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Lab 12: Clinical Microbiology Part II- Immunology; Transmission of

Microorganisms
Objectives:

Learn about the principles behind the ELISA test, and perform an experiment that simulates an ELISA test
for infectious mononucleosis
Learn how to use an agglutination test to identify pathogenic strains of Staphylococcus aureus

Key Words: Enzyme-linked immunosorbent assay (ELISA), antigen, antibody, colorimetric detection,
secondary antibody, Epstein-Barr virus, infectious mononucleosis, Protein A, coagulase, agglutination

Introduction: The rapid and accurate diagnosis of infectious disease is often crucial to determining patient
treatment, supportive care, and precautions for the health care worker. However, some infectious diseases are
more easily diagnosed than others. For example, although many types of bacteria may be easily identified by
appearance on differential media, gram staining, metabolic testing, etc. these identification methods may be
time-consuming and are not always practical. In addition, not all pathogens (ex: viruses) can be cultured in the
laboratory, and so other types of diagnostic tests are necessary.
Our immune systems respond to the presence of antigens (a foreign substance that stimulates an immune
response) by producing antibodies (globular proteins produced and secreted by B lymphocytes) that bind
specifically to them. Many diagnostic tests take advantage of the specificity of antigen-antibody binding to
detect exposure to a particular pathogen. For example, some diagnostic tests detect antibodies in a patients
serum- their presence indicates exposure to a specific pathogen, as these antibodies will only be present if the
patient has been exposed to that pathogen. Other diagnostic tests may look for specific antigens in serum (or in
a cultured clinical sample) by reacting them with known antibodies in clinical laboratory tests.
In this lab, you will learn how to use two standard immunological tests - an ELISA that detects serum antibodies
to diagnose infectious disease, and the SURE-VUE Staph ID test, which uses antibodies bound to latex beads to
identify pathogenic strains of Staphylococcus aureus.
ELISA TEST: The Enzyme-linked Immunosorbent Assay (ELISA) is used to detect several types of
pathogens and is particularly useful for the diagnosis of viral infections. Although there are many variations of
this test, it is most commonly used to detect serum antibodies to a particular viral pathogen. ELISAs are used to
diagnose exposure to human immunodeficiency virus (HIV), Epstein-Barr virus (EBV) and several other
pathogens. The test typically uses 96-well plates that have viral antigens bound to the wells. When serum is
added, only antibodies that are specific to the antigen in the well will bind to it- all other serum components
(including other types of antibodies) will be removed during washing steps. A secondary antibody (one that

recognizes the first antibody) is then added. This secondary antibody is conjugated (attached) to an enzyme
(horseradish peroxide is one commonly used enzyme). After washing away unbound secondary antibodies, the
enzymes substrate is added, and produces a color reaction in the well (this is known as colorimetric
detection). A color change is an indication of serum antibody, which means that the patient has been exposed to
that particular pathogen. Further testing may be done to confirm a positive ELISA test.

A typical ELISA 96-well plate

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How the ELISA test works

(From Pommerville)

In this lab, you will be using a kit that simulates the ELISA test using non-pathogenic materials. Our simulation
will show how ELISAs are used to detect infectious mononucleosis, commonly called kissing disease
because it is transmitted by saliva. Although young children are the largest population of people affected by this
viral pathogen, they generally exhibit very few symptoms. Adolescents and young adults are more likely to have
symptoms of infection, including fatigue, sore throat, fever, swollen lymph nodes and, in severe cases, and
enlarged spleen. The disease is caused by the Epstein-Barr virus (EBV) which is also associated with Chronic
Fatigue Syndrome, and, in some geographic locations, with certain types of cancers (ex: Burkitt Lymphoma).
SURE-VUE Staph ID test: Bacteria in the genus Staphylococcus are very common on the skin and mucous
membranes. Although they do not always cause disease, they are associated with many human and animal
infections, including food poisoning, toxic shock syndrome, skin abscesses and boils, and other types of
infections. Pathogenic strains of Staphylococcus aureus typically possess 1) Protein A, an antigenic protein
found in the cell wall, and 2) a coagulase enzyme that forms fibrin clots around the cells and protects them
from phagocytosis. These bacteria can be identified using tests that detect the presence of these two markers.
The SURE-VUE Staph ID test uses latex particles coated with antibodies to Protein A and coagulase that will
react with Protein A and/or coagulase enzyme and produce a visible clumping (or agglutination) reaction.
Note: other organisms may also give weakly positive results due to non-specific binding. Therefore the SUREVUE Staph ID test should only be used for gram positive, catalase positive cultures to confirm that they are
Staphylococcus aureus.

