PPT on ELISA covering different types of ELISA, their applications and their advantages and disadvantages.

1. ENZYME LINKED
IMMUNOSORBENT ASSAY
(ELISA)
Arshdeep Singh
M.Sc. Biochemistry
PGIMER Chandigarh

2. ENZYME IMMUNOASSAY
• EIA is a term used to describe all the tests that detect either antigen or antibodies or haptens in the
specimen, by using enzyme-substrate system for detection.
• They can be classified into homogenous or heterogeneous EIA.
Homogenous EIA:
• Performed for detection of haptens such as
drugs (e.g. opiates, cocaine), but not for
microbial antigens and antibodies.
• One step: The test can be completed in one step,
with all the reagents added simultaneously.
• There is no need to separate the bound and free
fractions of haptens, hence, there is no washing
step needed.
• Example of homogeneous assay is enzyme
multiplied immunoassay techniques (EMIT).
Heterogenous EIA:
• Used for detection of antigens and antibodies.
• Multiple steps: It requires the separation of
the free and bound fractions which is carried
out by absorption of the test Ag or Ab on to the
solid surface followed by washing.
• It involves multiple steps with different
reagents being added at every step.
• ELISA is a classical example of heterogeneous
assay.

3. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
• ELISA is so named because of its two components:
Enzyme is used to label one of the components of immunoassay (i.e. antigen or antibody).
Immunosorbent: Here, an absorbing material is used ( e.g. polystyrene, polyvinyl) that
specifically absorbs the antigen antibody present in serum.
Substrate-chromogen system: A substrate-chromogen system is added at the final step of ELISA.
• The enzyme reacts with the substrate, which in turn activates the chromogen to produce a colour.
• Sometimes, the substrate is chromogenic in nature (e.g. pNPP). On reaction with the enzyme, it
changes its colour.
• The color change is detected by spectrophotometry in an ELISA reader.
• Intensity of the color is directly proportional to the amount of (Ag or Ab) present in test serum.
(Ag-Ab Complex) – Enzyme + Substrate  activates the chromogen  Colour change  detected
by spectrophotometry

4. Enzymes used in ELISA and their substrate –
chromogen system
Enzyme Substrate Chromogen
Horseradish Peroxidase H2O2 Tetramethyl Benzidine (TMB)
Urease Urea Bromocresol
β Galactosidase ONPG ONPG
Alkaline Phosphatase pNPP pNPP
ONPG = o-nitrophenyl-D-galactopyranoside  Galactose + o-nitrophenol
pNPP = p-nitrophenyl phosphate  p-nitrophenol (color product)

5. Microtiter Plate
• ELISA is usually performed on a microtiter plate containing 96 wells (micro-ELISA) or less
commonly performed in cubes (macro-ELISA).
• The microtiter place or the cubes are made up of polystyrene, polyvinyl or polycarbonate material.
• ELISA kits are commercially available; contain all the necessary reagents (such as enzyme
conjugate, dilution buffer, substrate/chromogen, etc.).
• The procedure involves a series of steps done sequentially; at each step, a reagent is being added,
and then incubated followed by washing of the wells (manually or by an automated ELISA
washer).

6. PRINCIPLES OF DIFFERENT ELISA TYPES
ELISA type Used for detection of Enzyme is labelled with
Direct ELISA Antigen Primary antibody
Indirect ELISA Antibody or antigen Secondary antibody
Sandwich ELISA Antigen • Primary antibody in direct
sandwich ELISA
• Secondary antibody in indirect
sandwich ELISA
Competitive ELISA Antigen or antibody Secondary antibody

7. Direct ELISA
It is used for detection of antigen in test serum. Here, the primary antibody (targeted against the serum antigen) is
labelled with the enzyme.
Step 1: Wells of microtiter place are empty, not precoated with Ag or Ab.
Step 2: Test serum (containing antigen) is added into the wells. Antigen becomes attached to the solid phase by
passive adsorption.
Step 3: After washing, the enzyme-labeled primary antibodies (raised in rabbits) are added.
Step 4: After washing, a substrate-chromogen system is added and color is measured.
Well + Ag (test serum) + primary antibody-enzyme + substrate-chromogen  Colour change

8. Indirect ELISA
• Used for detection of antibody or less commonly antigen in serum
• Unlike direct ELISA, the secondary antibody is labelled with enzyme
• The secondary antibody is anti – species antibody e.g. anti human Ig. (an antibody
targeted to Fc region of any human Ig)

9. Indirect ELISA for antibody detection
Step 1: The solid phase of wells are precoated with Ag
Step 2: Test serum containing primary antibodies specific to the Ag is added to the wells. Ab attaches
to the Ag coated on the well.
Step 3: After washing, enzyme – labelled secondary Ab (anti – human Ig) is added
Step 4: After washing, a substrate – chromogen system is added and color is developed.
Wells are coated with Ag + 10 Ab (Serum) + 20 Ab-enzyme + Substrate chromogen system
Colour Development

10. Indirect ELISA for Antigen Detection
Here, the wells are empty, not coated with Ag or Ab.
Step 1: The test Ag (serum) is added to the well. The Ag gets adsorbed onto the well.
Step 2: The primary Ab raised in rabbits (reagent) is added. The Ag binds the primary Ab.
Step 3: After washing, enzyme – labelled secondary Ab (anti rabbit Ig) is added.
Step 4: After washing, a substrate – chromogen system is added and color is developed.
Ag (test serum) added, adsorbed onto the wells + 10 Ab + 20 Ab-Enzyme + substrate-chromogen
Colour Development

