ELISA

ELISA
(Enzyme-linked immunosorbent
assay)
Sandip .C. Babar

MSc Biotechnology
INTRODUCTION TO
ELISA
A test that uses antibodies and color

change to identify a substance.
ELISA is a popular format of a

"wet-lab" type analyticbiochemistry
assay.
ELISA involves at least one antibody

with specificity for a particular
antigen.
ELISA can perform other forms of

ligand binding assaysinstead of
strictly "immuno" assays.
A 96 - wellmicrotiter
plate
being used for ELISA.
PRINCIPLE
Wet lab" analytic biochemistry assay, ELISA involves detection of an
"analyte"
in a liquid sample by a method that continues to use liquid reagents
during the "analysis.
The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab
binding (antigen- antibody binding). The enzyme converts a colorless
substrate (chromogen) to a colored product, indicating the presence of
Ag:Ab binding.
Secondary antibody
substra
te
ym
z
n
E
e
Colored
product
Primary
antibody
Different antigen in
HOW DOES ELISA WORK ?
There are variations of the ELISA test but the most basic type
consist of an antibody attached to the solid surface. This antibody
has affinity for (will latch onto) the substance of interest.
For example Human Chorionic Gonadotropin (HCG), the

commonly measured protein which indicates pregnancy.
A mixture of purified HCG linked to an enzyme and sample (blood,

urine, etc.) under test are added to the test system. If no HCG is
present in the test sample the only HCG with linked enzyme will
bind. The more HCG which is present in the test sample the less
enzyme linked HCG will bind.
TYPES
OF
INDIRECT ELISA
DIRECT ELISA
SANDWICH ELISA
COMPETETIVE ELISA
ELISA
NON -COMPETETIVE
ELISA
INDIRECT
ELISA
Antigen is added to plate.
Added Blocking buffer.
Suitable primary antibody is added.
Secondary antibody- HRPO is then

added which recognizes and binds
to primary antibody.
TMB substrate is added, is

converted
to detectable form.
Under standard condition ,the enzyme activity measured is

proportional to amount of specific antibody in the original
serum.
ADVANTAGES OF INDIRECT
DETECTION
Wide variety of labeled secondary antibodies are available
commercially.
Versatile, since many primary antibodies can be made in one species
and the
Same labeled secondary antibody can be used for detection.
Immunoreactivity of the primary antibody is not affected by labeling.
Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing for
signal amplification.
DISADVANTAGES OF INDIRECT
DETECTION
in
Cross-reactivity may occur with the secondary antibody, resulting

nonspecific signal.
An extra incubation step is required in the procedure.
DIRECT
ELISA
1. Apply a sample of known

antigen to a surface.
2. Enzyme linked primary antibody
is applied to the plate.
3. Washed, After this wash, only
the antibody-antigen complexes
remain attached.
4. Apply a substrate which is
converted by the enzyme to
elicit a chromogenic signal.
Under standard condition ,the

enzyme activity measured is
proportional to amount of
specific antibody in the
original serum.
ADVANTAGES OF DIRECT
DETECTION
Quick methodology since only one antibody is used.
Cross-reactivity of secondary antibody is eliminated.
DISADVANTAGES OF DIRECT
DETECTION
Immunoreactivity of the primary antibody may be reduced as a

result of labeling.
Labeling of every primary antibody is time-consuming and

expensive.
No flexibility in choice of primary antibody label from one

experiment to another.
Little signal amplification.
SANDWICH
ELISA
1. a.
Plate is coated with suitable antibody.
b.
Blocking buffer is added.
2. Sample is added to plate so antigen is bounded by capture antibody.
3. A suitable biotin labeled detection antibody is added to plate.
4. Enzyme HRPO is added and binds the biotin labeled detection
antibody.
5. TMB substrate is added and converted by HRPO to colored product.
Under standard condition ,the enzyme activity measured is

proportional to the Amount of specific antigen in the original
serum.
PROCEDURE
ELISA
Wells are
coated
with 0.2
g
primary
antibody
Wash
Wash
Wash
Wash
3X
4X
4X
4X
Diluted
plasma
is added to
coated
wells,
which bind
to
antibodies
2h
Incubated
overnight at
4C
OF
0.1 g of
biotinylate
d (biotin =
)
antihuman
secondary
antibody
2h
Add 1.2000
dilution of
streptavidi
n
conjugate
to alkaline
phosphatas
e ( E)
1h
Incubated at room temperature

