A

S
I
EL

m
m
I
d
e
k
n
i
L
e
)
m
y
y
z
a
s
n
s
(E
A

t
n
e
b
r
unoSo

HISTORY
First developed in the early 1970s as a
replacement for radioimmunoassays.
Avramais and Pierce developed
methods to chemically link antibodies
to biological enzymes whose activities
produce a measurable signal with
solutions containing appropriate
substrates.

PRINCIPLE
A test that used to identify a substance using
antibodies and change in color.
Used to detect if there is a certain antigen or
antibody is present in a patient using an
enzyme
An immunologic test to determine levels of
protein in a biological sample.

ELISA
EL

Enzyme-linked

: Enzymes activity is used as a

reporter
: The enzymatic reaction will
produce a colored species

ELISA
EL

Enzyme-linked

IS

Immunosorbent

: An antibody or antigen (immuno) is

adsorbed (sorbent) onto the
polystyrene wells in which we conduct the
test.

ELISA
EL

Enzyme-linked

IS

Immunosorbent

Assay

: We will could determine the amount of NPTII

(quantitative assay).
: We will use NPTII concentration standards to
construct a standard curve

ENZYMES USED IN ELISA

Horseradish Peroxidase

ENZYMES USED IN ELISA

Alkaline Phosphatase

MICROTITER PLATE

Is a flat plate made of polystyrene with multiple wells used

as small test tubes. It has become a standard tool in
analytical research and clinical diagnostic testing
laboratories.

ELISA READER / MICROPLATE

READERS

- Is integrally installed in a personal computer

- It is where the plate is stably positioned in the
carrier and between the light source board and the
scanning assembly, and scanned by the scanning

Materials needed
for ELISA Kit:
ELISA Plate
Positive Control
Negative Control
Dilution buffer
Conjugate
TMB substrate
Stop solution

A
S
I
L
E
F
O
S
E
P
TY

DIRECT
ELISA
Involve attachment
of the antigen to
the polystyrene
plate followed by
an enzyme labeledantibody

ADVANTAGES
Quick methodology
Cross-reactivity of secondary
anitbody is eliminated

DISADVANTAGES
Must be labeled individually
Time- consuming
Inflexible when performing multiple experiments
The signal is less amplified (lower sensitivity)
Immunoreactivity of the primary anitbody may be
reduced as a result of labeling

INDIRECT
ELISA
Involves attachment of
the antigen to the
polystyrene plate, but in
this case, the primary
antibody is not labeled.
An enzyme conjugated
secondary antibody,
directed at the first
antibody, is then added.

ADVANTAGES
Immunoreactivity of primary antibody is
not affected by labeling
Wide variety of secondary antibodies
available on the market
More signal amplification (Sensitivity is
increased)

DISADVANTAGES
Cross-reactivity may occur with the secondary
antibody, resulting in nonspecific signal.
An extra incubation step is required in the
procedure.

SANDWICH ELISA
Measures the amount of
antigen between two
layers of antibodies. The
antigens to be
measured must contain
at least two antigenic
sites, capable of binding
to the antibody, since at
least two antibodies act
in the sandwich.

4 factors where
sensitivity depends:
1. Number of molecules of the first antibody
that are bound to the solid phase
2. Avidity of the second antibody to the
antigen
3. Avidity of the second antibody to the
antigen
4. Specific activity of the second antibody

ADVANTAGES
High specificity, since two antibodies are used
the antigen/analyte is specifically captured and
detected
Suitable for complex samples, since the antigen
does not require purification prior to
measurement
Flexibility and sensitivity, since both direct and
indirect detection methods can be used

DOT ELISA

COMPETITIVE
ELISA
This is the most complex
ELISA, and is used to
measure the
concentration of an
antigen (or antibody) in a
sample by observing
interference in an
expected signal output.
Hence, it is also referred
to as an inhibition ELISA.

REVERSE ELISA
A new tecnique that uses a strong stage created up of an
immunosorbent polystyrene rod with 4-12 sticking out
ogives.
The whole system is engrossed in a test tube containing
the gathered example and the following actions (incubation
in conjugate, washing and incubation in chromogenous) are
performed by dropping the ogives in microwells of
conventional micro plates pre-filled with reagents.

ADVANTAGES
The ogives can every be sharpened to an alternate reagent,
permitting the concurrent identification of distinctive antibodies
and diverse antigens for multi-target assays
The specimen volume could be expanded to enhance the test
affectability in clinical, sustenance and ecological tests; One
ogive is left unsensitized to measure the non-particular
responses of the example
The utilization of research center supplies for apportioning
specimen aliquots, washing result and reagents in microwells is
not needed, expediting prepared to-utilize lab-kits.

MULTIPLEX
ELISA
Logical progression of
the widely used
microtiter plate ELISA is
toward a protein array
format that allows
simultaneous detection
of multiple analytes a
multiple array addresses
withtin a single well.

ADVANTAGES (in general)

More accurate
High sensitivity
High specificity
Does not need radioisotopes
can be used for a variety of infections
No radiation hazards occur during labelling and
disposal of waste

DISADVANTAGES
It requires skilled laboratory professionals and
specific research facility types of equipments
More complex measurements of enzyme
activity
Plasma constituents effect on enzyme activity

Applications of ELISA:
Useful device both for identifying serum
antibody levels (such as with the HIV analyze or
West Nile Virus) and also for discovering the use
of antigen.
It has also found applications in the food
industry in detecting potential food substances
such as dairy products, walnuts, peanuts,
almonds, and egg.

Measuring hormone levels (HCG; LH; TSH, T3,

and T4)
Detects many bacterial and viral antigens
Used in toxicology as a rapid presumptive
screen for certain classes of drugs.
Detect various kind of diseases, such as
malaria, Chagas disease, and Johne's disease.

Other applications:
detection of Mycobacterium antibodies in
tuberculosis
detection of rotavirus in feces
detection of hepatitis B markers in serum
detection of enterotoxin of E. coli in feces

Sources:
http://history.cpet.ufl.edu/lm/elisa/types01.html
https://labtestsonline.org/understanding/analytes/alp/tab/te
st
/
http://www.elisa-antibody.com/ELISA-Introduction/elisaadvantages
https://lifescienceproduct.wordpress.com/2013/09/14/elisa/
http://www.slideshare.net/AnitaSingh13/elisa-ppt