Procedures
A. ELISA
Per Table: 1 strip of test wells; viral antigen solution (EBV), positive and negative control solutions, anti- IgG
peroxidase conjugate (second antibody solution), Phosphate buffered saline (PBS, for washing steps), substrate
solution, 4 unknown donor serum samples.
Note: follow all directions carefully. Be particularly careful to always use the appropriate transfer pipet to avoid
cross-contamination of the solutions.
1.
2.
3.
4.
5.
6.

Label the first 6 wells of your strip 1-6.

Add 3 drops of EBV (viral antigens) with the transfer pipet labeled EBV to each of the 6 wells.
Incubate for 5 minutes at room temperature.
Remove the liquid using the same transfer pipet. Discard the liquid in the beaker labeled liquid waste.
Add PBS to each of the 6 wells until almost full (do not allow them to spill over),
Remove the liquid from the well as demonstrated by your instructor (Important: do NOT use the PBS
transfer pipet to remove the liquid- this will result in contamination of the PBS with antigen.
7. Add 3 drops of the appropriate test reagent to each well as outlined in the table below.
Well #
1
2
3
4
5
6

Solution to add (3 drops for each using

appropriate transfer pipet)
(-) PBS
(+)
DS 1
DS 2
DS 3
DS 4

Description
Negative control
Positive control
Donor serum 1
Donor serum 2
Donor serum 3
Donor serum 4

8. Incubate at 37C for 15 minutes.

9. Remove all the liquid from each well as instructed.
10. Wash each well once with PBS.
11. Add 3 drops of anti-IgG peroxidase conjugate (secondary antibody) to each of the 6 wells.
12. Incubate at 37C for 15 minutes.
13. Remove the liquid from each well.
14. Wash each well once with PBS.
15. Add 3 drops of the substrate to each of the 6 wells.
16. Incubate at 37C for 5 minutes.
17. Remove the test strip from the incubator and examine each well. Development of a color reaction is an
indication of a positive result.
Note: wells may be incubated for a longer period of time if the color reaction does not develop after 5 minutes.

B. Sure-Vue Staph ID test

Per pair of students: 2 unknown bacterial species (A and B), Staph ID test cards, test latex reagent, mixing sticks
Note: All supplies and reagents for this test are set up at a station on the side of the room (positive and negative
controls may be set up by your instructor).
1. Add one drop of the Test Latex reagent (white cap) directly to 2 available wells (circles) on the card.
The test latex reagent contains latex particles coated with proteins that will bind to Protein A/coagulase.
2. Use a fresh mixing stick to apply one fresh colony (unknown A) to a reaction well containing the test latex
reagent. Mix for approximately 10 seconds, breaking up the colony in the latex reagent. Spread to cover the
entire circle.
3. Repeat this procedure with your other bacterial sample (unknown B).
4. Rotate the card using a rocking motion for 30 seconds.
5. Read the results as follows:
A positive result: red agglutination with change of the background color from purple to blue.
Negative result: no agglutination, latex stays purple throughout the well.
Note: false positives may result if the cards are left too long. Read immediately after 30 seconds.
6. Compare your results to the positive and negative controls to confirm your results.

Results
A. ELISA test
Record the appearance of each of the wells here.

Well

Contents

Color appearance

1
2
3
4
5
6

B. Sure-Vue Staph ID test

Interpretation

Record the appearance of the following tests below

Sample
1

Contents
-

Color/ appearance

Interpretation

Control

+ control

Unknown A

Unknown B

Review Questions
1. Why are the washing steps necessary in the ELISA test?

2. Why are positive and negative controls always required for immunological tests? (be specific)

3. What is the purpose of the enzyme conjugated to the second antibody in the ELISA test?

4. How does the production of the coagulase enzyme contribute to the virulence of Staphylococcus aureus?