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 @ 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
• 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
Std 1 Std 2 Dcs PGE2 LPS LPS + -5 -6 -7 -8 Neg Ctrl
y = 0 .0 2 7x + 0 .10 4 6
6.125 0.331 0.275 0.099 0.094 2 0.315 0.168 0.268 0.289 0.319 0.098
R = 0 .9 8 79
3.0625 0.183 0.18 0.1 0.095 0.31 0.172 0.268 0.285 0.297 0.095
1.53125 0.155 0.136 0.106 0.099 0.286 0.179 0.263 0.263 0.266 0.104
0.765625 0.139 0 .3 0.13 0.105 0.105 0.322 0.205 0.278 0.298 0.279 0.102
0.382813 0.127 0.12 0.111 0.106 0.324 0.204 0.309 0.353 0.292 0.12
0.191406 0.118 0.112 0.112 0.12 0.31 0.204 0.326 0.308 0.324 0.108
0.116 0 .2 5 0.11 0.045 0.042 0.052 0.052 0.053 0.051 0.042 0.042
0.123 0.123 0.044 0.052 0.051 0.052 0.054 0.052 0.052 0.053
0 .2

Dcs PGE2 LPS LPS + -5 -6 -7 -8

-0.207
0 .15 -0.393 7.793 2.348 6.052 6.830 7.941
-0.170 -0.356 7.607 2.496 6.052 6.681 7.126
0.052 -0.207 6.719 2.756 5.867 5.867 5.978 LPS+ NS398 .01m icroM
0.0150 .1 0.015 8.052 3.719 6.422 7.163 6.459
0.237 0.052 8.126 3.681 7.570 9.200 6.941 LPS+ NS398 0.1m icroM
0.274 0.570 7.607 3.681 8.200 7.533 8.126
0 .0 5 LPS+ NS398 1m icroM

LPS + NS398 10m icroM

0
Med PGE2 100nM LPS
LPS + NS398 10m
LPS+icroM
NS398LPS+
1m icroM
NS398 LPS+
0.1m icroM
NS398 .01m icroM
Av 0
0.033 -0.0532 7.651 4 3.114 6
6.694 8
7.212 7.095 LPS
SEM 0.082 0.145 0.206 0.265 0.392 0.458 0.339
PGE2 100nM

Med

0.00 2
 Graph Plotting

10

8
TNF- (ng/mL)

0
Medium 10 1 0.1 0.01 NS398 M

LPS (1 g/mL)