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 immunoelectrop oresis double/radial diffusion i I fl (c l (c r sc r) il ss c

Qg/ l -

g/ l

. .

Qg/ l g/ l g/ l g/ l

sc

c )

. .

i i

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

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 25 10 5 50 25 10 5 50 25 10 5 3 3 3 2 2 2 1 1 1

= standard = unknown = blank

2.5 2.5 2.5 1 1 1

0.5 0.5 0.5 0 0 0

Data Analysis
Standard Curve in Excel
Insert chart Insert trendline (logarithmic)
.
Ab rbance (490 nm)

Sampl Standard Curv

. . . . .2 .2 .1 .1 . y a sor ance og. (a sor ance) .1 1 - .
2

n(x) + . . 1 1

T-test
Ttest(array1, array2, tails, type)

C ncentrati n (ug/mL)