A path breaking technology which has made it possible for us to detect HIV. ELISA is an immunological assay nowadays even used to detect food proteins & is the science behind pregnancy color test. This presentation unlocks the working of this assay.

1. ELISA
MOKSHA CHIB
13FET1003

2. ELISA
Enzyme Linked ImmunoSorbent Assay
It is a lab technique, used to measure the
concentration of antibodies or antigens (‘ analyte’)
in solution by color change.
This technique is used in immunology and even in
the food industry and was developed in 1970.

3. BRIEF REVIEW
 ELISA involves coating (binding) an antigen or a
protein to a solid support as a membrane or a 96-
well micro plate or ELISA plate.
 Antigens from the sample are attached to a
surface.
 Further, an antibody, an enzyme and then a
substrate is added to the adsorbed antigen.
 This reaction produces a detectable color
change. 8×12 cm plate
Well- 1cm deep and 0.7 in diameter

4. PRINCIPLE
 It combines the specificity of antibodies with the sensitivity of
simple enzyme assays, by using antibodies or antigens
coupled to an easily assayed enzyme.
 It involves the usage of an enzyme to detect the binding of an
Antigen (Ag)and an Antibody (Ab).
 The enzyme converts a colorless substrate (chromogen) to a
colored product, indicating the presence of Ag : Ab binding.
 Therefore, ELISA can be used to detect either the presence of
antigens or antibodies in a sample depending upon how the
test is designed.

5. STEPS INVOLVED in ELISA
Antigen Binding
 The sample antigen is binded or immobilized to the micro plate via
adsorption to the surface.
 Binding is achieved by incubating the wells with a solution containing the
antigen for 2hrs at RT or overnight at 4°C.
 The protein adheres due to hydrophobic interactions between the protein
& the plastic.
 Coating is done using carbonate/bicarbonate buffer at a pH 9.6.

6. STEPS INVOLVED in ELISA
Blocking
 All unbound sites on the solid support are blocked to prevent nonspecific
binding of the antibodies.
 Blocking buffers like BSA, Non fat dry milk powder (casein) in PBS (
Phosphate buffered saline) or TBS ( Tris buffered saline) at a pH 7.4 with
minute percentage of Tween 20 are used to block the free sites.
 Protein in the blocking solution will attach to membranes in places where
the target proteins have not attached.
 Excess blocking agent is removed by washing the plate membrane with
washing buffer.

7. STEPS INVOLVED in ELISA
Primary Antibody
 The 1° antibody is added and will be bound only if there is a recognized
epitope within sample antigen.
Secondary Antibody
 An enzyme-linked 2° antibody is added with suitable dilutions which will
bind to any available 1° antibody (i.e. it is bound to the antigen).
 2° antibodies are linked to the enzyme through biconjugation.
 Enzymes generally used are Horse Radish Peroxidase (HRP) & Alkaline
Phosphatase (AP).
 Plate is washed with buffer or a mild detergent to remove any unbound
antibodies or proteins.

8. STEPS INVOLVED in ELISA
Detection/ Developing
 After the final wash step, the plate is developed by adding an enzymatic
chromogenic substrate to give color.
 The entire plate is placed into a plate reader and the OD is determined for
each well.
 Intensity of color reflects the amount of specific 2° antibody bound to the
target.
 A spectrophotometer is designed to read each well in the 96 well plate. It
is interfaced with a computer for data management.
 Substrates used are pNPP ( p- nitro phenyl phosphate), BCIP (5-bromo,4-
chloro,3-indoyl phosphate), NBT (nitro blue tetrazolium), TMB (3,3´,5,5´-
tetra methyl benzidine), DAB (3,3-diamino benzidine).

9. ELISA Assay to determine antigen concentration

11. TYPES OF ELISA
 3 types of ELISA :
1. Direct ELISA : Only 1° antibody is used.
2. Indirect ELISA : 1° antibody used is then detected by labeled
2° antibody.
3. Sandwich ELISA : A capture antibody is used and bound to the
solid surface. A solution containing the antigen is added followed by
washing.

13. ADVANTAGES OF SANDWICH OVER
INDIRECT ELISA
 A major disadvantage of the indirect ELISA is the method of antigen
immobilization is not specific.
 Without the first layer of "capture" antibody, any proteins in the sample
(including serum proteins) may competitively adsorb to the plate surface,
lowering the quantity of antigen immobilized.
 Use of the purified specific antibody to attach the antigen to the plastic
eliminates a need to purify the antigen from complicated mixtures before
the measurement.
 Simplifies the assay.
 Increases the specificity and the sensitivity of the assay.

