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 binding of the Ag or Ab depending on the type of the ELISA test.  Think of blocking agents as a way to “cover up” areas on the solid substrate (card or plastic) where there is nothing to detect.  For some rapid detection ELISA tests such as home pregnancy tests, the blocking agent has already been applied by the manufacturer.
• Washing buffer is a solution that is buffered to prevent any cross-reaction with either the Ag or Ab(s).  Washes are often used after the addition of Ag or Ab to remove any unbound materials.  Note that if only one sample is being added, as in the home pregnancy test mentioned above, wash steps were completed during manufacture and thus are not usually needed though each test may vary based so always read the instructions when conducting a home test.

The three most common types of ELISA tests fall into the following groups.  All of them depend on Ag-Ab interactions but there are subtill changes in how Ag and Ab are used and in what order.

• In direct ELISAs, Ag are fixed onto some sort of solid substrate such as card or plastic dish to prevent the sample from washing away throughout the steps of the ELISA reaction.  A primary Ab conjugated (cross linked or fixed) to an indicator molecule or compound is added to bind to and thus detect the Ag being tested.  A color substrate is commonly used for detection so the substrate would be added to the solution to produce a color change.  Note that some ELISA test will use a fluorescent or radiographic indicator; in such cases, no color change would visually be detected without the aid of radiographic film or fluorescent detection methods (e.g., fluorescent microscopy).
• An indirect ELISA is very much like a direct ELISA.  The Ag is bound to the solid substrate then a primary Ab is added to bind to the Ag.  The difference between direct and indirect ELISAs are that indirect ELISA has the indicator molecules bound to the secondary Ab rather than the primary Ab so there is an “extra” step.  Indirect ELISAs are commonly used clinical and microbiological labs and offer an added layer of sensitivity while direct ELISAs are used for rapid tests and “at home” tests.
• The sandwich ELISA makes an Ag “sandwich” by trapping the Ag of interest between two Ab.  One Ab, typically the primary Ab, is put on the solid substrate then the Ag of interest is added, followed by adding the secondary Ab carrying the indicator molecule.  Sandwich ELISAs are one way to determine if someone has produced Ab against a specific pathogen, rather than detecting if a person has a specific pathogen.  Of course, Ab titers can also be detected by serological methods in addition to ELISA testing.