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«ELISA Simulation Kit TEACHER’S MANUAL WITH STUDENT GUIDE ELISA Simulation Kit Teacher’s Manual Overview..................... ...»

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ELISA

Simulation Kit

TEACHER’S MANUAL WITH STUDENT GUIDE

ELISA Simulation Kit

Teacher’s Manual

Overview..................................................................... 3

Objectives.................................................................... 3

Background................................................................... 3 Materials..................................................................... 7 Teacher Tips.................................................................. 7 Teacher Preparation............................................................ 8 Instructions................................................................... 8 Expected Results............................................................... 8 Answers to Questions in the Student Guide......................................... 8 Photocopy Masters Patient Profiles Patient Profiles for HIV Testing.................................................. P-1 Patient Profiles for Lyme Disease Testing.......................................... P-2 Patient Profiles for Avian Influenza Testing......................................... P-3 Patient Profiles for West Nile Virus Testing......................................... P-4 Student Guide............................................................ S-1 Note: Upon receipt, store samples A–F and the positive and negative controls in a freezer (approximately –20°C). If a freezer is unavailable, these materials can be stored in a refrigerator (approximately 4°C) for up to one month. All other materials may be stored at room temperature (approximately 25°C).

©2006 Carolina Biological Supply Company Printed in USA ELISA Simulation Kit Overview Using the ELISA Simulation Kit, students explore how the principles of antibody- based human immunity apply to a common laboratory test called ELISA (enzyme- linked immunoabsorbant assay). ELISA is commonly used to test blood serum for the presence of antibodies against disease-causing pathogens such as viruses and bacteria.

In this way, the assay indirectly detects infection by particular disease-causing agents.

Students work in pairs to perform a hands-on simulation of an indirect ELISA using simulated reagents. Students take an investigative approach to diagnose six fictitious patients for either HIV, Lyme Disease, Avian Influenza (Bird Flu), or West Nile virus.

Hypothetical scenarios are provided for each patient being tested for each disease.

This kit is a simulated ELISA and does not include actual serum samples, antibodies, antigens, or disease agents. As such, there is no risk of infection from the materials included in this kit.

–  –  –

The Body’s Defense System Background The body possesses several lines of defense against infection by pathogenic organisms.

Pathogens are tiny, disease-causing agents including viruses, bacteria, protozoa, molds, and other microorganisms. Pathogens invade the body and multiply; they can cause sickness or even the death of the invaded individual. The body employs three lines of defense to prevent and fight off such dangerous intrusions. The first two defense modes are nonspecific. They include the body’s physical barriers and the nonspecific immune system. These defenses function without regard to the type of pathogenic intruder. The third layer of defense is the body’s specific immune system.

Specific immune responses are tailored to the type of invading pathogen.

Nonspecific Barriers The primary defense against intruding pathogens is the external protective covering of the body, the skin, and the mucous membranes that line the mouth, nostrils, and other potential gateways. This armor presents a nonspecific physical barrier to foreign invaders. Secretions from the skin and mucous membranes, such as sweat, tears, and saliva, also inhibit entry into the body.

Nonspecific Immune Response If the body’s exterior barriers are breached, several internal, nonspecific immune defenses are launched. One such response is a fever, a rise in body temperature. Many pathogens cannot function well at body temperatures even slightly higher than normal. A fever, therefore, can slow the spread of an infection.

–  –  –

When an antibody forms an antigen-antibody complex, generally it marks the invading organism/antigen for destruction or for clearance from the bloodstream by phagocytic cells. This removal is designed to prevent the organism/antigen from infecting the cell. Antigen-antibody complexes also stimulate additional immune responses to aid the body in clearing an infection.





The specific immune system has an incredible ability to learn and remember. The first time the body is exposed to an antigen, a primary immune response is launched and antibody-producing B cells and T cells work steadily over time to eliminate the infection. If the same antigen is encountered a second time, the body is primed and remembers how to respond. It launches a more potent secondary immune response that can rid the body of the invading antigen more quickly. This is known as immunological memory.

ELISA Scientists have applied the basic principles of antibody-mediated immunity to an assay for detecting infection by specific organisms. This assay is called an ELISA (enzyme-linked immunoabsorbant assay) and is based on the principle that antibodies produced in response to pathogens attach to their antigen targets with great specificity to form antigen-antibody complexes. ELISAs can be used to test for infection by HIV, influenza virus, the bacterium that causes Lyme Disease, smallpox virus, SARS coronavirus, West Nile virus, and other disease agents.

There are two types of ELISA tests—direct ELISA and indirect ELISA. Indirect ELISA is used to detect infection by testing patients’ blood for the presence or absence of antibodies against a particular pathogen. The presence of such antibodies indicates that the individual has been infected and that their body has launched an immune response against the disease-causing agent. Direct ELISA assays for the presence or absence of certain antigens in patients’ blood. The lab performed in this kit simulates the indirect ELISA test.

In the first step of an indirect ELISA, antigen proteins purified from the infectious agent, or genetically engineered versions of the antigens, are added to the wells of plastic microtiter plates. These antigen proteins bind to the bottom of the well by forming hydrophobic associations with the plastic surface (see Figure 2a). The wells of the plate are then washed with a buffer to remove any unbound material.

Next, blood serum from the patient(s) being tested is added to the treated wells. If these serum samples contain antibodies against the bound antigen, the antibodies will attach to the antigens, forming tight complexes (see Figure 2b). Such antigenantibody complexes are not visible by eye, so detection steps (described in the next paragraph) must be employed to visualize them. The wells are again washed to remove any unbound proteins.

