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ELISA

(aka Enzyme-Linked
Immunosorbent Assay)
Professor C. Roth
125:315: BME Measurements and
Analysis Laboratory
Spring 2003
What is an ELISA?
Enzyme-linked immunosorbent assay
Name suggests three components
Antibody
Allows for specific detection of analyte of interest
Solid phase (sorbent)
Allows one to wash away all the material that is not
specifically captured
Enzymatic amplification
Allows you to turn a little capture into a visible color
change that can be quantified using an
absorbance plate reader
What is it used for?
Measure antibody levels (allergies,
vaccines)
Detect viruses (hepatitis, HIV, venereal
diseases)
Detect hormonal changes (pregnancy)
Detect circulatory inflammatory markers
(cytokines)
Advantages
Sensitivity
Quantitative
Reproducible
Kit format
Relative sensitivities of tests (approx)
Usual operating range
[Ab] or [Ag]
precipitation
immunoelectrophoresis
double/radial diffusion
10 g/ml - 1 mg/ml
immunofluorescence 0.1 - 10 g/ml
ELISA (colour) 0.1 - 10 ng/ml
(chemiluminescence) 0.01 - 10 ng/ml
radioimmunoassay 0.01 - 10 ng/ml
Enzymes with Chromogenic
Substrates
High molar extinction coefficient (i.e.,
strong color change)
Strong binding between enzyme and
substrate (low K
M
)
Linear relationship between color intensity
and [enzyme]
Antibodies
Specificity
Diversity hypervariable region (20
20
~
10
26
combinations; human make ~ 10
8
)
Affinity range 10
5
< K < 10
9
M
-1
Capture and Detection Antibodies
Sandwich ELISA
Competitive ELISA
Less is more. More antigen in your
sample will mean more antibody competed
away, which will lead to less signal
Todays Lab
Our antigen = human albumin
Our antibody = rabbit anti-human
Our enzyme = horseradish peroxidase

You will develop (i.e. perform enzymatic
reaction) using o-phenylene diamine
(OPD). It is hazardous. Please wear
gloves and treat with respect.
Antibody Steps
Antigen (purified albumin) is already coated onto
microwell plates
You will add standards and samples in triplicate
You will incubate for 60 minutes at 37 degrees C
to allow for Ab-Ag binding
50 50 50
25 25 25
10 10 10
5 5 5
2.5 2.5 2.5
1 1 1
0.5 0.5 0.5
0 0 0
= standard
U
= unknown
U1 U1 U1
U2 U2 U2
U3 U3 U3
U4 U4 U4
= blank
Data Analysis
Standard Curve in Excel
Insert chart
Insert trendline (logarithmic)




T-test
Ttest(array1, array2, tails, type)
Sample Standard Curve
y = -0.0583Ln(x) + 0.3858
R
2
= 0.9919
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.1 1 10 100
Concentration (ug/mL)
A
b
o
r
b
a
n
c
e

(
4
9
0

n
m
)
absorbance
Log. (absorbance)

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