ADVANCED CLINICAL
BIOCHEMISTRY
By: Hussein Mohamed
Definition:
The Enzyme-Linked Immunosorbent Assay
(ELISA) is a common laboratory technique
which is used to measure the concentration of
an analyte (usually antibodies or antigens) in
solution.
History:
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Basic Terms
Solid Phase
Usually a microtiter plate well, having
8 X 12 well format.
Basic Terms
Adsorption
The process of adding an antigen/antibody,
diluted in buffer, so it attaches to the solid phase on
incubation.
Washing
The simple flooding & emptying of wells with a
buffered solution to separate bound from unbound
reagents in ELISA.
Basic Terms
Antigen
Any molecule that elicits the production of
antibodies when introduced into body.
Antibodies
Proteins produced in response to antigenic
stimuli.
Enzyme conjugate
An enzyme that is attached irreversibly to an
antibody.
e.g: Horse-redish peroxidase (HRPO).
Basic Terms
Chromogen:
A chemical alters color as a result of an
enzyme interaction with substrate (color reaction
used as signal) e.g Trimethyl benzidine (TMB).
Stopping:
The process of stopping the action of an enzyme
on a substrate.
Reading:
Spectrophotometric measurement of color
developed in ELISA.
Principle of ELISA
Based on Basic Immunology Response
Lock and Key Concept:
1) Antigen (key) 2) Antibody (lock):
–Key fits into the lock
Enzyme conjugate substrates
Bound to a secondary antibody that binds with the
antibody-antigen complex.
Equipment
1) Microwell Plate
Flat bottom polystyrene plate, contains 8 x 12
wells holding 350 μL each.
Equipment
2) Multipipette
An 8-channel 100 μL pipette is a good help for
even small-scale work.
Equipment
3) Washing Device:
Manually operated washing devices.
May be of use particularly when there is a risk that
the samples tested in ELISA contain infectious
material, so must be collected for subsequent
disinfection.
Reagents Used:
Reagent Composition
Coating Buffer 0.01 M Phosphate Buffer
+ 0.15 M NaCl (PBS)
Diluting/Washing Buffer 0.01 M Phosphate Buffer
+ 0.50 M NaCl + 0.1% Tween 20
Blocking Buffer Bovine Serum Albumin
(BSA)
Enzyme Horse-redish peroxidase
(HRPO)
Chromogenic Substrate Trimethyl benzidine
(TMB)
Stop Solution 0.5 M H₂SO₄
General Procedure:
Types of ELISA:
On the Basis of Detection
1) Colorimetric ELISA:
Assay to Determine the Antibody Concentration.
Types of ELISA:
2) Chem-iluminescent ELISA:
Assay for the Quantitation of an Antigen in a
biological Sample.
Types of ELISA:
On the Basis of Procedure
Direct
Non-
Indirect
Competitive
Types Competitive Sandwich
Multiple &
Portable
Non- Competitive ELISA
Direct ELISA
It uses a primary labeled anti-body that react
directly with the antigen.
It can be performed with the antigen that is directly
immobilized on assay plate.
Not widely used but common for immuno-
histochemical staining of cells & tissues.
Non- Competitive ELISA
Indirect ELISA
It utilizes a primary un-labeled antibody in
conjunction with a labeled secondary antibody.
Secondary antibody has specificity for primary
antibody.
Non- Competitive ELISA
Sandwich ELISA
Antigens like Tumor markers, hormones, serum
proteins may be determined.
Antigens in the sample bind with the capture
antibody & become immobilized.
The antibody of the enzyme conjugate bind with
the immobilized antigen to form a sandwich of Ab-
Ag-Ab/ enzyme bound to microwell.
