ELISA_LECTURE.ppt
- 1. Enzyme Linked Immunosorbent Assay (ELISA)
- 2. 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
- 4. • 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) Sandwich ELISA
- 5. • 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 ELISA Plate
- 6. • 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) Standard Curve
- 7. • 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 Sensitivity Of Elisa
- 8. • 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 4C General Protocol
- 9. • 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). Binding Solution
- 10. • 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? Blocking
- 11. • 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? After Blocking
- 12. • 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 Standard Preparation
- 13. Assume You Have A Stock Tube @ 2ng/L, Volume 5 L Usually Remaining Standard Cytokine Is Thrown Away Thawing-Unthawing Affects Cytokine
- 14. • 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 After Standard Preparation
- 16. • 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 Of Detection Ab
- 17. • 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) Addition Substrate
- 18. Read Plate At Appropriate Wavelength (=450 nm)
- 19. Data Analysis Std 1 Std 2 Dcs PGE2 LPS LPS + -5 -6 -7 -8 Neg Ctrl 6.125 0.331 0.275 0.099 0.094 0.315 0.168 0.268 0.289 0.319 0.098 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 0.13 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 0.11 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 Dcs PGE2 LPS LPS + -5 -6 -7 -8 -0.207 -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 0.015 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 0.274 0.570 7.607 3.681 8.200 7.533 8.126 Med PGE2 100nM LPS LPS + NS398 10microM LPS+ NS398 1microM LPS+ NS398 0.1microM LPS+ NS398 .01microM Av 0.033 -0.053 7.651 3.114 6.694 7.212 7.095 SEM 0.082 0.145 0.206 0.265 0.392 0.458 0.339 0.00 2 Med PGE2 100nM LPS LPS + NS398 10microM LPS+ NS398 1microM LPS+ NS398 0.1microM LPS+ NS398 .01microM y = 0.027x + 0.1046 R2 = 0.9879 0 0.05 0.1 0.15 0.2 0.25 0.3 0 2 4 6 8
- 20. Graph Plotting 0 2 4 6 8 10 TNF- (ng/mL) Medium LPS (1 g/mL) 10 1 0.1 0.01 NS398 M