Enzyme Linked

ImmunoSorbent Assay

 Used to detect the presence of Ag, Ab

from the Serum
 By measuring the enzymatic activity which
interacts with substrate that chemically
changed to produce color.
 The density of color is either directly or
indirectly proportional to target according
to the type of ELISA.
 This Enzyme may be linked to Ag or Ab.
 Performed as a solid phase assay in which
the target to be detected is bound to solid
 Non competitive assay to detect Ag
(HBsAg).
 Non competitive assay to detect Ab (HCV-
Ab)
 Competitive assay using labeled Ag (for
Ags. with one Receptor).
 Competitive assay using labeled Ab (HDV
Ab)
 Non competitive Capture ELISA: for
detection of polyclonal Ab. Molecules
(HAV IgM, HBcIgM, Toxo.IgM….).
 Competitive Neutralizing ELISA: an
The target is captured by the coating
well and it is directly bonded by the
conjugate. The intensity of color is
directly proportional to the amount of
target to be measured.
can be used only to those Ag or Ab
which can be conjugated with enzyme
and attached to the micotitieration
well
 Sample 1 Sample 2
positive negative

Antigen-specific antibody is attached to a solid-phase

surface
 Sample 1 Sample 2

TestWashing
specimen
willisremove
added, excess
which may orbounded
or un may not contain
Ag
the antigen
 Sample 1 Sample 2

An enzyme-labeled
Washing will
antibody
removespecific
excess to
Conjugate
the antigen is
)added )conjugate
 s s
s s
s s
s s s s s s

Sample 1 Sample 2

Chromogenic
Stopping substrate
solution isisadded
added,towhich in the
stop any
.presence of the enzyme,
reactionchanges its color
 Optical Density )color)

Concentration of Analyte
The amount of color that develops is proportional
.to the amount of antigen in the test specimen
 Sample 1 Sample 2

Specific antigen is attached to a solid-phase surface

 Sample 1 Sample 2

Test
Washing
specimen
willisremove
added, excess
which may
serum
or may
with not
un bounded
contain the
Ab antibody
 Sample 1 Sample 2

An enzyme-labeled
Washing will remove
antibody
excess
specific
or un
tobounded
the serum
)antibody isLabeled
added Ab
)conjugate
 s s
s s
s s
s s s s s s

Sample 1 Sample 2

Chromogenic
Stoppingsubstrate
solution is added
added,to
which
stop in
anythe
reaction
presence
.of the enzyme, changes its color
 Optical Density )color)

Concentration of Analyte

The amount of color that develops is proportional

.to the amount of antibody in the specimen
 To detect Ab usually IgM ( HAV IgM)
which can not be labeled due to its
large molecule.
 The amount of IgM is indirectly
attached by conjugate and the
intensity of color is directly
proptional to the amount of target
 Sample 1 Sample 2

Antibody-specific to human IgM antibody is

attached to a solid-phase surface
 Sample 1 Sample 2

Test sample is added which may or may not

IgM
Washing
Ab Bindwill
to the
remove
well excess
coating Ab
Ab
contain IgM Ab
 Sample 1 Sample 2
Conjugate is prepared out side the well by
Washing
Agcombing Agwill
–conj complex remove
bind
)HAV) excess
to captured
with Ab Ag-conj
IgM
Labeled Ab
with
complex
enzyme
 s s
s s
s s
s s s s s s

Sample 1 Sample 2

Chromogenic substrate
Stopping solution is added,
is added which
to stop in the
any
.presence of the enzyme, changes color
reaction
 Optical Density )color)

Concentration of Analyte

The amount of color that develops is proportional

.to the amount of antibody in the test 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

Competition
An enzyme-labeled to remove
bindtracer
antigen
Washing will to attached
any)conjugate)
un
Ab antibody
specific to the attached
bounded Ag is added
after with serum
 s s
s s
s s
s s s s s s

Sample 1 Sample 2

Chromogenic
Stopping substrate
solution isisadded
added,towhich in the
stop any
.presence of the enzyme,
reactionchange its color
 Optical Density )color)

Concentration of Analyte

The amount of color that develops is 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

Washing Antibody
An enzyme-labeled
Competition will remove
to bind any
tracer
to unAg
)conjugate)
attached
specific to the attached antigen
bounded Agare added after
with serum
 s s
s s
s s
s s s s s s

Sample 1 Sample 2

Chromogenic
Stoppingsubstrate is added
solution is added,towhich
stop in
anythe
.presence of the enzyme,
reactionchange its color
 Optical Density )color)

Concentration of Analyte

The amount of color that develops is 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 HBe Ag
 Sample 1 Sample 2

Competition
Serum is addedbetween
Washing the
will it
which serum
remove
may anyAb andto
unAb
contain
coating
boundedAbAgto tracer
bind toSol
.Neutralizing Ag serum
and tracer Ab
 Sample 1 Sample 2

An enzyme-labeled antibody
Washing will remove specific
excess to the
or un
)antigenbounded
tracer is Labeled
added )conjugate
Ab
 s s
s s
s s

s s s s
s s

Sample 1 Sample 2

Chromogenic
Stopping substrate
solution isisadded
added,towhich in the
stop any
.presence of the enzyme,
reactionchange its color
 Optical Density )color)

Concentration of Analyte

The amount of color that develops is proportional

.to the amount of antibody in the test specimen