ELISA%20(aka%20Enzyme-Linked%20Immunosorbent%20Assay)

1
ELISA(aka Enzyme-Linked Immunosorbent Assay)

• Professor C. Roth
• 125315 BME Measurements and Analysis Laboratory
• Spring 2003

2
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

3
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)

4
Advantages

• Sensitivity
• Quantitative
• Reproducible
• Kit format

5
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

6
Antibodies

• Specificity
• Diversity hypervariable region (2020 1026
  combinations human make 108)
• Affinity range 105 lt K lt 109 M-1

7
Capture and Detection Antibodies
8
Sandwich ELISA
9
Competitive ELISA

• Less is more. More antigen in your sample will
  mean more antibody competed away, which will lead
  to less signal

10
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.

11
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
standard
25
25
25
unknown
U
10
10
10
U2
U2
U2
blank
5
5
5
2.5
2.5
2.5
U3
U3
U3
1
1
1
0.5
0.5
0.5
U4
U4
U4
0
0
0
12
Data Analysis

• Standard Curve in Excel
• Insert chart
• Insert trendline (logarithmic)
• T-test
• Ttest(array1, array2, tails, type)