ELISA

Enzyme Linked
ImmunoSorbent Assay
Group 03
Anudhi Dangampala
Contents
Important Words to Know 3

What is ELISA? 4-5

Steps of ELISA 6-9

Advantages and Disadvantages of ELISA 10

Video Representation of ELISA 11

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Important Words to Know
• Immunoassay – A highly selective analytical method that
measures the presence or concentration of analytes. Eg:
antigen
• Reporter enzyme- Produces a colour change when it acts on a
specific substrate.
• Immunohistochemical staining- It is a method for detecting
antigens in cells of a tissue section.

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What is ELISA?

• ELISA stands for Enzyme Linked ImmunoSorbent Assay.

• It is a labelled immunoassay where antigen or antibody is labelled with a

reporter enzyme.

• This is used for detection and quantification of substances such as peptides,

proteins, antibodies and hormones.

• This is commonly used for immunohistochemical staining of tissues and cells.

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What is ELISA?
• There are 3 types of ELISA as,
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
Direct ELISA Indirect ELISA

Enzyme linked antibody directly binds to the antigen Enzyme linked antibody is not directly bound to the
present in the sample. enzyme, it is bound to the antibody already bound to
the antigen.

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Steps of Direct
ELISA
Step 01: Plate Coating
• Antigens present in serum sample
are passively adsorbed and
immobilized in microtiter well.

• Walls are rinsed to remove unbound

antigens.

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Step 02: Antibody Incubation
• Enzyme linked antibodies specific to
the target antigen are added.
• Antibodies will bind to the antigen if
they are present in the serum
sample.
• Walls are rinsed to remove unbound
antibodies.

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Step 03: Detection
• Substrate (chromogen) that is specific for
the enzyme (linked to the antibody) is
added.
• Colour develops when the enzyme acts on
the substrate added and a coloured end
product is formed.
• Coloured sample – positive test result
• Non-coloured sample – negative result
• Quantification can be done by measuring
the output by a spectrophotometer.
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Advantages and Disadvantages of
ELISA
Advantages Disadvantages
• Fast and simple procedure • Less specific as only one antibody is used.

• Less reagents and fewer steps used. • Antigen immobilization is not specific resulting
in potentially high background interference.

• Cross reactivity of secondary antibody is • Primary antibody must be labeled individually

eliminated. which is slow and time consuming.

• A specific linked antibody is needed for each

target protein.

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Video Representation of ELISA

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Thank you