1. How Does ELISA Work? By: Ian Lin, Marko Cavar, Justin Chiu & Lily Hou Sep 15, 2015 http://www.randoxfooddiagnostics.com/technology/elisa-tech PHM142 Fall 2015 Instructor: Dr. Jeffrey Henderson

2. Background  ELISA = Enzyme-linked immunosorbent assay  Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971;8:871-874.  It is used as a diagnostic tool to detect and quantify antigen or antibody in samples  Performed in multi-well plate (usually 96 wells)  4 types of ELISA: direct, indirect, sandwich, competitive http://www.apsnet.org/edcente r/intropp/HungryPlanet/Chapte r13/Pages/ImageGallery.aspx

3. How does an ELISA work? Basic Concept of ELISA You have something you want to detect and measure? Antigen in sample You use an antibody to specifically detect the antigen in sample The primary or secondary antibody has an enzyme (HRP or AP) attached to it Colourless substrate is added to each well and the enzyme will catalyze it producing a colour The intensity of the colour is directly related to the number of antigen-antibody complexes

4. Direct ELISA 1.Antigen 2.Purified antibody (Enzyme-linked) *measuring Ag in sample (e.g. serum)

5. 1.Antigen TWO kinds of antibodies 1.Primary antibody of interest (red) 2.Secondary antibody (Enzyme-linked) *More than one labeled antibody per antigen *can measure Ab or Ag Indirect ELISA

6. Direct vs Indirect Direct:  Faster (less steps)  Need to label (enzyme-link) antibody  Weaker signal (less sensitive) Indirect:  Amplified signal (more sensitive)  Flexible setups (can purchase pre-made antibody)  Secondary antibody cross-reactivity In general, INDIRECT is preferred

7. Sandwich ELISA Capture and detection antibody, hence “sandwich” Also requires enzyme-linked secondary antibody ➔ Doesn’t require sample purification ➔ Highly sensitive ➔ Antigen requires two epitopes

8. Competitive ELISA Variations on this type of ELISA are used to determine if a person contains antibodies against an antigen

9. Applications Disease diagnostics (disease markers) Antibody screening and quantification Virus Test (HIV, West Nile, New Castle Disease.) Detect Food Allergens Home Pregnancy Test Clinical Testing http://www.sinobiological.com/Disease- Therapeutic-Targets-a-876.html http://www.rkm.com.au/VIRUS/HIV/HIV- virion-laevo.html http://www.rkm.com.au/VIRUS/WEST-NILE- VIRUS/West-Nile-Virus-Structure.html http://pathology5.pathology.jhmi.edu/micro/v 20n47.htm

10. SUMMARY Key Points:  Antigen-Antibody interaction  Enzyme-linked antibody  Enzyme converts substrate to a colour  Antigen is meaured by the intensity of colour (i.e: Strong dark colour = lots of antigen in sample) Types of ELISA: 1.Direct 2.Indirect 3.Sandwich/Capture ELISA 4.Competitive ELISA Limitations: 1.Can be costly 2.Can be labour intensive Strengths: 1.Easy-to-use 2.Highly specific and sensitive 3.Detect and even quantify 4.Multi-well → multiple samples on one plate Applications: 1.Point of Care (e.g. home pregnancy test) 2.Diagnostics (e.g. viral test) via pre- made kits 3.Measuring response to drugs

12. Direct vs Indirect Direct:  Faster (less steps)  Need to label (enzyme-link) antibody  Weaker signal (less sensitive) Indirect:  Amplified signal (more sensitive)  Flexible setups (can purchase pre-made antibody)  Secondary antibody cross-reactivity In general, INDIRECT is preferred SUMMARY

13. References 1.Engvall E, Perlmann P. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 8:871-874. 2.Gaastra W. 1984. Enzyme-linked immunosorbant assay (ELISA). Methods Mol Biol. 1:349-55. 3.Lequin RM. 2005. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA).Clin Chem. 51(12):2415-8. 4.Sino Biological Inc. 2004. ELISA encyclopedia. Beijing, P.R. China. (http://www.elisa-antibody.com)http://www.elisa-antibody.com 5.Murphy. K. 2012. Janeway’s Immunobiology 8 th Ed. Garland Science, Taylor & Francis Group, LLC: New York