(24C)
Alkaline
phosphatase
substrate is added
and developed
colour is read at
405 nm
wavelength to
measure plasma
cencentration
FINAL PLATE OF
ELISA
COMPETETIVE ELISA
COMPETETIVE
ELISA
Solid phase coated with
antibody
Add unknown amount of unlabeled
antigen and known amount of
labeled antigen
Free and labeled antigen are captured
Color formation by oxidation of substrate

into a colored compound
Under standard condition ,the enzyme activity measured

is proportional to the proportion of labeled antigen in the
mixture of labeled and unlabled antigen.
ADVANTAGES:
Suitable for complex (crude or impure) samples,

since the antigen does not require purification
prior to measurement.
DISADVANTAGES
Each antigen may require a different method to couple

it to the enzyme.
COMPARISON BETWEEN VARIOUS TYPES

OF ELISA
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA
ELISA
The RESULTS
ELISA assay yields three different types of data output:
Quantitative:
ELISA data can be interpreted in comparison to a

standard
curve in order to precisely calculate
the concentrations of
antigen in various
samples.
Qualitative:
ELISAs can also be used to achieve a yes or no answer

indicating whether a particular antigen is
present in a sample,
as compared to a blank well containing no antigen or an
unrelated
control antigen.
Semi-quantitative:
ELISAs can be used to compare the relative levels
of antigen
in assay samples, since the intensity of signal will vary
directly with antigen concentration.
PRECAUTIONS
Negative control with strong signal
The excessive background signal can be caused by inadequate rinsing of plates,
reagents not sufficiently diluted, inadequate blocking of plates or non-specific
binding of enzyme conjugate. The appearance of color in negative control wells
may also indicate cross-reactivity of secondary antibody with components in the
antigen sample.
Positive control with no signal
Microwell plates not coated properly.

Reagents applied in wrong order or step omitted.
Secondary antibody not matched to the species of primary antibody.
Enzyme conjugate defective or inhibited by contaminant.
ELISA with weak signal
Wash buffer not adequately drained after every wash step.

Inadequate incubation times.
Enzyme conjugate defective or inhibited by contaminant.
Substrate defective or contaminated.
Microwell plates poorly coated.
Loss of capture antibody during blocking/washing.
APPLICATIONS
Screening donated blood for evidence of viral

contamination by
HIV-1 and HIV-2 (presence of anti-HIV antibodies)

Hepatitis C (presence of antibodies)
Hepatitis B (testing for both antibodies and a viral
antigen)
Measuring hormone levels
HCG (as a test for pregnancy)

LH (determining the time of ovulation)
TSH, T3 and T4 (for thyroid function)
Detecting infections
Sexually-transmitted agents like HIV, syphilis and

chlamydia
Hepatitis B and C
Toxoplasma gondii
Detecting illicit drugs.
Detecting allergens in food and house dust
Gluten
Gluten (from Latin gluten, "glue") is a mixture of
proteins found in wheat and related grains, including

barley and rye.
Gluten contains hundreds of proteins, which have low
biological and nutritional value and high contents of

prolamins (glutamines ), as opposed to the grains of
pseudocereals (gluten free), which are rich in proteins
with high biological value .
Product with Gluten ae as follow Bread products,
Wheat , Beef, Chicken , Beer , Soya sauce , Ice

cream,ketchup , Animal feed .
Adverse Effect of gluten are celiac disease , wheat
allergy , fatigue , Chronic dosorder etc .
Folic Acid
Folic acid or folate is also referred to as vitamin B9.
Folic acid is synthetically produced, and used in fortified foods

and supplements on the theory that it is converted into folate.
Vitamin B9 is essential for numerous bodily functions.

