyme-labeled antibody specific to the serum antibody is added (conjugate)
Washing will remove excess or un bounded Labeled Ab
Sample 1 Sample 2Chromogenic substrate is added, which in the presence of the enzyme, changes its color.
Stopping solution is added to stop any reaction
ss
s
ss
s
ssss s s
Opti
cal D
ensi
ty (c
olor
)
Concentration of Analyte
The amount of color that develops is directly proportional to the amount of antibody in the specimen.
The competition is between the serum Ag and the tracer on the Ab that coat the well.
Sample 1 Sample 2
Antigen-specific antibody is attached to a solid-phase surface
Sample 1 Sample 2
Test specimen is added, which may or may not contain the antigen
Sample 1 Sample 2
An enzyme-labeled antigen( tracer ) is added with serum.Competition on coated Ab.Washing will remove any un bounded Ag
Sample 1 Sample 2
Chromogenic substrate is added, which in the presence of the enzyme, change its color.
Stopping solution is added to stop any reaction
ss
s
ss
s
ssss s s
Opti
cal D
ensi
ty (c
olor
)
Concentration of Analyte
The amount of color that develops is inversely proportional to the amount of antigen in the test specimen.
The competition is between the serum Ab and the tracer on the Ag that coat the well.
Sample 1 Sample 2
Antibody -specific antigen is attached to a solid-phase surface
Sample 1 Sample 2
Test specimen is added, which may or may not contain the antibody
Sample 1 Sample 2
An enzyme-labeled Antibody (conjugate) specific to the attached antigen are added with serum.
Washing will remove any un bounded Ag
Sample 1 Sample 2
Chromogenic substrate is added, which in the presence of the enzyme, change its color. Stopping solution is added to stop any reaction
ss
s
ss
s
ssss s s
Opti
cal D
ensi
ty (c
olor
)
Concentration of Analyte
The color that develops is inversely proportional to the amount of antibody in the test specimen.
The competition is on neutralizing solution between the well’s Ab and the serum Ab.
Sample 1 Sample 2
Antibody specific to HBe Ag is attached to a solid-phase surface
Sample 1 Sample 2
Neutralizing solution is added, which contains the Ag
Sample 1 Sample 2
Serum is added which it may contain Ab to Neutralizing Sol.
Competition between the serum Ab and coating Ab to bind to Neutral.Ag
Washing will remove any un bounded Ag tracer and serum Ab
Sample 1 Sample 2
An enzyme-labeled antibody specific to the antigen is added (conjugate)
Washing will remove excess or un bounded Labeled Ab
Sample 1 Sample 2
Chromogenic substrate is added, which in the presence of the enzyme, change its color.
Stopping solution is added to stop any reaction
ss
s
ss
s
sss s s s
Opti
cal D
ensi
ty (c
olor
)
Concentration of Analyte
The color that develops is inversely proportional to the amount of antibody in the test specimen.
