Enzyme Linked Immunosorbent

Assay (ELISA)
• Enzyme Linked Immunosorbent Assay (ELISA)
• Term Was Coined By Engvall and Pearlmann in 1971
• Similar To RIA, Except No Radiolabel
• Can Be Used To Detect Both Antibody and Antigen
• Very Sensitive, pg/mL
• Relies on Monoclonal Abs
• ELISA may be run in a qualitative or
quantitative format.
• ELISA results are reported as a
number using a
spectrophotometer,
spectrofluorometer, or other
optical device. This test is
determining the "cut-off" point
between a positive and negative
result.
• Unknowns that generate a signal
that is stronger than the known
sample are called "positive";
those that generate weaker signal
are called "negative.
• Most commonly, ELISAs are performed in 96-
well (or 384-well) usually polystyrene microtiter
plates, which will passively bind antibodies and
proteins
Advantages of ELISA

 Less costly and safest.

 Easy visualization of results with high level of accuracy.
 Specific and highly sensitive assay that can detect protein at the
picomolar to nanomolar range.
 Easily automated for performance of large numbers of tests.
 Require minimal reagents.
 Qualitative detection or Quantitative measurement of either antigen or
antibody.
 Wells can be coated with antigens or antibodies.
 Can be done by personnel with only minimal training.
Applications of ELISA

 Analysis of hormones, vitamins, metabolites,

and diagnostic markers.

 Therapeutic drug monitoring.

 Diagnostic procedures for detecting infection.

Requirements for ELISA test

 Purified antigen (if you want to detect or quantify antibody).

 Purified antibody (if you want to detect or quantify antigen).
 Standard solutions (positive and negative controls).
 Sample to be tested.
 Micro-titer plates: plastic trays with small wells in which the assay is
done.
 Wash fluid (buffer).
 Enzyme-labeled antibody and enzyme substrate.
• ELISA reader (spectrophotometer) for quantitative measurements.
Enzyme labels

• Enzyme labels are used to detect the binding of antigen-

antibody complex.
• It should have high specific reactivity.
• It should be easily coupled to antigen-antibody complex and
must stable.
• Enzymes used in labelling should not be normally present in
the patient samples.
• Examples of enzyme labels are Horse radish peroxidase,
Alkaline phosphatase, and Glucose oxidase.
Stages in ELISA

1. The adsorption of either antigen or antibody to the micro-titer plate.

2. The addition of the test sample and subsequent reagents.
3. The incubation of reactants (formation of antigen-antibody complex).
4. The separation of bound and free reactants by washing.
5. The binding of enzyme to the target antigen or antibody.
6. The addition of substrate (production of a visible signal).
7. The visual or spectrophotometric reading of the assay.
Types of ELISA assay

– Direct ELISA
– Indirect
– Sandwich
– Competitive
 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 = 0.027x + 0.1046
6.125 0.331 0.275 0.099 0.094 2 0.315 0.168 0.268 0.289 0.319 0.098
R = 0.9879
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 0.25 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 .01microM
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 0.1microM
0.274 0.570 7.607 3.681 8.200 7.533 8.126
0.05 LPS+ NS398 1microM

LPS + NS398 10microM

0
Med PGE2 100nM LPS
LPS + NS398 10microM
LPS+ NS398LPS+
1microM
NS398 LPS+
0.1microM
NS398 .01microM
Av 0
0.033 -0.0532 7.651 4 3.114 6
6.694 7.2128 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)