Humans cannot synthesize folates therefore, folic acid has to
be supplied through the diet to meet their daily requirements.
Sources are Folate naturally occurs in a wide variety of foods,

including vegetables (particularly dark green leafy
vegetables), fruits and fruit juices, nuts, beans, peas, dairy
products, poultry and meat, eggs, seafood, grains, and some
beers.Avocado,spinach, liver, yeast, asparagus, and Brussels
sprouts are among the foods with the highest levels of folate.
Adverse effect with less intake are pregnancy , fertilit y,

cancer , heart disease ,stroke etc .
Melamine
Melamine is an organic base and a trimer of cyanamide, with

a 1,3,5-triazine skeleton.
Melamine is combined with formaldehyde to produce
melamine resin, a very durable thermosetting plastic used in
high pressure decorative laminates such as Formica,
melamine dinnerware, laminate flooring, and dry erase
boards.
Melamine foam is used as insulation, soundproofing material
and in polymeric cleaning products, such as Magic Eraser.
The use of melamine as fertilizer for crops had been used
because of its high nitrogen content
Sources are Animal feed , adulterated milk ,melamine dishes
etc.
Adverse Effect are toxicity & chronic toxicity .
Aflatoxin
Aflatoxins are poisonous and cancer-causing chemicals that are

produced by certain molds (Aspergillus species ) which grow in
soil, decaying vegetation, hay, and grains.
Types of Aflatoxin
Aflatoxin B1 and B2, produced by Aspergillus flavus and A.
parasiticus
Aflatoxin G1 and G2, produced by Aspergillus parasiticus
Aflatoxin M1, metabolite of aflatoxin B1 in humans and animals
(exposure in ng levels may come from a mother's milk)
Aflatoxin M2, metabolite of aflatoxin B2 in milk of cattle fed on
contaminated foods .
Sources are cassava, chili peppers, corn, cotton seed, millet,
peanuts, rice, sorghum, sunflower seeds, tree nuts, wheat, and a
variety of spices, milk , animal feed , egg , meat etc
Adverse effect is liverdisease , chronic disease , hepatic
necrosis , cancer etc.
THANK YOU

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What is ELISA?

What is ELISA?

What are the basic principles of ELISA?

What are the basic principles of ELISA?

ELISA

Sandip .C. Babar

A test that uses antibodies and color

ELISA is a popular format of a

ELISA involves at least one antibody

ELISA can perform other forms of

HOW DOES ELISA WORK ?

For example Human Chorionic Gonadotropin (HCG), the

A mixture of purified HCG linked to an enzyme and sample (blood,

Antigen is added to plate.

Added Blocking buffer.

Suitable primary antibody is added.

Secondary antibody- HRPO is then

TMB substrate is added, is

Under standard condition ,the enzyme activity measured is

Cross-reactivity may occur with the secondary antibody, resulting

1. Apply a sample of known

Under standard condition ,the

Immunoreactivity of the primary antibody may be reduced as a

Labeling of every primary antibody is time-consuming and

No flexibility in choice of primary antibody label from one

Little signal amplification.

Under standard condition ,the enzyme activity measured is

Incubated at room temperature

Free and labeled antigen are captured

Color formation by oxidation of substrate

Under standard condition ,the enzyme activity measured

Suitable for complex (crude or impure) samples,

Each antigen may require a different method to couple

COMPARISON BETWEEN VARIOUS TYPES

ELISA data can be interpreted in comparison to a

ELISAs can also be used to achieve a yes or no answer

ELISAs can be used to compare the relative levels

Microwell plates not coated properly.

Wash buffer not adequately drained after every wash step.

Screening donated blood for evidence of viral

HIV-1 and HIV-2 (presence of anti-HIV antibodies)

HCG (as a test for pregnancy)

Sexually-transmitted agents like HIV, syphilis and

Detecting illicit drugs.

Detecting allergens in food and house dust

proteins found in wheat and related grains, including

biological and nutritional value and high contents of

Wheat , Beef, Chicken , Beer , Soya sauce , Ice

allergy , fatigue , Chronic dosorder etc .

Folic acid or folate is also referred to as vitamin B9.

Folic acid is synthetically produced, and used in fortified foods

Vitamin B9 is essential for numerous bodily functions.

Sources are Folate naturally occurs in a wide variety of foods,

Adverse effect with less intake are pregnancy , fertilit y,

Melamine is an organic base and a trimer of cyanamide, with

Aflatoxins are poisonous and cancer-causing chemicals that are

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