Materials Included In Kit

Additional Materials Required

Prelab Preparation

Prior to the laboratory, separate chemical test sets should be prepared for each lab group. This will prevent congestion at a central dispensing area, and it will also decrease the probability that someone will contaminate the entire bottle of a specific chemical.

The ten solutions used in the lab should be dispensed into the covered microtubes provided in the kit. Fill each tube with its appropriate solution up to the 0.5 mL mark. This will be an adequate amount to run all the individual tests. Use abbreviations to label each tube as follows: “HIV-A,” “2°A,” “Sub,” “+,” “–,” “1,” “2,” “3,” “4,” “5.”

Safety Precautions

Note: This kit contains no actual blood or blood products or anything that could be considered an infectious agent. The lab is a chemical simulation only. Unknown Sera 2, Sera 3 and Simulated known HIV positive solutions contain phenolphthalein. The Substrate Color Indicator contains sodium hydroxide solution. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions from this laboratory exercise can be disposed of down the drain following Flinn Suggested Disposal Method #26b.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. The laboratory can reasonably be completed in one 50-minute class period.

• A dark violet or purple color is the indication of a positive ELISA test in this simulation. The difference between a negative test (clear) vs. positive (purple) should be obvious.
• Unknown sera 1, 4 and 5 should test negative and unknowns 2 and 3 positive. These results are reproducible if the proper solutions are added in the proper order and, most importantly, if the transfer pipet is rinsed thoroughly between the various steps. If these results are not achieved it is an indication of cross contamination of the key chemical components.

Sample Data

II. Testing Unknowns

Answers to Questions

I. The ELISA Test

Describe a positive ELISA Test result for this simulation:

A color change to purple occurred.

Describe a negative ELISA Test result for this simulation:

The sera remained colorless.

II. Testing Unknowns

1. Would an infected person have a positive ELISA test the day after infection? Explain.

No, it usually takes from two weeks to three months before antibodies can be detected.

2. Which individuals 1–5 should definitely be tested further (Western Blot Analysis) for AIDS infection?

Individuals 2 and 3 should be tested further since a positive ELISA test indicates the presence of antibodies against HIV.

Introduction

AIDS is a continuing human health problem. Although there is no known cure for AIDS, the early detection can result in treatment with drugs—which can often slow the spread of the virus and extend the life of the victim. How can we test for AIDS so it is detected early?

Concepts

• Antibody

• Antigen
• AIDS (Acquired immune deficiency syndrome)
• ELISA (Enzyme linked immunosorbent assay)

Background

Acquired immune deficiency syndrome (AIDS) was first recognized in 1981 and its known incidence has roughly doubled every year since. The disease is characterized by the progressive deterioration of an individual’s immune system. Because the virus attacks the individual’s immune system, the person is in danger from invasion of any infectious agent. Once the immune system has been sufficiently weakened, the individual will suffer the consequences of the worst symptoms of the attacking agent. The result of AIDS infection takes on many forms and often results in a complex of many different symptoms from multiple infecting agents.

AIDS is caused by a retrovirus (a virus that contains RNA instead of DNA as its genetic material). The virus is called human immunodeficiency virus (HIV–1) and is widespread throughout the world. The HIV virus infects the T-helper cells, the cells required to activate B-lymphocytes and to induce the production of antibodies and other defense structures so critical in fighting outside invaders.

When an AIDS virus infects a T-helper cell, its viral RNA is used to make viral DNA (viral genes), which in turn may be incorporated into the host cell’s DNA. Then the virus may become dormant, failing to produce any noticeable effects for some time. During the latency period, which may last for several years, the infected person may show no symptoms of AIDS, but can probably transmit the virus to uninfected persons. The transmission of the disease seems to require direct introduction of the virus into the blood stream, as may occur during certain sexual activities. The virus can also be transmitted by contaminated hypodermic needles or by the transfusion of virus-containing blood or blood products. Many other unusual blood contacts between individuals have resulted in infection. Though AIDS is apparently not spread by casual contact with AIDS patients, each year a small percentage of AIDS cases cannot be easily traced to the original source of the infection.

