ELISA (enzyme-linked immunosorbent assay) is an analytical biochemistry technique used to detect the presence of an antibody or antigen in a sample using antibodies specific to the target antigen or antibody. There are four main types of ELISA: direct, indirect, sandwich, and competitive. ELISA uses an enzyme-linked antibody or antigen to detect the target molecule and a chromogenic substrate to produce a detectable color change that corresponds to the amount of target molecule present. ELISA has many applications in clinical diagnostics, pharmaceutical analysis, and food safety due to its high sensitivity and specificity.

1. ELISA ( ENZYME LINKED
IMMUNOSORBENT ASSAY)
Minu Treeza M
1st Mpharm

2. IMMUNOASSAY
• Immunoassays are bioanalytical methods in which the quantification of
the unknown antigen/antibody(analyte)based on antigen-antibody
reaction.
• Quantification is done by using a specific known labelled
antigen/antibody.
• Label used- Enzymes , radioactive compounds, chemiluminiscent agents,
etc.
• Widely used – diagnosis of diseases, TDM, clinical pharmacokinetic and
bioequivalence studies,etc.

3. ENZYME LINKED IMMUNO SORBENT ASSAY
(ELISA)
Principle :
• Based on the formation of Ag- Ab complex ,which is detected by
chromogenic detection using enzyme conjugated secondary
antibody/antigen.
• An enzyme conjugated with an antibody/antigen reacts with a colourless
substrate to generate a coloured reaction product . Such a substrate is called a
chromogenic substrate.
• The amount of compound corresponds to the substrate converted to coloured
product by the enzyme linked antibody.
• Light absorption of the product formed after substrate addition is measured
and converted to numeric values.

5. DIRECT ELISA
• Used for the detection of antigen in
the given biological sample.
• Microtiter wells are initially coated
with Ag to be detected which is
followed by an Ab linked to an
enzyme conjugate
• This follows addition of substrate
which produces color detected using
ELISA detector.

6. Advantages:
• Simple and quick to perform due to minimal steps required
Disadvantages:
• Specificity of the primary antibody may be affected by the enzyme-
linking
• Linking 1' antibody for each specific ELISA experiment is expensive
and time-consuming
• Minimal signal amplification

7. INDIRECT ELISA
Used for the detection of antibody in the given sample.

8. • Microtiter wells are initially coated with antigen specific for antibody to be
detected.
• Serum or some other sample containing primary antibody is added to the
microtiter well and allowed to react with the coated antigen
• Any free primary antibody is washed away and the bound antibody to the
antigen is detected by adding an enzyme conjugated secondary antibody
that binds to the primary antibody.
• Unbound secondary antibody is then washed away and a specific
substrate for the enzyme is added.
• Enzyme hydrolyzes the substrate to form colored products.
• The amount of colored end product is measured by spectrophotometric
plate readers that can measure the absorbance of all the wells of 96-well
plate.

10. Advantages:
• Specificity of the 1' antibody is retained
• Many enzyme-linked 2' antibodies are commercially available
• Better signal amplification since multiple polyclonal 2' antibodies can bind
to each 1' antibody
Disadvantages:
• Potential cross reactivity with 2' antibody leading to non-specific signal

11. SANDWICH ELISA
Used for the detection of antigen in the given biological sample.

12. • Antibody is coated on the microtiter well.
• A sample containing antigen is added to the well and allowed to react with
the antibody attached to the well, forming antigen-antibody complex.
• After the well is washed, a second enzyme-linked antibody specific for a
different epitope on the antigen is added and allowed to react with the
bound antigen.
• Then after unbound secondary antibody is removed by washing. Finally
substrate is added to the plate which is hydrolyzed by enzyme to form
colored products.

13. Advantages:
• High specificity since the signal detection requires the binding of two 1'
antibodies
• Crude sample can be used: target antigen-antibody complex is
immobilized to the plate, and everything else can be washed off
Disadvantages:
• Commercially-prepared kits may not be available

14. COMPETITIVE ELISA
• This test is used to measure the concentration of an antigen in a sample.

15. • Antibody is first incubated in solution with a sample containing antigen
• The antigen-antibody mixture is then added to the microtitre well which is
coated with antigen.
• The more the antigen present in the sample, the less free antibody will be
available to bind to the antigen-coated well.
• After the well is washed, enzyme conjugated secondary antibody specific
for isotope of the primary antibody is added to determine the amount of
primary antibody bound to the well.
• The higher the concentration of antigen in the sample, the lower the
absorbance.

16. Advantages:
• High sensitivity
• Crude sample can be used
• Signal can be quantified by comparing to a serial dilution standard curve
Disadvantages:
• Requires 1' antibodies with high specificity to the antigen

17. Components of ELISA kit

19. ENZYMES AND SUBSTRATES
• ABTS – 2,2’-azino-(3-
ethylbenzothiazoline-6-sulphonic
acid)
• TMB (3,3',5,5'-
Tetramethylbenzidine)
TMB is one of the most versatile,
most widely used substrates for
ELISA assays. It is typically used in
conjunction with HRP and results
in a blue color reaction.
• pNPP (Para-
Nitrophenylphosphate)
pNPP is used to detect alkaline
phosphatase in ELISA kits.

20. Data analysis
• After adding stop solution, keep
the microtiter plate in microtiter
reader.
• Standard curve of the known
concentration of the sample is
plotted.

21. • For quantitative and semi-quantitative ELISAs, a Standard Curve is
generated using a serial dilution of Standard with a known concentration.
• The signal reading (optical density, fluorescence) of each concentration is
graphed vs log concentration to produce a sigmoidal curve .
• The relatively long linear region of the curve is most accurate and
reproducible and is used to generate a linear equation.
• The intensity of a sample with an unknown analyte concentration can then
be applied to this linear equation to determine analyte concentration.

23. ELISAs can be designed to yields three different types of results, quantitative,
qualitative and semi-quantitative.
 Quantitative
The precise amount of analyte in a test sample can be determined by
comparing its values to those generated from a serial dilution of a known,
purified analyte (a Standard Curve). This type of result is particularly useful
when measuring the effect on a system in response to changing
experimental conditions.
 Qualitative
Qualitative assessment simply indicates whether an analyte is present in
the sample or not, as compared to a negative and positive control run in
parallel. This type of result is commonly used for diagnostic tests, such as
detecting the presence of immune response to a particular pathogen.
 Semi-quantitative
Semi-quantitative results indicate the relative amount of analyte within a
sample because the strength of the signal obtained will vary
proportionately with analyte concentration.

24. • ADVANTAGES
 Reagents are relatively cheap &
have long shelf life.
 Highly specific and sensitive
 No radiation hazards occuring
during labelling or disposal of
waste.
 Easy to perform
 Equipment can be inexpensive
and widely available.
• DISADVANTAGES
 Mesurement of enzyme activity
can be more complex than
measurement of some types of
radioisotopes.
 Enzyme activity may be affected
by plasma constituents.

25. APPLICATIONS
• Presence of antigen or the presence of antibody in a sample can be
evaluated.
• Determination of serum antibody concentrations in a virus test.
• Used in food industry when detecting potential food allergens.
• Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird
flu, common, colds, cholera, STD etc.

26. REFERENCE
• https://microbiologynotes.com/elisa-principle-types-and-applications
• https://www.lsbio.com/resources/devkitfaq#competitive-elisa
• https://blog.benchsci.com/elisa-principle
• Darwish I. A (2006) Immunoassay methods and their applications in pharmaceutical analysis:
Basic methodology and recent advances. International Journal of Biomedical Science.