14. DIRECT ELISA INDIRECT ELISA SANDWICH ELISA
Only primary antibody is used. Both primary and secondary antibodies
are used.
A capture antibody is used which is
bound to the solid surface before the
antigen is added.
Antigen immobilisation is not specific. Antigen immobilisation is not specific. Antigen immobilisation is specific.
Any protein in the sample may attach to
the plate.
Any protein in the sample may attach to
the plate.
A specific will be attached as a ‘capture’
antibody is used. This increases the
specific antigen immobilisation.
There is a competition between the
serum protein and the other proteins.
As any protein can bind to the surface,
there is a competition between the
serum and the other proteins.
No competition as only the specific
serum proteins will be attached due to
the inclusion of a ‘capture’ antibody.
It requires purification of the sample to
be tested.
It also requires the purification of the
sample to be tested.
There is no need of sample purification
as only the specific antigen will bind.
The selectivity is questionable. The selectivity and specificity is a major
disadvantage.
Thus it simplifies the assay and
increases the selectivity.
Less sensitive but a faster method Highly sensitive Highly sensitive

15. COMPETITIVE ELISA
 Plate is precoated with an antigen to which
Ag:Ab complex is added.
 The more antigens in the sample, the fewer
antibodies will be able to bind to the antigen in
the well, hence “competition”.
 After washing, the unbound Ag:Ab complex is
removed.
 The 2° antibody that is specific to the primary
antibody and conjugated with an enzyme is
added followed by addition of a substrate.
 For Competitive ELISA, the higher the sample
antigen concentration, the weaker the eventual
signal. The major advantage of this ELISA is the
ability to use crude or impure samples and still
selectively bind any antigen that may be present.

16. WHAT CAN BE TESTED USING ELISA
 Protein with multiple epitope can be detected using
Sandwich ELISA because it requires two antibodies which
can react with different epitopes.
 Pure form of an antigen can be detected using
Competitive ELISA as the purified analyte is immobilized
and the other antigen competes.
 Small target molecules which do not adsorb by
themselves can be attached to a larger protein which
provides the means to attach to a desired epitope for
detection.
 Membrane proteins from the cells can be adsorbed and
thus detected by reducing the concentration of the
detergents in which these cells are generally maintained.

17. APPLICATIONS OF ELISA
 Detection of HIV antibodies
 Detection of microorganisms and the toxins produced by them. For example:
Salmonella, Listeria, Bacillus cereus enterotoxin.
 Food Allergens- It has also found applications in the food industry in detecting
potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs.
 Serological blood test for coeliac disease.
 ELISA can also be used in toxicology as a rapid presumptive screen for certain
classes of drugs.
 ELISA can be used as a detection method for detection of Mycobacterium
antibodies in tuberculosis, detection of rotavirus in faeces, detection of
hepatitis B markers in serum, detection of enterotoxin of E. coli in faeces,
detection of HIV antibodies in blood samples.

19. COMPONENETS IN ELISA KIT
 Immunosorbent: Solid supporter which has been coated with antigens &
antibodies, can be stored in low temperature of about 2-8°C. Polystyrene is
generally used in ELISA
 Conjugate: Conjugate is the antibody (antigen) linked with the enzyme,
and is the key substance for ELISA. A good conjugate possess not only
catalytic activity of enzyme but also immunological competence of
antibody. It should be stable and must be least contaminated.
 Substrate: the chemical activity of the substrate will be compromised if it is
exposed to light or comes into contact with the metal.
 Controls: Need to be diluted and then added to the plate.
 Sample Diluents & Wash Solution
 Stop solution

21. ELISA DEVICE
ELISA pipettes
Can be single or multi channeled.
Adjustable -volume (1–20 μL, 10–
100 μL, 20–200 μL, etc.)
Washer systems
Manual or semi/fully automated
systems that wash one row or
column at a time
ELISA plate readers
Manual or semi/fully automated
readers that read one row or
well at a time

22. ELISA DETECTION STRATERGIES
 Chromogenic Assay - The substrate forms a colored product under the
catalysis of the enzyme catalysed, which is directly related to the amount
of test substance. The Optical Density is proportional to the concentration
of the analyte to be measured.
 Chemiflourescent Assay- An enzyme converts a substrate to a reaction
product that fluoresces when excited by light of a particular wavelength.
 Chemiluminescent Assay- The chemiluminescent substance is excited by
the oxidation and catalysis forming intermediates. When the intermediates
return back to their stable ground state, a photon is released, which is
detected by the luminescent signal instrument

