Elisappt 151008062236 Lva1 App6891

ELISA
By:
Dr. Saba Ahmed
M.Phil. Pharmacology
UNIVERSITY OF SARGODHA
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:
n
Basic Terms:
• Solid Phase:
Usually a microtiter plate well, having 8 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 un-bound
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.
Equipments:
1) Microwell Plate:
Flat bottom
polystyrene
plate,
contains 8 x 12
wells holding
350 μL each.
Equipments:
2) Multipipette :
An 8-channel 100 μL
pipette is a good help
for even small-scale
work.
Equipments:
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.
Equipments:
4) Microplate washer:
• These are very efficient
with unusually low
carry-over
contamination.
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) Chemiluminescent ELISA:
Assay for the Quantitation of an Antigen in a Biological
Sample.
Types of ELISA:
3) Competitive Fluorescence ELISA:

Types of ELISA:
(on the basis of procedure)
Direct
Non-
Indirect
Competitive
Types Competitive Sandwich
Multiple &
Portable
Non-Competitive:
1) 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:
2) 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:
3) 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.
Competitive:
• Antibody coated microwell.
• Serum antigen & labeled antigen added together .... Competition
• Ab-Ag enzyme complex bound is inversely related to the conc. of

antigen present in sample.
• Increased serum antigen results in reduced binding of Ag-

enzyme conjugate with the antibody producing less enzyme
activity & (yellow) color formation.
• Used to determine small molecules like T₃ , T₄ & Progesterone.

Modified ELISA:
• Enzyme interfere with Ag-Ab interaction.
• Second antibody is often labeled with a very small molecular
substance, biotin (MW=244.31), and a specific
binding protein for biotin, avidin is conjugated with enzyme
such as HRP.
Modified ELISA:
Reading:
• Measure the absorbance at 450nm with the help of ELISA
reader.
• Calculate the absorbance for each sample and reference.
• Ascent software for the calculation of results can be used.
Results:
Troubleshooting
in ELISA
Precautions:
1) Use of Exchange type pipette:
(always use new tip)
Precautions:
2) Washing:
Precautions:
3) Reagents:
Precautions:
3) Reagents:
Precautions:
4) Plate cover:
• During incubation, well plate should be covered using the
plate cover
• Plate cover is effective only under suitable conditions i.e room

temp. humidity > 50%, air steam <0.2 m/sec.
Precautions:
5) Coating of wells:
• Coating of wells should be proper with the addition of

Blocking solution.
• Improper coating False positive results

Advantages of ELISA:
• Reagents are relatively cheap & ‘ve long shelf life.
• It is highly specific & sensitive (<1pg/ml).
• No radiation hazards occur during labeling or disposal of
waste.
• Easy to perform & quick procedures.
• Equipment is widely available.
• It can be used to a variety of infections.
• It can be used on most type of biological samples like plasma,
serum, urine, cell extracts.
Disadvantages of ELISA:
• Measurement of enzyme activity can be more complex
than the measurement of activity of some type of
radioisotopes.
• Enzyme activity may be affected by plasma constituents.
• Kits are not cheap.
• Very specific to particular antigen but won’t recognize
other antigens.
• False positive/ negative possible, especially with
mutated/ altered antigen.
Limitations:
• Results may not be absolute.
• Antibody must be available(poor producer,
interference).
• Concentration may be unclear.
• False positive possible (Ab already present).
• False negative possible.

References:
• Gen. procedure of ELISA by DAKO A/S • Produktionsvej 42 •
DK-2600 Glostrup • Denmark, www.dako.com
• ENDOCRINE MANUAL FOR REPRODUCTIVE ASSESSMENT OF
DOMESTIC AND NON-DOMESTIC SPECIES by Janine Brown,
Ph.D , Sue Walker, M.S.
• ELISA -A to Z .....from introduction to practice by Katsumi
WAKABAYASHI, Ph.D , Prof. Emer. Gunma University.
• www.wikipedia.com.
• www.googleimages.com
• www.slideshare.net
• www.hhmi.org/biointeractive/immunology-virtual-lab.
• www.ncbi.nlm.nih.gov/pubmed/25926946.
Thank
You

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ELISA

The process of adding an antigen/antibody, diluted in

buffer, so it attaches to the solid phase on incubation.

The simple flooding & emptying of wells with a

buffered solution to separate bound from un-bound

signal) e.g Trimethyl benzidine (TMB).

1) Antigen (key) 2) Antibody (lock):

–Key fits into the lock

Enzyme conjugate substrates

• Bound to a secondary antibody that binds with the

Coating Buffer 0.01 M Phosphate Buffer

3) Competitive Fluorescence ELISA:

Types Competitive Sandwich

• It uses a primary labeled anti-body that react directly

• It can be performed with the antigen that is directly

• Not widely used but common for immuno-histochemical

• It utilizes a primary un-labeled antibody in conjunction

• Secondary antibody has specificity for primary antibody.

• Antigens like Tumor markers, hormones, serum proteins

• Antigens in the sample bind with the capture antibody &

• The antibody of the enzyme conjugate bind with the

• Serum antigen & labeled antigen added together .... Competition

• Ab-Ag enzyme complex bound is inversely related to the conc. of

• Increased serum antigen results in reduced binding of Ag-

• Used to determine small molecules like T₃ , T₄ & Progesterone.

• Enzyme interfere with Ag-Ab interaction.

• Second antibody is often labeled with a very small molecular

substance, biotin (MW=244.31), and a specific

binding protein for biotin, avidin is conjugated with enzyme

• Plate cover is effective only under suitable conditions i.e room

• Coating of wells should be proper with the addition of

• Improper coating False positive results

• Antibody must be available(poor producer,

• Concentration may be unclear.

• False positive possible (Ab already present).

• False negative possible.

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