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    Elisa Lecture

    Uploaded by

    Ankit Narula
    The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA uses antibodies and color changing enzymes to detect the presence of antigens or antibodies in a biological sample. There are different types of ELISA including sandwich ELISA, which uses two antibodies, one to capture the antigen and another enzyme-linked antibody for detection. The protocol involves coating an ELISA plate with capture antibodies, adding samples and standards, detecting with a secondary antibody, and measuring color change to quantify results.

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    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd

    Elisa Lecture

    Uploaded by

    Ankit Narula
    135 views20 pages
    The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA uses antibodies and color changing enzymes to detect the presence of antigens or antibodies in a biological sample. There are different types of ELISA including sandwich ELISA, which uses two antibodies, one to capture the antigen and another enzyme-linked antibody for detection. The protocol involves coating an ELISA plate with capture antibodies, adding samples and standards, detecting with a secondary antibody, and measuring color change to quantify results.

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    © Attribution Non-Commercial (BY-NC)

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    The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA uses antibodies and color changing enzymes to detect the presence of antigens or antibodies in a biological sample. There are different types of ELISA including sandwich ELISA, which uses two antibodies, one to capture the antigen and another enzyme-linked antibody for detection. The protocol involves coating an ELISA plate with capture antibodies, adding samples and standards, detecting with a secondary antibody, and measuring color change to quantify results.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    135 views20 pages

    Elisa Lecture

    Uploaded by

    Ankit Narula
    The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA uses antibodies and color changing enzymes to detect the presence of antigens or antibodies in a biological sample. There are different types of ELISA including sandwich ELISA, which uses two antibodies, one to capture the antigen and another enzyme-linked antibody for detection. The protocol involves coating an ELISA plate with capture antibodies, adding samples and standards, detecting with a secondary antibody, and measuring color change to quantify results.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 20

    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)

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