using the elisa to identify west nile virus outbreaks

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Using the ELISA to Identify West Nile Virus Outbreaks

Danielle R. Snowflack, Ph.D. Edvotek ®

Washington, D.C.

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West Nile Virus (WNV) is a Contagious Mosquito-borne Disease

West Nile Virus infections are asymptomatic in most healthy adults.

20% of people exhibit flu-like symptoms for 3 – 6 days.

A very small number of cases (<1%) result in neurological diseases resembling encephalitis, meningitis, or acute paralysis.

Anopheles sp.

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Incidence of West Nile Virus Infection

The 2012 outbreak of the WNV was the largest ever documented in the United States – over 1,100 cases were reported to the Centers for Disease Control (CDC).

There is no vaccine currently available, but controlling mosquito populations and protecting oneself from mosquito bites can prevent WNV infections.

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Diagnosing West Nile Virus Infections

Clinical symptoms

Dates of travel

History of epidemic in area

Enzyme-Linked ImmunoSorbent Assay (ELISA)

Polymerase Chain Reaction (PCR)

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Antibodies Distinguish Between “Self” and “Non-self”

Antibody molecules comprise four linked polypeptide chains: two “heavy chains” and two “light chains” that are connected by disulfide bonds.

The amino acid sequence of the antigen binding site is variable, allowing each antibody to recognize a unique epitope (a particular location within an antigen).

Antibodies can be polyclonal (heterogeneous mixture) or monoclonal (directed against a single epitope).

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Antibodies Detect Specific Proteins in Complex Mixtures

Western blotting allows us to detect the presence of a specific protein separated by SDS-PAGE.

Fluorescently-labeled antibodies are used to identify specific proteins within a cell or tissue.

Cat. #317 Budding Yeast Fluorescent Ab for Cell Surface Protein

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Enzyme Linked Immuno-Sorbent Assay (ELISA)

Scientists use the ELISA to quantitatively detect the presence of molecules within a sample. Allergen detection – milk,

peanut, walnut, egg proteins Hormones – Pregnancy tests Toxicology – drug tests

The ELISA is commonly used for medical diagnostics, as it is can be used to identify antigens in blood and other biological samples. Disease detection – WNV, HIV,

TB, Infectious Mononucleosis

Cat. #279 Cat. #274

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Principles of ELISA The primary antibody recognizes the

antigen of interest.

The secondary antibody recognizes the primary antibody.

The secondary antibody is covalently linked to an enzyme that lets us detect the presence of the antibody-antigen complex. Chromogenic detection

Fluorogenic detection

ELISA is very sensitive – Low antigen concentrations can be detected because each enzyme can produce many product molecules.

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ELISAs can be Quantitative or Qualitative

Qualitative: “Yes or No?” Results are determined by

comparing to a known standard.

Quantitative: “How much?” A “standard curve” is

generated using known concentrations of the target molecule

The experimental sample is assayed and the result is compared to standard curve

Cat. #279

Quantitative ELISA #278

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Single Antibody ELISA Diagnostics

The primary antibody is directly conjugated to the Horseradish Peroxidase (HRP) enzyme.

By eliminating the secondary antibody, we have decreased the time necessary to perform the assay.

Background from non-specific interactions of the secondary antibody is reduced.

Cat. # 267

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Performing the Single Antibody ELISA

Step 1: The sample to be analyzed is added to the wells of the microtiter plate, where it non-specifically adheres to the plastic through hydrophobic and electrostatic interactions.

Step 2: After washing away any unadsorbed sample, the wells are “blocked” with a protein-containing buffer to prevent non-specific interactions between the antibody (added in Step 3) and the plastic wells.

Cat. # 267

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Step 3: The enzyme-linked antibody is added to the wells. Excess antibody is removed from the wells by washing several times with buffer. If the antibody can bind with the adsorbed antigen, it will remain in the well.

Step 4: The substrate is added to all the wells. A positive signal (brown color in wells) is observed in wells with antigen-antibody interactions.

Cat. # 267

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1. Label the plate.

2. Label the pipets.

3. Add three drops of the Negative Control to the three wells in the first row.

4. Add three drops of the Positive Control to the three wells in the second row.

5. Add three drops of Simulated Donor Serum 1 the three wells in the third row.

6. Add three drops of Simulated Donor Serum 2 the three wells in the third row.

7. Incubate for 10 minutes.

8. Remove liquid using the transfer pipet designated for each row.

9. Wash each well by adding the PBS buffer to each well until it is almost full (~ 8 drops). Remove all the PBS from the wells. Do not allow buffers to spill over into adjacent wells.

10. Add five drops of the substrate solution into each well

11. Incubate for 10 minutes.

12. Analyze your results. Cat. # 267

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Identification of West Nile Virus with the ELISA

Row 1 – Negative Control

Row 2 – Positive Control

Row 3 –Patient Sample 1

Row 4 – Patient Sample 2

Results from Patient 2 would be confirmed using PCR. Results would be reported to the CDC.

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West Nile Virus is a potentially serious disease that is transmitted by mosquitoes.

The Enzyme-Linked ImmunoSorbant Assay is used to detect WNV in biological samples.

Quick and easy ELISA can be performed by students in under 30 minutes.

Using the ELISA to Identify West Nile Virus Outbreaks

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Antigen-Antibody Interactions(#270)

Incorporating Immunology into your Curriculum with Edvotek

HIV Detection by Western Blot (#275)

Quantitative ELISA #278 Introduction to ELISA (#269)

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using the elisa to identify west nile virus outbreaks

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