Maarg Manthan · Topic 10.8

Enzyme-Linked Immunosorbent Assay (ELISA)

Science & Technology › Biotechnology – Principles and Techniques · Topic 10.8

The enzyme-linked immunosorbent assay, or ELISA, is a test that detects and measures a specific protein, usually an antibody or an antigen, in a sample such as blood, using an antibody linked to an enzyme. When a substrate is added, the enzyme changes it to give a colour, and the strength of the colour shows how much of the target is present. The method was developed in 1971 by Eva Engvall and Peter Perlmann and, independently, by Anton Schuurs and Bauke van Weemen. ELISA is used to test for HIV, hepatitis, dengue, pregnancy hormone, allergies and many other conditions because it is sensitive, cheap and suitable for many samples at once.

5 min read · UPSC Prelims · Science & Technology · Biotechnology – Principles and Techniques

At a Glance

Full form Enzyme-Linked Immunosorbent Assay
Principle Antibody-antigen binding plus enzyme colour
Support Microtitre plate
Types Direct, indirect, sandwich, competitive
Use Screening of infections
Exam link UPSC Prelims, Science & Technology: Biotechnology – Principles and Techniques

Where Does It Fit?

This topic follows the polymerase chain reaction. PCR detects the genes of a germ, while ELISA detects the proteins and antibodies that the body or the germ makes. Previous topic: Polymerase Chain Reaction (PCR). Next topic: Antibody-Antigen Interaction and Detection Methods.

How Does ELISA Work and What Are Its Types?

Type	How it works
Direct ELISA	The antigen is fixed to the plate and an enzyme labelled antibody binds to it. Simple but less sensitive
Indirect ELISA	The antigen is fixed, a first antibody binds to it, and an enzyme labelled second antibody binds to the first. Commonly used to detect antibodies in a patient
Sandwich ELISA	A capture antibody fixed on the plate holds the antigen, and a second labelled antibody binds to another site. Very specific and used for hormones and proteins
Competitive ELISA	The sample antigen competes with a labelled antigen for limited antibody sites, so a weaker colour means more antigen in the sample
Substrate step	The enzyme, often horseradish peroxidase or alkaline phosphatase, turns a colourless substrate into a coloured product
Reading	A plate reader measures colour intensity, which is compared with known standards

Where Is ELISA Used?

HIV screening: fourth generation tests detect antibodies and the p24 antigen
Hepatitis: tests for surface antigen and antibodies of hepatitis B and C
Dengue: detects the NS1 antigen in early illness and antibodies later
Pregnancy tests: detect the hormone human chorionic gonadotropin
Allergies: measures immunoglobulin E against allergens
Food safety: finds allergens, toxins and residues in food
Veterinary use: tests for animal diseases such as brucellosis
Research: measures proteins such as cytokines and hormones in blood or cell culture

What Else Should You Remember?

Microtitre plate: a plastic plate with 96 small wells so that many samples can be tested together
Washing steps: unbound material is washed away between steps to avoid false signals
Blocking: empty spots on the plate are covered with a neutral protein to stop non-specific sticking
Sensitivity and specificity: sensitivity is the ability to catch true positives, while specificity is the ability to avoid false positives
Window period: in early infection antibodies may not yet be present, so an ELISA for antibodies can be negative even though the person is infected
Confirmation: a positive screening result is usually confirmed with another method such as a Western blot, an antigen test or PCR
Comparison with PCR: PCR finds genetic material and ELISA finds proteins or antibodies, as in Polymerase Chain Reaction (PCR)
Rapid tests: many point of care strip tests use the same antibody antigen idea in a simpler form, as in Diagnostics in Developing Countries
Monoclonal antibodies: highly specific antibodies from a single clone improve accuracy, as in Antibody-Antigen Interaction and Detection Methods
Cost and scale: reagents are cheap and plate readers are common, which makes ELISA a workhorse in district laboratories
Enzyme labels: horseradish peroxidase and alkaline phosphatase are the most common enzymes because they give strong colour signals
Standard curve: known amounts of the target are run on the same plate so that unknown samples can be read from a graph
Blood banks: all blood donations in India are screened for HIV, hepatitis B and C, syphilis and malaria using approved tests
Nobel link: the immunoassay idea goes back to Rosalyn Yalow and Solomon Berson, whose radioimmunoassay won the 1977 Nobel Prize in Physiology or Medicine
Antigen and antibody tests in dengue: the NS1 antigen shows up in the first days, while antibodies appear later, so the type of test depends on the day of illness

Why Does It Matter for the Exam?

