Elisa (Enzyme Linked Immuno Sorbent Assay)

ENZYME LINKED IMMUNO
SORBENT ASSAY
(ELISA)
SIVASANGARI SHANMUGAM

CONTENTS
 Introduction
 History
 Principle of ELISA
 Materials ,Sample & Reagents
 Equipments
 Micro titre plate (or) 96 well Plate
 ELISA plate washer
 Liquid dispensing system
 Micro plate incubator
 Micro plate reader (or) ELISA reader
• Principle
• Procedure
 Application
 Advantages
 Disadvantages
 Types of ELISA
 Qualitative ELISA
 Quantitative ELISA

ELISA
 Biochemical assay technique.
 Also, called as enzyme immunoassay (EIA).
 Used in immunology.
 For detecting and quantifying substances.
 EX; Peptides, Proteins, Aminoacids and Hormones

HISTORY OF ELISA
 1900 - Antibody formation theory (Paul Ehrlich)
 1938 - Antigen-Antibody binding hypothesis (John Marrack)
 1948 - antibody production in plasma B cells
 1959-1962 - Discovery of antibody structure
 1960 - Radioimmunoassay was first described by Rosalyn Sussman Yalow and
Solomon Berson
 1971 - Peter Perlmann and Eva Engvall at Stockholm University invented ELISA
 1975 - Generation of the first monoclonal antibodies (George Kohler and Cesar
Milstein)

PRINCIPLE OF ELISA
 In ELISA, various antigen-antibody combinations are used, always including an
enzyme-labeled antigen or antibody, and enzyme activity is measured
colorimetrically.
 The enzyme activity is measured using a substrate that changes color when
modified by the enzyme.
 Light absorption of the product formed after substrate addition is measured
and converted to numeric values.
 Depending on the antigen-antibody combination, the assay is called a direct
ELISA, indirect ELISA, sandwich ELISA, competitive ELISA etc.
 Follow LOCK(ab) & KEY(ag) method.

MATERIALS
1. Testing sample
2. Antibody ( 1st,2nd)/Antigen
3. Polystyrene microtiter plate
4. Blocking buffer
5. Washing buffer
6. Substrate
7. Enzyme

TESTING SAMPLES
1. Serum
2. Sputum
3. Urine
4. Semen
5. Stool
6. Supernatant of culture

REAGENTS
1. COATING BUFFER - 0.1M Phosphate buffer+ 0.15M Nacl
2. WASHING BUFFER - 0.1M Phosphate buffer+ 0. 50M Nacl + 0.1%
Tween20
3. BLOCKING BUFFER – Bovine Serum Albumin (BSA)
4. ENZYME - Horse Radish Peroxidase (HRPO)
5. CHROMOGENIC SUBSTRATE – Trimethyl benzidine
6. STOP SOLUTION - 0.5M H2So4

LAB EQUIPMENTS
1. Micro plate strips
2. Pipettes
3. Pipette tips
4. Transfer pipette
5. Paper towels
6. Marking Pen

EQUIPMENTS
In order to perform the ELISA technique, the following equipments are
required :
1. ELISA 96 well Micro plate
2. Micro plate washer
3. Liquid dispensing system ( multi-channel pipettes)
4. Incubator (to incubate the plate)
5. Micro plate reader

ELISA PLATE
• Microtitre wells.
• Generally 96 wells.
• Marked on one side
alphabetically.
• Numerical on the other side.
• 8cm*12cm plastic plate it
contains 8*12 matrix of 96 wells,
each of which are about 1cm
high & 0.7cm in diameter.
• Available in both black & white
color.

ELISA PLATE WASHER
 Very efficient with low carry over contamination.
 Used to wash the well plate.

LIQUID DISPENSING SYSTEM
MULTIPIPETTE:
• An 8 channel 100 pipette is a
good help for even small scale
work.
• Used to fill sample to each
wells.
• Make work to easy.

MICRO PLATE INCUBATOR
Used to incubate the
plate.
Maintain temperature.
Important for good result.

IMPORTANCE OF WASHINNG AND
INCUBATION
WASHING :
 For the removal of unbound Ab/Ag proper washing.
 No proper wash we will get false results.
INCUBATION :
 Maintain temperature which is must for Ag – Ab binding.

ELISA READER
 Also known as photometric micro
plate reader (or) ELISA reader.
 Principle – “Photoelectri
clorimetry”.
 Wavelength is 400 – 700nm
(spectrophotometer).
 Some reader operate UV (340 –
700nm).

PRINCIPLE OF READER
Based on the Ag – Ab complex,which is detected by
chromogenic detection using enzyme conjugated
secondary Ab.
The conjugated enzyme acts on a specific
substrate,generate a coloured reaction products.
This product is qualitatively (or) quantitatively read using
ELISA plate reader.

