ELISA

( Enzyme-Linked Immunosorbent Assay)

• Presented by: Samira Algamati

• Presented to: Dr. Fawzi Irshaid

 What is an ELISA?
• Enzyme-linked Immunosorbent Assay
• Name suggests three components
– Antibody
• Allows for specific detection of analyte of interest
– Solid phase (sorbent)
• Allows one to wash away all the material that is not
specifically captured
– Enzymatic amplification
• Allows you to turn a little capture into a visible color
change that can be quantified using an absorbance
plate reader
 Definitions
• Antibodies (Ig) are gamma globulin proteins that
are found in blood and are used by the immune
system to identify and neutralize foreign objects,
such as bacteria and viruses

• Antigens:- a substance that when introduced

into the body stimulates the production of an
antibody.

Analyte:-the sample being analyzed and in

immunoassays the analyte is either Antibody or
Antigen
 Definitions
Primary antibody (Serum Samples): Polyclonal anti-
chicken antibody made by rabbits

Secondary antibody (enzyme-linked): Polyclonal anti-

rabbit antibody made by goats linked (conjugated) to
horseradish peroxidase (HRP) binds to primary
antibody. It catalyzes substrate turning it blue.

Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine

(TMB) – a colorless solution that when oxidized by
HRP turns blue
 Types of ELISA
1- Indirect ELISA

2- Competitive ELISA

3- Sandwich ELISA
 1-Indirect ELISA
• Prepare antigen (dilute to 1ug/ml in coating
buffer (0.1M carbonate)
• Vortex to mix
• Coating antigen to microplate
• Incubate at room temperature for 3 hours
or 4C overnight
• Remove the coating solution
• wash the plate by filling the walls with
200ul 0.2% PBST
 1-Indirect ELISA
• Block the remaining protein-binding sites
by adding 200ul blocking buffer (5%skim
milk in 0.2% PBST)
• Incubate at room temperature for 1 hour or
4C overnight
• Remove the blocking buffer
• Wash the plate twice with PBST
• Add 100ul of diluted primary antibody
 1-Indirect ELISA
• Incubate at room temperature for 2hours
or 4C overnight
• Remove the solution
• wash the plate three time with PBST
• Add 100ul of conjugated secondary
antibody
• Incubate at room temperature for 1hour
 1-Indirect ELISA
• Wash the plate four time with PBST

• Dispense 100ul of the substrate (OPD,O-

phenylenediamine dihydrochloride)

• After sufficient color development read the

absorbance (450nm) with a ELISA reader
 8. Observe
colour
development
7. Add substrate
1. Add antigen for enzyme

2. Wash with 6. Wash with

PBST PBST

4. Wash with
PBST
3. Add primary 5. Add secondary
antibody antibody
ELISA Kit
Results

• Clear
• Determination
• Of Positive
• And Negative
• Results
 2-Competitive ELISA
• Coating 100ul of diluted antibody to each
well
• Incubate at room temperature for 3 hours
or 4C overnight
• Wash the plate by filling the wells with
200ul 0.2%PBST
• Blocking with 200ul 1% BSA
• Incubate at room temperature 3hours
 Competitive ELISA
• Wash the plate twice with PBST
• Antibody is incubate in the presence of its
antigen
• Apply bound antibody/antigen complexes to
coated well
• Incubate at room temperature for 2 hours
• Wash the plate three times with PBST (the more
antigen in the sample, the less antibody will able
to bind to the antigen in the well, hence
<competition>)
 Competitive ELISA
• Add 100ul of conjugated secondary
antibody
• Incubate at R. Tem for 1 hour
• Wash the plate four time with PBST
• Dispense 150ul of the substrate( OPD)
After sufficient color development, read
the adsorbance 450nm with a ELISA
reader
Competitive ELISA
 3-Sandwich ELISA
-Add 100ul coating buffer per well (137mM
Nacl, 2.7mM Kcl, 8.1mM Na2Hpo4,1.5mM
KH2PO4)
-Incubate at room temperature for 3hours or 4C
overnight
-Wash by 200ul 1x PBST once
-Add 200ul blocking buffer per well (5% skim
milk in PBST (O.O5% Tween_20)
 Sandwich ELISA
• Incubate at room temperature for 1hour
• Wash by 200ul 1xPBS twice
• Prepare the analytes
• Add 100ul analyte to each well
• Incubate at room temperature for 2 hours
• Wash by 200ul 1xPBS three times
• Add 100ul detect antibody to each well
 Sandwich ELISA
• Incubate at room temperature for 2hours
• Wash by 200ul xPBS four times
• Add 80ul secondary antibody per well
• Wash by 200ul 1xPBS five times
• Add 150ul OPD per well
• Incubate at room temperature for 15-30
minutes (according to the OPD
manufacturer’s recommedation Avoid light
exposure during incubation
 Sandwich ELISA
• Turn on the ELISA reader and put plate
into reader

• Read the plate at OD 450 (OD 490 for

adding the stop solution).
Sandwich ELISA
 Results

• After reading the results the standard

curve is drawn were the concentration
is blotted on the X-axis and the
absorbance on the Y-axis

Absorption
mn

Concentration
ng/ml
 Result
• The standards concentrations is specified
on the x-axis and the reading of each
standard is specified on the y-axis and the
standard curve is drawn
 Result
• This standard curve is used to determine
the unknown concentration of each
sample by finding the opposite
concentration to the absorbance

• Absorption nm

Concentrati
on ng/ml
 Advantages of ELISA
1-Reagents are relatively cheap & have a long
shelf life.
2- ELISA is highly specific and sensitive .
3-No radiation hazards occur during labeling or
disposal of waste.
4-easy to perform and quick procedures.
5-Equipment can be inexpensive and widely
available.
6-ELISA can be used to a variety of infections.
 Disadvantages of ELISA
1-Measurement of enzyme activity can be more
complex than measurement of activity of some
of radioisotopes
2-Enzyme activity may be affected by plasma
constituents
3-Kits are commercially available, but not cheap
4-Very specific to a particular antigen. won’t
recognize any other antigen.
5-False positives /negatives possible, especially
with mutated/ altered antigen .
 Applications of ELISA
1-Hormones
2-Protein
3-infectious agent (viral, bacterial, parasitic
fungal)
4-drug markers
5-tumor markers
6-serum proteins
7-vaccine quality control
8-For GMO (Genetically Modified
Organism)

9-For rapid test

10-IgG, IgM, IgA

11-In new born screening

12-Inclinical research