Transcript ELISA Lab - Rutgers University School of Engineering

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
immunofluorescence
10 g/ml - 1 mg/ml
0.1 - 10 g/ml
ELISA (colour)
(chemiluminescence)
0.1 - 10 ng/ml
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 KM)
• Linear relationship between color intensity
and [enzyme]
Antibodies
• Specificity
• Diversity – hypervariable region (2020 ~
1026 combinations; human make ~ 108)
• Affinity – range 105 < K < 109 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
Today’s 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
U1 U1 U1
= standard
U2 U2 U2
2.5 2.5 2.5 U3 U3 U3
1
1
1
0.5 0.5 0.5 U4 U4 U4
0
0
0
U
= unknown
= blank
Data Analysis
• Standard Curve in Excel
– Insert chart
– Insert trendline (logarithmic)
Sample Standard Curve
0.5
Aborbance (490 nm)
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
y = -0.0583Ln(x) + 0.3858
R2 = 0.9919
Log. (absorbance)
absorbance
0.05
0
• T-test
– Ttest(array1, array2, tails, type)
0.1
1
10
Concentration (ug/mL)
100