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
Relative sensitivities of tests (approx)

Usual operating range

• Quantitative
[Ab] or [Ag]

precipitation

• Reproducible immunoelectrophoresis
double/radial diffusion
10 g/ml - 1 mg/ml

• Kit format 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 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 U1 U1 U1

25 25 25 = standard
10 10 10 U2 U2 U2 U = unknown
5 5 5
= blank
2.5 2.5 2.5 U3 U3 U3

1 1 1

0.5 0.5 0.5 U4 U4 U4

0 0 0
 Data Analysis
• Standard Curve in Excel
– Insert chart Sample Standard Curve

– Insert trendline (logarithmic) 0.5

0.45
0.4

Aborbance (490 nm)

0.35
0.3
0.25
0.2
0.15
0.1
absorbance y = -0.0583Ln(x) + 0.3858
0.05
Log. (absorbance) R2 = 0.9919
0

• T-test
0.1 1 10 100
Concentration (ug/mL)

– Ttest(array1, array2, tails, type)