PROCEDURE OF READER
Plate is placed into the plate reader.
Optical density (amount of colored product)is determined
for each well.
The amount of color produced is proportional to the
amount of primary Ab bound to the proteins on the
bottom of the wells.

APPLICATION OF ELISA
1. Detection of retrovirus in feces.
2. Detection of Hepatitis markers in serum.
3. Detection HIV antibodies in blood.
4. Detection of mycobacterium antibodies in
Tuberclosis.

ADVANTAGES OF ELISA
1. Reagents are long self life time.
2. Highly specific & sensitive.
3. Easy to perform & Quick procedure.
4. Used to detect a variety of infection.

DISADVANTAGES OF ELISA
1. Result may not be absolute.
2. Antibody must be available.
3. Concentration is not clear.
4. False positive possible.
5. False negative possible.
6. Requires trained person.
7. Labs requires a special license to handle radioactive materials.

TYPES OF ELISA
1. Qualitative ELISA :
 Positive (or) Negative result
 Ag / Ab presence or absence
 It include direct, indirect and sandwich ELISA.
2. Quantitative ELISA :
 Determine the quantity of antibody.

METHODS OF ELISA
Direct Indirect
Sandwich Competitive

DIRECT ELISA
 Primary labelled Ab is directly binds to the Ag.
 Not require secondary Ab.
 Perform with ag which is directly immobilized on assay plate.

PROCEDURE
Detect
Substrate added
Primary Ab is added
Add blocking buffer
Ag added to the plate

DIRECT ELISA
Advantages :
1. Faster than other ELISA – the technique has fewer steps.
2. Less prone to error – as less reagents and fewer steps are required
3. Only one Ab is needed.
4. Require less time.
Disadvantages :
1. Antigen immobilization is not specific - may cause higher background noise than
indirect ELISA.
2. Mainly because all proteins in the sample, including the target protein, will bind to the
plate.
3.
Less flexible - each target protein needs a specific conjugated primary antibody.
4.
No signal amplification - reduces assay sensitivity.

INDIRECT ELISA
 Micro-well plates are incubated with antigens, washed up and blocked
with BSA.

Samples with antibodies are added and washed.

Enzyme linked secondary antibody are added and washed.

A substrate is added, and enzymes on the antibody elicit a chromogenic or
fluorescent signal.

PROCEDURE
Detect
Substrate is added
HRPO is added (which recognize & bind to the 1°Ab
2° Ab is added
1° Ab is added
Add blocking buffer
Ag added to the plate

INDIREST ELISA
ADVANTAGES :
1. High flexibility
2. Signal amplification
DISADVANTAGES :
1. Complex protocol.
2. Cross reactive from secondary Ab

SANDWICH ELISA
• Ag in the sample bind with the capture Ab become immobilized.
• The ab of the enzyme conjugate bind with the immobilized ag to form
sandwich of Ab-Ag-Ab.
• Antigens like tumor markers,serum protein,hormones.

PROCEDURE
Color change observed
Substrate added
Enzyme linked Ab is added
Ag bounded to the capture Ab
Sample is added
Blocking buffer added
Plate coated with Ab (Capture Ab)

SANDWICH ELISA
ADVANTAGES :
1. High flexibility
2. High sensitivity
3. high specificity
Disadvantages :
1. Optimization in terms of antibody becomes problematic due to
cross-reactivity issues.

COMPETITIVE ELISA
• Inhibition ELISA, Blocking ELISA, Plate/surface based assay.
• Ab is first incubated in the solution with a sample containing Ag.
• Ag-Ab mixture is added to the Ag coated micrititer well.
• The more Ag present in the sample ,the less free Ab is available to bind
to the Ag coated well.

PROCEDURE
Detect
Substrate is added
2°Ab with enzyme added
Wash & Remove unbound Ab
Bound Ag-Ab
Incubate unlabelled Ab with Ag

COMPETITIVE ELISA
Advantages :
1. Negligible sample processing is required and can be applicable to
crude samples
2. Less sensitive to experimental errors.
3. Good reproducibility and flexibility.
Disadvantages :
1. Basic ELISA limitations apply.

REFERENCE
1. http://ruo.mbl.co.jp/bio/e/support/method/elisa.html
2. https://www.bosterbio.com/protocol-and-troubleshooting/elisa-
principle
3. https://www.healthline.com/health/elisa#more-information-about-elisa
4. https://www.sinobiological.com/elisa-introduction.html
5. https://www.sinobiological.com/elisa-history.html
6. http://www.cusabio.com/m-264.html
7. https://www.mybiosource.com/learn/ELISA

Elisa (Enzyme Linked Immuno Sorbent Assay)

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Elisa (Enzyme Linked Immuno Sorbent Assay)