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

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

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 @ 4C
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)
Lets Say 4 mL Needed
You will need 16 L of capture Ab
Add capture Antibody, Seal plate (minimize evaporation)
Incubate overnight at 4C

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

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

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
PGE2
-0.207
0.15 -0.393
-0.170
-0.356
0.052
-0.207
0.0150.1 0.015
0.237
0.052
0.274
0.570
0.05

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 .01m icroM

LPS+ NS398 0.1m icroM
LPS+ NS398 1m icroM
LPS + NS398 10m icroM

Av
SEM

0
Med
PGE2 100nM
0
0.033
-0.0532

0.082

0.145

LPS
LPS + NS398 10m
LPS+icroM
NS398LPS+
1m icroM
NS398 LPS+
0.1m icroM
NS398 .01m icroM
4
6
8
7.651
3.114
6.694
7.212
7.095

0.206

0.265

0.392

0.458

0.339

LPS
PGE2 100nM
Med

0.00

Graph Plotting

10

TNF- (ng/mL)

Medium

10

0.1

LPS (1 g/mL)

0.01

NS398 M