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ELISA
B. Pharm 8th semester
Advanced instrumentation techniques
INTRODUCTION ELIZA
• Enzyme linked immunosorbent
assay is a biochemical assay that
involves an enzyme used for
measuring a wide variety of
samples, especially body fluids

QUANTITATIVE QUALITATIVE
• Detects substances with
antigenic properties • Quantity of Antigen or • Type of antigen or
Antibody antibody
(concentration)
HISTORY
• 1960 - First described by Rosalin Sussman Yalow, an
American Medical physicist and Solomon Berson ,
American physician
• 1971- Peter Perlmann and Eva Engvall, Swedish
scientists at Stockholm university, Sweden
• Anton Schuurs and Bauke van Weemen in Solomon Berson
R.S. Yalow
Netherlands – independent publications
• 1971 - Van Weemen BK, Schuurs AH - Immunoassay
using antigen-enzyme conjugates
• 1974 – A. Voller, D. Bidwell, G. Huldt, E. Engwall –
Microplate method of Enzyme-Linked Immunosorbent
Assay and its application to malaria (96-well
polystyrene plate)
Peter Pearlmann
• 1977 – Nobel Prize for Medicine to Rosalin Yalow for Eva Engvall
the development of the RIA for peptide hormones –
displacement of antibody-bound -insulin by serum-
derived insulin – separation of antibodies and
antigen-antibodies complex
 COMPONENTS OF ELISA
ANTIBODY ANTIGEN LABELLING MATERIALS
Protein produced by immune Molecules that induces Market that labels either
system production of antibodies when antigen or antibody.
introduced in the body Donot interfere with binding.
Have variable sites for
recognition and binding with Can be enzyme or substrate.
antigen

Monoclonal
Polyclonal
Recombinant
Synthetic

Primary Coating Tracer

Secondary Conjugate
1. Primary Antibody
• interacts with antigen
• un/labeled

2. Secondary Antibody
• interacts with primary antibody
• labeled

3. Coating
• interacts with antigen
• unlabeled

4. Conjugate
• labeled secondary antibody
• conjugated enzyme

5. Tracer
• labelled antigen in competitive assays
PRINCIPLES

 1. To use an enzyme to detect the Ag-Ab binding. The enzyme converts a

Competitive TYPES Indirect

Sandwiched
Elisa
 DIRECT ELISA
Only an Enzyme labelled primary antibody is
used.
No requirement of Secondary antibodies.
Enzyme Labelled primary antibody directly
binds to the target antigen- immobilized to
the plate.
Enzyme linked to the primary antibody reacts
with substrate to produce visible signal- can
be measured
INDIRECT ELISA
• Both primary and secondary antibody are
used
• Unlabelled primary antibody used- not
labelled with enzyme
• Secondary antibody labelled with enzyme
• Antigen fixed on plate
• Primary antibody binds to antigen
• Labelled secondary antibody binds to
primary antibody
• Secondary antibody reacts with substrate-
produce visible color change signal
SANDWICHED ELISA
• Antibody immobilized on solid surface- called
CAPTURE ANTIBODY
• Involves use of detection antibodies-
i) unlabelled primary detection antibody
ii) enzyme labelled secondary detection
antibody
• Target antigen binds to capture antibody
• Primary detection antibody binds to antigen
• Secondary detection antibody binds to primary
detection antibody
• Reaction of enzyme with substrate to produce
visible signal- colour change
COMPETITIVE ELISA
• Excess of Unlabelled primary antibody
incubated with sample containing target
antigen
• Forms Ag-Ab complex
• Ag-Ab mixture added to plate coated with
inhibitor antigen- binds to primary antibody
• Free primary antibody in the mixture binds
to inhibitor antigen
• Ag-Ab complexes already formed donot bind
to inhibitor antigen
• Enzyme labelled secondary antibody added
to plate- binds to primary antibody-inhibitor
antigen complex
• Substrate added-reacts with enzyme- visible
coloured signal
• Labelled antigen competes for primary
antibody binding site with unlabelled
antigen.
• More antigen in the sample, less labelled
antigen retained in the plate-weaker
intensity in coloured signal
REQUIREMENTS
1. Coated Plates : 96 well microtitre
plates
2. Sample Diluents: specific sample
diluent
3. Controls : both positive and negative
4. Wash concentrate :basically phosphate
buffer solution containing detergent Microtitre plate

5. Substrate : Specific substrate according

Washer system
ENZYMES AND SUBSTRATES USED
FOR ELISA
1. PNPP ( p-Nitrophenyl phosphate)-
detect alkaline phosphatise and
yellow water product –at 405 nm
2. ABTS (2,2-Azinobis[3-
ethylbenzothiazoline]-6-sulfonic
acid)- detect HRP enzyme and
green colour product formation – at
ABTS
410 and 650 nm
PNPP
3. OPD (o-phenylenediamine) – to
detect HRP and yellow orange
colour product formed – at 492 nm
4. TMB ( 3,3,5,5-
tetramethylbenzidine)- to detect
HRP and blue colour product –
detected at 370 and 652 nm

OPD
TMB
ADVANTAGES AND DISADVANTAGES OF
ELISA

ADVANTAGES DISADVANTAGES
 Fast results- 90 samples tested in 2-3 hr  Measurement of enzyme activity can be more
complex than measurement of activity of some
 Sensititivity ( upto 10 pg/ml) type of radioisotopes
 Specificity ( samples with high concentration  Enzyme activity may be affected by plasma
contaminants constituents
 Many samples can be processed at once  Kits are commercially available but expensive
 Small sample size required  Very specific to a particular antigen
 Colorimetric results ( easily observed and
measured through spectrophotometer)
 Test for presence of Ag or Ab
 Flexible usage of research design
 Simple procedure