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 K M ) Linear relationship between color intensity and [enzyme]

6. Antibodies Specificity Diversity – hypervariable region (20 20 ~ 10 26 combinations; human make ~ 10 8 ) Affinity – range 10 5 < K < 10 9 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 25 10 5 55 2.5 111 0.5 00 0 = standard U = unknown U1 U2 U3 U4 = blank

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