8 Lab 8: ELISA
Lab Objectives
After completing this lab, the student should be able to:
- Use aseptic technique to carry out an ELISA reaction.
- Examine how antibodies can be used to detect specific diseases such as Covid-19 or influenza in patient specimens.
- Identify if a patient has a disease based on ELISA test results.
Introduction
ELISA tests depend upon antigen (Ag) – antibody (Ab) interactions. These reactions can be used to diagnose illnesses such as Covid-19, influenza, strep throat or even changes in health states such as the secretion of pregnancy related hormones. ELISA technology with in the clinical environment serves as a rapid identification test. While not all tests are ELISA based, several common illnesses or states can be detected within a few seconds to a few minutes and new ELISA tests are being developed by medical researchers. An example of new ELISA based tests include PSA (prostate specific antigen) and TAA (tumor associated antigen) tests that are linked to the early detection of cancer, even before cancer can be detected by imaging. This is great news for patients because it means that lifestyle and chemotherapeutic changes can be made before cancer becomes problematic yet these tests do not require genetic analysis of patients thus avoiding complicated ethical and economic discussions that genetic analysis may uncover.
The ELISA reaction is fairly standard but it is helpful to identify the different reagents that are used before talking about the different types of ELISAs. There are five key components of an ELISA reaction:
- An antigen – This is a cell (or virus’) identification card. All cells and viruses have them and for the most part, they are rather unique. The epitope on an antigen is a unique set of molecules, typically amino acids or simple sugar chains, that can be used to identify that organism. For example, E. coli O157:H7 has epitopes that would mark it as having O polysaccharide 157 and flagellar protein H7. Often times epitopes are just called antigens (which can make discussions in microbiology and immunology a little confusing). For the purposes of this class, we will refer to an epitope as an antigen.
- A primary antibody is the Ab that will bind to the Ag in the reaction. The purpose of the primary Ab is to detect the Ag of choice, depending on what the ELISA is detecting. Some ELISA methods use only one antibody in which case the indicator molecule is attached to the primary antibody. Other ELISA methods will use a secondary Ab which is another antibody that binds either to the primary Ab and carries the indicator molecule.
- Indicator molecules are used to produce some type of change or signal that an Ag has been detected. Often, indicator molecules will produce a color change that can easily be detected while other indicator molecules give off fluorescent light or radiation.
- Blocking agents include any large, non-reactive molecule that can be used to bind to a surface and prevent non-specific