ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and...
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Transcript of ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and...
![Page 1: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/1.jpg)
ELISA(aka Enzyme-Linked
Immunosorbent Assay)
Professor C. Roth125:315: BME Measurements and
Analysis LaboratorySpring 2003
![Page 2: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/2.jpg)
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
![Page 3: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/3.jpg)
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)
![Page 4: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/4.jpg)
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
![Page 5: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/5.jpg)
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]
![Page 6: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/6.jpg)
Antibodies
• Specificity
• Diversity – hypervariable region (2020 ~ 1026 combinations; human make ~ 108)
• Affinity – range 105 < K < 109 M-1
![Page 7: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/7.jpg)
Capture and Detection Antibodies
![Page 8: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/8.jpg)
Sandwich ELISA
![Page 9: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/9.jpg)
Competitive ELISA
• Less is more. More antigen in your sample will mean more antibody competed away, which will lead to less signal
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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.
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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
![Page 12: ELISA (aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: BME Measurements and Analysis Laboratory Spring 2003.](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649ea15503460f94ba4d00/html5/thumbnails/12.jpg)
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)