23. FOOD ALLERGIES
 Food allergies are reaction to food proteins.
They may be categorized as
 Immunoglobulin E (IgE)–mediated (immediate) reactions- Cause flushing,
itchy skin, wheezing, vomiting, throat swelling, and even anaphylaxis
immediately after exposure.
 Non–IgE-mediated (delayed) hypersensitivity reactions- Cause localized
(e.g., contact dermatitis) or generalized reactions, which are usually
gastrointestinal or dermatological in nature.
 Some allergic disorders are both IgE and non-IgE or mixed reactions.

24. ELISA FOR FOOD INDUSTRY
 Certain allergens are responsible for 90% food
allergen cases.
 ELISA can detect potential allergic reactions
in the body.
 Traditional food allergen detection uses ELISA
to find protein.
 Because the ELISA is protein-based, it is
proficient at determining the exact source of
an allergen, such as dairy or wheat.

25. EXAMPLES IN FOOD INDUSTRY
 CASEIN:
Casein is a source of food allergies and must be excluded from the diet of susceptible individuals.
Because it is present in high levels in milk, casein is a useful indicator of the presence of milk residue.
This has applications in Non Dairy Products which can be tested for any presence of milk protein. Takes
45 minutes.
 HAZELNUTS :
These nuts contain several allergenic proteins that can pose a serious threat to allergic
consumers. Hazelnut proteins in the presence or absence of soluble wheat proteins are modified
with glucose via the Maillard reaction. Changes in hazelnut proteins, such as the formation of
protein-bound carbonyls, losses of reactive lysine residues and free amino groups, and severe
aggregation dramatically affects the hazelnut protein detection by the ELISA kits. The observed
impact was highly dependent on the type of ELISA kit used.

26. EXAMPLES IN FOOD INDUSTRY
 GLUTEN:
Gluten contains proteins like gliadin and glutenin found in seeds of wheat,
barley and rye. Around 1% of the population of North America & Europe is
sensitive to gluten. Therefore, adoption of gluten free diet is mandatory
avoiding foods containing wheat, rye, barley and other related cereal
grains. There are possibilities that traces of these seeds are present in the
food. Thus, ELISA kits are used to determine the traces of gluten.
ELISA Technologies also offers a rapid, lateral flow
gluten test — EZ Gluten.

27. HOW GLUTEN TEST WORKS ??
1. Food sample is ground to a fine
consistency.
2. Then added to gluten extraction
solution and then mixed.
3. Few drops of sample extract are
placed in a test tube.
4. EZ Gluten test strip is placed in a test
tube to absorb the sample extract.
5. After 10 mins, strip can be read.

28. ADVANTAGES
 Highly sensitive due to enzyme ‘linkage’. Small amount of enzyme can
induce large amount of catalytic reactions.
 Strongly specific due to the selectivity of the antibody or the antigen.
 Detects the actual allergen protein molecule.
 Flexible; more than one systems can be used to measure the same
analyte.

29. DISADVANTAGES
 Some matrices can interfere with the ELISA method, such as chocolate.
 Can cause cross reactivity as seen between different types of nuts.
 This method is also not the most suitable for cooked or heated products
because the protein molecules are denatured or broken down and the
allergen is no longer detectable.

30. RECENT INVENTION
 Scientists at Imperial College London in 2012 developed
PLASMONIC ELISA technology for detecting a variety of
pathogens that needs nothing but a eye to read the results.
 Prostate specific antigen (PSA) and HIV-1 capsid antigen p24
were detected in whole serum at the ultralow concentration of
1 × 10−18 g ml−1.
 When blood serum is introduced into a small plastic bottle
containing a special mix that includes gold nanoparticles, they
combine in specific ways depending on whether an analyte
being investigated is present or not. If it is, the solution turns a
shade of blue; if not, it turns red.

31. REFERENCES
 Research paper : DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT
ASSAY (ELISA) FOR THE DETECTION OF PECAN RESIDUES IN PROCESSED
FOODS by Denise Tran
 ELISA Encyclopedia by Sino Biological Inc.
 ELISA Technologies, Laboratory Testing services and Diagnostic Kits
website.
 Wikipedia website