Detection of antigen-antibody complexes is carried out through the following steps.

A secondary antibody that recognizes antibodies produced by humans (anti-human antibody) is added to the wells. If antigen-antibody complexes formed in the wells, this secondary antibody recognizes and binds to the primary antibodies from the patients’ serum (see Figure 2c). The secondary antibody is attached to an enzyme that will facilitate the final detection. (This antibody/enzyme combination is called a conjugate.) The wells are rinsed one last time to remove unbound molecules.

–  –  –

3. What is the function of the secondary antibody and chromogen in an ELISA?

Antigen-antibody complexes formed in the initial steps of an ELISA are not visible to the unaided eye. Therefore, a colorimetric detection system involving a secondary antibody and chromogen is employed. The secondary antibody, which is conjugated to an enzyme, recognizes and binds to primary antibodies of antigen-antibody complexes, if they are present. Chromogen substrate is then added. If present, the enzyme linked to the secondary antibody changes the color of the chromogen. A color change indicates that the patient has antibodies against the pathogen and has been infected.

4. Why did you perform three identical tests for each control and patient sample?

Each assay was performed in triplicate to ensure reproducibility of the results.

5. What might cause some positive results to be lighter in color than others?

Weak positive results may be an indication that the patient’s blood serum carries few antibodies against the disease-causing agent. The patient’s exposure to the pathogen may be recent and the body may not have launched a full immune response yet.

Alternatively, the infection may have occurred long ago, and the level of antibodies in the patient’s bloodstream is declining. (Note: Other explanations are possible and acceptable, but these are most likely.)

6. Describe the disease-causing pathogen for the disease you assayed for, including its mode of transmission to people.

Answers will vary. The disease-causing agent for HIV, West Nile virus, and Influenza are different viruses. The disease-causing pathogen for Lyme Disease is the bacterium, Borrelia bergdorferi. HIV can be transmitted from person to person through exchange of body fluids. Lyme Disease can be transmitted to people through the bite of an infected tick. West Nile virus can be transmitted to people through the bite of an infected mosquito. Although there have been few cases as of early 2006, Avian Influenza can be transmitted to people through close contact with infected domestic poultry.

7. Write your reaction to the diagnosis of the patients you tested. Were you surprised by the outcome of the test based on the scenarios provided? How do you think infection could have been prevented in the patients who tested positive?

Answers will vary.

9 Teacher’s Manual ELISA Simulation

Patient Profiles for HIV Testing HIV stands for Human Immunodeficiency Virus. It is the virus responsible for causing the Acquired Immune Deficiency Syndrome, or AIDS. HIV attacks and destroys a type of T lymphocyte of the immune system called CD4+ cells, and thereby severely weakens immune defense. Shortly after infection by HIV, the body begins producing antibodies against the virus, and the infected person may feel flu-like symptoms. The virus then enters a latent phase where the infected person does not experience symptoms but the virus and the immune cells continue to attack each other. Eventually, the HIV virus gains the upper hand. The immune system loses its ability to fight off the opportunistic infections it could normally combat, and advanced AIDS results. HIV is communicable and can be transmitted from person to person through blood, pregnancy, breast milk, and other bodily fluids.

–  –  –

Patient A Patient A has just learned that her ex-boyfriend was diagnosed with HIV. Although they often used protection when sexually active, she is concerned that she could have contracted the virus from him.

Patient B Patient B is the current boyfriend of Patient A. Although they are not sexually active, they have engaged in deep kissing. Patient B is concerned for his girlfriend and for himself, and decides to get tested along with her.

Patient C Patient C is an EMT who treated a bleeding car accident victim who was HIV positive. After attending to the accident victim’s injuries, he realized that his glove was torn and his hand cut.

Patient D Patient D just broke up with her boyfriend after learning about several lies he told her. She worries that he has not been monogamous and decides to be tested for HIV.

Patient E Patient E is happily married with young children when he gets a call from an old college girlfriend who is HIV positive. She does not know when she contracted the virus and is contacting all her old partners so that they can be tested as well.

Patient F Patient F experimented with drugs during college and used some intravenous substances. Although he has been clean and sober for years, he remembers sharing a needle with a friend at a social function and decides to be tested for HIV.

–  –  –

Patient Profiles for Lyme Disease Testing The pathogenic agent of Lyme Disease is a spirochete bacterium, Borrelia bergdorferi. The bacterium is transmitted to people through the bite of infected ticks, such as deer ticks and black-legged ticks. It cannot be transferred from person to person. The life cycle stages of ticks require meals of blood obtained from warm-blooded animals. When an infected tick bites a victim, it transmits the infectious bacteria after 36–48 hours of attachment. An immune response is then launched in the infected individual. Early removal of ticks can prevent such transmission of infection. Early symptoms of Lyme Disease include a characteristic bull’s eye rash (erythema migrans) surrounding the bite, and flu-like symptoms such as fever, headache, chills, muscle aches, and fatigue. If the disease goes untreated, it can result in more serious symptoms such as severe muscle pain, extreme fatigue, neurological difficulties, and respiratory and gastrointestinal problems.

–  –  –

Patient A Patient A went on a hike with her hiking club and has since felt feverish and achy. She is an avid hiker who knows the risks of Lyme Disease and always uses insect spray and wears protective clothing. She thinks she probably has the flu.



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