Enzyme Linked Immunosorbent Assay
(ELISA)
ELISA
• Enzyme Linked Immunosorbent Assay (ELISA)
• Term Was Coined By Engvall and Pearlmann in 1971
• Different Types
– Sandwich
– Indirect
– Competitive
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Similar To RIA, Except No Radiolabel
Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal Abs
Sandwich ELISA
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2 Antibodies Required
Must Recognize Different Epitopes
1st Antibody Is Referred To As Capture Ab
2nd Antibody Detection Ab
2nd Antibody Is Biotinylated
Enzymes Commonly Used: HRP (Horse Radish
Peroxidase) And AKP (Alkaline Phosphatase)
• Substrate is TMB (Chromogen)
ELISA Plate
• 96 well plate
• Made of plastic on which protein can be adsorbed
(bind) easily
• Usually done overnight @ 4C
• Special buffer used that will not denature Ab and
maximize binding
• Blocking step ensures no empty spaces are left
• Blocking reagent is often 10% FBS
Standard Curve
• Serial dilutions of the cytokine being
measured
• Exact concentration is needed
• A plot of concentration (pg/mL or ng/mL) is
plotted against OD (optical density)
Sensitivity Of Elisa
• Typically the lowest cytokine concentration
that can be detected above negative control
• 2-3 S.D Above Mean Background Signal
• Depending On Antibody Pair Used
Sensitivity Varies
• Ex. 10 pg/mL
General Protocol
• Dilute capture Ab @ 1-4 g/mL In Binding Solution
• Ex. Stock Solution Of Capture Ab: 0.5 mg/mL And Capture Ab
Recommended Conc. 2 g/mL
• First Question To Ask Yourself ?
– How much volume would I use?
– Count 16 wells for S.C+
– 3 wells for Negative Controls
– Your Samples (usually in triplicates)
– Add them up and multiply by 100 L (typical volume used per well)
• Let’s Say 4 mL Needed
– You will need 16 L of capture Ab
• Add capture Antibody, Seal plate (minimize evaporation)
• Incubate overnight at 4C
Binding Solution
• Pharmingen Recommended Reagent
• 0.1 M Na HPO4, adjust to pH 9.0 or to pH
6.0 with 0.1 M NaH2PO4
• pH Is Very Important, If Wrong No Binding
• Some Antibodies Require pH 6.0
– Ex. Antibodies for mIL-10, mMCP-1, mTNF,
rGM-CSF).
Blocking
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Blocking Reagent 10% FBS in PBS
Alternatively 1% BSA (Immunoassay Grade)
Filter To Remove Particulates
Plate Is Brought To R.T
Add 200 L per well Blocking Buffer
Wait For 2 Hours At R.T
Why Do We Block?
After Blocking
• Wash x3 With PBS/Tween (detergent)
• Add Standards + Samples
• Samples Are Typically Supernatants From
Cultures Or Patient Serum/Plasma
• Use 100 L
• Often Dilution Is Required If Signal Is Too
Strong
• Standards?
Standard Preparation
• Standards Are Diluted in Blocking
Buffer/Tween
• Start By Labeling eight, 1 mL Eppendorf
Tubes
• Prepare Highest Conc. Tube (1 mL)
• Fill The Remaining Tubes with 0.5 mL
Blocking Buffer
• Serially Dilute From Top To Lowest
Assume You Have A Stock Tube @ 2ng/L, Volume 5 L
Usually Remaining Standard Cytokine Is Thrown Away
Thawing-Unthawing Affects Cytokine
After Standard Preparation
• Add Samples, Standards, Negative Control
– Negative Control Should Be The Buffer You
Use Dilute Standard or Culture Medium
• Incubate For 2 Hrs at R.T
• Aspirate And Wash 5x
Addition Of Detection Ab
• Avidin is a Hen Oviduct Protein
• Avidin has very high affinity for biotin (B
vitamin)
• B vitamin is conjugated on the detection Ab
• Add Working Detector @ 100 L/well
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Ex. Stock Detection Antibody=0.5mg/mL
You need to prepare 5 mL @ 1  g/mL
Use 10  L of Stock Antibody
Add 5 L of Enzyme (Avidin-HRP)
Dilution is 1:1000
• Incubate for 60 mins @ R.T
• Wash 6x
Addition Substrate
• Prepare Substrate by Mixing 1:1 volume
• Add 100 L/well
• Incubate for 10 mins, Avoid Formation of
Excessively Bright Color (Spec will not be
able to read)
• Terminate Reaction by Adding 0.5 M
H2SO4 (color changes from blue to yellow)
Read Plate At Appropriate
Wavelength (=450 nm)
Data Analysis
6.125
3.0625
1.53125
0.765625
0.382813
0.191406
Std 1
Std 2
0.331
0.275
0.183
0.18
0.155
0.136
0.139 0.3 0.13
0.127
0.12
0.118
0.112
0.116 0.25 0.11
0.123
0.123
Dcs
0.099
0.1
0.106
0.105
0.111
0.112
0.045
0.044
PGE2
LPS
LPS + -5
y = 0.027x + 0.1046
0.094 2
0.315
0.168
R = 0.9879
0.095
0.31
0.172
0.099
0.286
0.179
0.105
0.322
0.205
0.106
0.324
0.204
0.12
0.31
0.204
0.042
0.052
0.052
0.052
0.051
0.052
-6
0.268
0.268
0.263
0.278
0.309
0.326
0.053
0.054
-7
0.289
0.285
0.263
0.298
0.353
0.308
0.051
0.052
-8
Neg Ctrl
0.319
0.098
0.297
0.095
0.266
0.104
0.279
0.102
0.292
0.12
0.324
0.108
0.042
0.042
0.052
0.053
0.2
Dcs
-0.207
0.15
-0.170
0.052
0.0150.1
0.237
0.274
0.05
PGE2
-0.393
-0.356
-0.207
0.015
0.052
0.570
LPS
7.793
7.607
6.719
8.052
8.126
7.607
LPS + -5
2.348
2.496
2.756
3.719
3.681
3.681
-6
6.052
6.052
5.867
6.422
7.570
8.200
-7
6.830
6.681
5.867
7.163
9.200
7.533
-8
7.941
7.126
5.978
6.459
6.941
8.126
LPS+ NS398 .01microM
LPS+ NS398 0.1microM
LPS+ NS398 1microM
LPS + NS398 10microM
Av
SEM
0
Med
PGE2 100nM
0
0.033
-0.0532
0.082
0.145
LPS
LPS + NS398 10microM
LPS+ NS398LPS+
1microM
NS398 LPS+
0.1microM
NS398 .01microM
8
6
7.651 4 3.114
6.694
7.212
7.095
0.206
0.265
0.392
0.458
0.339
LPS
PGE2 100nM
Med
0.00
2
Graph Plotting
10
TNF- (ng/mL)
8
6
4
2
0
Medium
10
1
0.1
LPS (1 g/mL)
0.01
NS398 M