11. Sandwich ELISA
• Detects Ag in serum
• Named so because the Ag gets sandwiched between a capture Ab and a
detector Ab.
Sandwich
ELISA
Direct
Detector Ab is
a 10 Ab
Indirect
Detector Ab is
a 20 Ab

12. Direct Sandwich ELISA
Step 1: The microtiter well is precoated with the capture Ab targeted against the test Ag.
Step 2: The test serum (Ag) is added which gets attached to the capture Ab coated on the well
Step 3: After washing, an enzyme labelled primary “detector Ab” specific for the Ag is added. The
detector Ab can be same as the capture Ab.
Step 4: After washing, a substrate-chromogen system is added and colour is developed.
Wells coated with capture Ab + Ag (test serum) + primary Ab-enzyme + substrate-chromogen
Colour Development

13. Indirect Sandwich ELISA
• The Primary and the capture Ab belong to different species.
• More so, the primary Ab is not labelled with enzyme.
• Another enzyme-labelled secondary Ab targeted against the primary Ab is added.
• Thus, it is more specific than the direct sandwich ELISA.
Wells coated with capture Ab + Ag (test serum) + primary Ab + secondary Ab-enzyme + substrate-chromogen
Colour Development

14. Competitive ELISA
• The competitive ELISA used for the detection of antigen in the test sample.
1. In this technique, at first, the antibody (primary antibody) is incubated with a sample
solution containing antigens.
2. After that this antigen-antibody complex is added to an antigen-coated microtiter well.
3. The more antigen present in the initial solution-phase sample, the less free antibody will be
available to bind to the antigen-coated well.
4. After that unbound antibodies are removed from the microtiter well by using ELISA
washer.
5. Then an enzyme-linked secondary antibody added to this microtiter well which is specific
for the isotope of primary antibody. It will help to determine the amount of primary
antibody bound to the well.
• In the competitive assay, the higher the concentration of antigen in the original sample, the
lower the final signal.
Named so because Ag in the test
serum competes with another Ag of
same type coated on well to bind to
the primary Ab.

15. ELISA Sensitivity
• The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng
to 0.1 ng, with sensitivity dependent upon the particular characteristics
of the antibody-antigen interaction.
• In addition, some substrates such as those yielding enhanced
chemiluminescent or fluorescent signal, can be used to improve
results.

16. Chemiluminescence
• Measurement of light
• Provides a convenient and highly sensitive alternative to absorbance
measurements in ELISA assays.
• Here a luxogenic (light-generating) substrate takes the place of the
chromogenic substrate in conventional ELISA reactions.
• For example, oxidation of the compound luminol by H2O2 and the
enzyme horseradish peroxidase (HRP) produces light:

17. Advantage of chemiluminescence
• Enhanced sensitivity.
• In general, the detection limit can be increased at least ten-fold by
switching from a chromogenic to a luxogenic substrate, and with the
addition of enhancing agents, more than 200-fold.
• As little as 5 attomoles (5 X 10 -18 moles) of target antigen have been
detected.

18. ELISPOT Assay
• A modification of the ELISA assay
• Allows the quantitative determination of the number of cells in a
population that are producing antibodies specific for a given antigen.

19. ELISPOT Assay (for Cytokine producing Cells)
• In the ELISPOT assay, a well is coated with antibody against
the antigen of interest, a cytokine in this example, and then a
suspension of a cell population thought to contain some
members synthesizing and secreting the cytokine are layered
onto the bottom of the well and incubated.
• Most of the cytokine molecules secreted by a particular cell
react with nearby well-bound antibodies.
• After the incubation period, the well is washed and an
enzyme-labeled anti-cytokine antibody is added.
• After washing away unbound antibody, a chromogenic
substrate that forms an insoluble colored product is added.
• The colored product (purple) precipitates and forms a spot
only on the areas of the well where cytokine-secreting cells
had been deposited.
• By counting the number of colored spots, it is possible to
determine how many cytokine-secreting cells were present in
the added cell suspension.

21. APPLICATIONS OF ELISA TEST
1.Used for the detection of HIV.
2.In the food industry, it used for the detection of potential food allergens, such as milk, peanuts,
walnuts, almonds, and eggs.
3.In toxicology ELISA used for rapid presumptive screening for certain classes of drugs.
4.ELISA used for the detection of different diseases, such as dengue, malaria, Chagas disease,
Johne’s disease, etc.
5.It is also being used as in-vitro diagnostics in medical laboratories.
6.ELISA also detects Mycobacterium antibodies, rotavirus in feces, hepatitis B markers in serum,
hepatitis C markers in serum, enterotoxin of E. coli in feces.

22. CONCLUSION
• ELISA is a novel technique useful in detecting (quantitatively or
qualitatively) an Ag or Ab present in the given biological sample.
• Besides its disadvantages the technique is being widely used in
diagnostics and drug screening.
• Chromogenic detection method used in ELISA is convenient and
sensitive for any assay and is hazard free.

23. References
• Textbook of microbiology Apurba Sankar Sastry 2nd edition
• Kuby immunology 6th edition
• https://microbenotes.com/competitive-elisa-protocol-and-
animation/#:~:text=Protocol%20and%20Animation-
,Competitive%20ELISA%20is%20a%20technique%20used%20for%20the
%20estimation%20of,for%20antibodies)%2C%20are%20used.
• https://medlineplus.gov/ency/article/003332.htm#:~:text=ELISA%20stands
%20for%20enzyme%2Dlinked,detects%20harmful%20substances%2C%20
called%20antigens.
• https://www.immunology.org/public-information/bitesized-
immunology/experimental-techniques/enzyme-linked-immunosorbent-assay
• https://www.cusabio.com/c-20659.html