ELISA(aka Enzyme-Linked

Immunosorbent Assay)

Professor C. Roth125:315: BME Measurements and

Analysis LaboratorySpring 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]

precipitationimmunoelectrophoresisdouble/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 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 binding50 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

R2 = 0.99190

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

bo

rban

ce (

490

nm

)

absorbance

Log. (absorbance)