Principle: antibody binding and enzyme colour change
Types: sandwich versus indirect
Uses: HIV, dengue, hepatitis
Current affairs link: disease screening programmes
Common errors: confusing ELISA with PCR, or saying that ELISA copies DNA

As an analytical point, ELISA allows mass screening at low cost, but results must be read with the window period and confirmation steps in mind. Training and quality assurance protect patients from wrong results.

Why Is This Topic Important?

Principle Immune binding with colour
Types Four main types
Uses Screening

What Came Next?

The next topic covers antibody-antigen interaction and detection methods. Next topic: Antibody-Antigen Interaction and Detection Methods.

Key People and Terms

Antigen Substance that triggers antibodies
Antibody Immune protein
Substrate Chemical changed by enzyme
Titre Measure of antibody amount
Microtitre plate 96 well plate
Capture antibody Holds the antigen

Exam Corner

Points to Remember

ELISA developed: 1971.
Plate usually has 96 wells.
Signal: colour from enzyme.
Sandwich ELISA: two antibodies.
Detects proteins, not DNA.

Do Not Confuse With

ELISA and PCR Protein or antibody test versus DNA copying test
Direct and indirect ELISA One labelled antibody versus two antibodies
Sensitivity and specificity Catching positives versus avoiding false ones

Memory Hook

Fix, bind, wash, colour; the deeper the colour, the more is there.

Mains Angle

Questions ask for the principle and use of ELISA. These points help.

Principle Binding and colour
Uses Screening
Limits Window period

A Question You May Face

An original practice question, not a past paper question.

“Explain the principle of ELISA and discuss its role and limitations in the screening of infectious diseases.”

How to Answer

Principle Antibody, enzyme, colour.
Types Direct to sandwich.
Uses Screening.
Limits False results, confirmation.

GS Relevance

Prelims: principle, types of ELISA, uses, difference from other tests. GS Paper 3: science and technology, biotechnology, health diagnostics.

Frequently Asked Questions

What is ELISA?

ELISA, or enzyme-linked immunosorbent assay, is a laboratory test that detects a specific antigen or antibody in a sample using an antibody linked to an enzyme. The enzyme changes a colourless substrate into a coloured product, and the colour strength shows how much of the target is present.

What are the main types of ELISA?

The main types are direct, indirect, sandwich and competitive ELISA. Direct ELISA uses one labelled antibody, indirect uses a labelled second antibody, sandwich uses a capture and a detection antibody for high specificity, and competitive ELISA shows lower colour when the sample contains more of the target.

How is ELISA different from PCR?

ELISA detects proteins, such as antigens and antibodies, using antibody binding and an enzyme colour reaction. PCR detects and copies nucleic acids, such as the DNA or RNA of a germ. ELISA shows the immune response or germ proteins, while PCR shows the presence of genetic material.

Which diseases are screened using ELISA?

ELISA is used to screen for HIV, hepatitis B and C, dengue, and many other infections, and to measure hormones such as the pregnancy hormone. It is cheap and can test many samples together on a 96 well plate, so it suits blood banks and district laboratories.

Why can an ELISA test be negative in early infection?

An antibody based ELISA can be negative in early infection because the body takes days to weeks to make detectable antibodies. This gap is called the window period. Newer tests detect antigens as well, which shortens the gap, and positive screening results are confirmed with another method.

PYQ Practice — Statement Analysis

1 ELISA works on the principle of antigen-antibody binding.

True

With an enzyme label.

2 A microtitre plate normally has 96 wells.

True

For many samples.

3 ELISA makes copies of DNA.

False

That is PCR.

4 In sandwich ELISA only one antibody is used.