Using the ELISA to Detect West Nile Virus

Looking for a way to keep your immunology or virology lessons fresh?  Consider teaching the lesson from a biomedical diagnostic point of view!  Here, we describe how the West Nile Virus can be used to teach the Enzyme Linked Immunosorbent Assay (or ELISA).

What is the West Nile Virus?

Every summer, we prepare ourselves for warm weather, sunshine, and (unfortunately) mosquitos.  Mosquitos are both a nuisance and a public health concern, as diseases can be transmitted through their bite.  West Nile Virus (WNV) is a mosquito-borne contagious

disease that was originally identified in Uganda.  The virus has spread all over the world, including the United States.  In fact, the 2012 outbreak of the WNV was the largest ever documented in the United States – over 1,100 cases were reported to the Center for Disease Control (CDC).  There is no vaccine currently available, but controlling mosquito populations and protecting oneself from mosquito bites can prevent WNV infections. West Nile disease is often asymptomatic in healthy adults.  Symptoms of West Nile disease last 3-6 days and are flu-like (fever and chills, sore throat, severe headaches, coughing, weakness, nausea, and muscle aches).  A very small number (<1%) of cases result in neurological diseases resembling encephalitis, meningitis, or poliomyelitis.

How is WNV diagnosed?

First, the medical professional will compile the patient’s medical history, including clinical symptoms and dates of travel to areas with a history of the epidemic.  Since the symptoms of WNV can mimic Influenza or encephalitis, the ELISA is used to rule out these diseases and to plan the best treatment plan.  The ELISA information is also important for epidemiologists to determine which virus is responsible for a given outbreak.   Positive ELISA results are further confirmed using PCR or immunohistochemistry.

How does the ELISA work?

The ELISA uses antibodies to detect the presence of specific biomolecules  (i.e. peptides, proteins, antigens and hormones) in a complex sample (for more about antibodies, see our previous post). These samples can be single proteins or complex mixtures like cellular lysates.  The ELISA is commonly used for medical diagnostics, as it is can be used to identify antigens in blood and other biological samples.

The traditional ELISA requires two antibodies.  One antibody, called the primary, recognizes the antigen of interest.  For example, an ELISA that detects the HIV virus would use an antibody that recognizes one of the virion’s coat proteins.  The secondary antibody recognizes the primary antibody – if a rabbit produced our primary antibody, we would use a secondary antibody that recognizes rabbit antibodies.  The secondary antibody is covalently linked to an enzyme (usually Alkaline Phosphatase or Horseradish Peroxidase) that lets us detect the presence of the antibody-antigen complex (Figure 2). To detect antibody-antigen interactions, we add hydrogen peroxide and amino salicylate to each well.  HRP converts hydrogen peroxide (its substrate) to H₂O + O₂, which in turn oxidizes the amino salicylate, turning the clear substrate solution to brown.   HRP has a high catalytic activity – its substrate turnover rates exceed 10⁶ per second – allowing us to quickly detect even the smallest amount of antigen.  Since the ELISA is performed in transparent plastic plates, we can easily visualize our the color change (Figure 3).

When developing an ELISA, scientists carefully choose the antibodies that produce the best signal to noise ratio while having a robust, reproducible interaction.  Antibodies that have a high affinity for non-specific antigens will have unwanted cross-reactions that can result in high backgrounds.  In contrast, an antibody with a weak affinity may not be sensitive enough for antigen detection.   These antibodies would produce results with a high false-positive or false-negative rate.

Which kit would I use to teach this lesson?

We have successfully used Kit 267, Single Antibody ELISA Diagnostics, to teach this lesson in many of our Professional Development courses  (for more inspiration for this lesson, here is our PowerPoint Presentation).  For other lessons, check out our Workshop Materials on our website!  Happy teaching!