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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
0.2

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

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 2 0.033 -0.053

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

LPS PGE2 100nM Med

0.082

0.145

0.206

0.265

0.392

0.458

0.339

0.00

Graph Plotting

10

TNF- (ng/mL)

Medium

10

0.1

0.01

NS398 M

LPS (1 g/mL)