Biostatistics (6)

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Enzyme Linked Immunosorbent Assay (ELISA):

Enzyme Linked Immunosorbent Assay (ELISA)

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 1 st Antibody Is Referred To As Capture Ab 2 nd Antibody Detection Ab 2 nd Antibody Is Biotinylated Enzymes Commonly Used: HRP (Horse Radish Peroxidase) And AKP (Alkaline Phosphatase) Substrate is TMB (Chromogen) Sandwich ELISA

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 ELISA Plate

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) Standard Curve

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 Sensitivity Of Elisa

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 General Protocol

Binding Solution:

Pharmingen Recommended Reagent 0.1 M Na HPO 4, adjust to pH 9.0 or to pH 6.0 with 0.1 M NaH 2 PO 4 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

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? Blocking

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? After Blocking

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 Standard Preparation

PowerPoint Presentation:

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 After Standard Preparation

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 Of Detection Ab

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 H 2 SO 4 (color changes from blue to yellow) Addition Substrate

Read Plate At Appropriate Wavelength (=450 nm):

Read Plate At Appropriate Wavelength ( = 450 nm)

Data Analysis:

Data Analysis

Graph Plotting:

Graph Plotting

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