Antibodies appear in the blood of persons infected by the AIDS virus, and these antibodies can usually be detected by means of a simple blood test performed between two weeks and three months following exposure to the virus. If antibodies do not develop within several months following exposure to the virus, it is assumed that the person has not been infected.

There are several diagnostic procedures used to determine if a person is infected by HIV-1. The enzyme-linked immunosorbent assay (ELISA) test is used as the initial screening test to detect HIV antibodies in blood samples. In an ELISA test, a series of steps are performed to determine if an individual has produced antibodies in response to the HIV virus. If the person has been infected by HIV and has formed antibodies against the virus, the virus can be detected, indirectly, by the presence of the antibodies. In the test, if an antigen-antibody reaction occurs, a color change occurs indicating the presence of antibodies. If an antigen–antibody complex does not form, the test solution remains colorless. If the antibody against the HIV virus is detected with the ELISA test, then another test, called Western blot analysis, is performed to confirm the HIV infection. If both tests are positive, the individual is 99% likely to be infected.

The ELISA test is done in a plastic well plate containing rows of very small wells that hold small volumes of chemicals. HIV antigen is bound to the bottom of each well. When blood is drawn from the person to be tested, the red and white blood cells are first removed and some of the remaining sera is added to a test well. If anti-HIV antibodies are present, they will bind to the HIV antigen in the well. Only individuals that have been infected with HIV will have antibodies that recognize the antigen. In between each step of the test procedure, the well is rinsed to remove any excess reagents. Only the bound materials remain after each rinsing. To detect whether any anti-HIV antibodies are bound in the well, a second antibody that recognizes human anti-HIV antibodies is added to the well. If any human anti-HIV antibodies are present, the anti-human antibody (often called a secondary antibody) will bind to them. Thus, a chain of molecules is linked to the bottom of the well and the HIV antigens.

The secondary antibody has an attached enzyme. The enzyme causes a color change in the presence of a color indicator. If the enzyme is present in the well because of the presence of anti-HIV antibodies in the test sera, there will be a color change when the indicator is added to the well. If there are no antibodies, the indicator remains colorless. The following diagram is a schematic representation of a positive and negative ELISA reaction:

Materials

Safety Precautions

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part I. The ELISA Test

1. ELISAs are done in specially prepared microwell plates. HIV antigens are added to the wells where they absorb to the plastic well walls.

Place one drop of HIV antigen into each of two wells on the well plate. Rinse the pipet thoroughly with distilled water.

2. Next the blood sera of the test individual is placed into the well with the HIV antigen. If the blood contains the anti-HIV antibodies, they will bind with the HIV antigens in the bottom of the well plate.

Place one drop of the known positive sera into one of the wells containing the HIV antigen. Note which well is used for the positive. Rinse the pipet thoroughly with distilled water. Add one drop of known negative sera into the other well containing the HIV antigen. Let the two drops set for a one-minute, room-temperature incubation period. Rinse the pipet thoroughly with distilled water.

3. A secondary antibody (from another organism) that will bind with the human antibody is added to the mixture. The secondary antibody contains an attached enzyme.

Add one drop of secondary antibody to both the positive and negative well. Rinse the pipet thoroughly with distilled water.

4. A substrate that will interact with the enzyme on the secondary antibody is then added.

Add one drop of the substrate indicator to each of the wells. Notice any color changes. Record the results on the ELISA Test Worksheet. Rinse the pipet thoroughly with distilled water.

1. Note the exact position of each unknown’s test well.
2. Place one drop of HIV antigen in each test well. Rinse the pipet thoroughly in distilled water.
3. Place one drop of each unknown sera into a test well with the HIV antigen, rinsing the pipet in between each unknown.
4. Add a drop of secondary antibody to each test well. Rinse the pipet thoroughly.
5. Add a drop of substrate test indicator to each well. Rinse the pipet thoroughly.
6. Record the test results on the ELISA Test Worksheet and answer the questions on the worksheet.
7. Consult with your instructor on proper disposal procedures.

Student Worksheet PDF

10339_Student1.pdf