1. ELISA(aka Enzyme-Linked Immunosorbent Assay) Professor C. Roth 125:315: 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 < K < 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. 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.

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 U1 U1 U1 50 = standard 25 25 25 = unknown U 10 10 10 U2 U2 U2 = blank 5 5 5 2.5 2.5 U3 U3 U3 2.5 1 1 1 0.5 0.5 U4 U4 U4 0.5 0 0 0

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