Biology Van

Science in Motion Wilkes University Rev 6/11

Immunology - Antigen/Antibody Reactions

ELISA Test

PA Standards:

PA S. T. & E.:

3.1.10.A7. Describe the relationship between the structure of organic

molecules and the function they serve in living organisms.

Introduction:

One of the body’s most important defense mechanisms against infection is the production of proteins called antibodies, or immunoglobulins. These proteins are produced by white blood cells and circulate in the bloodstream as part of the blood plasma. The production of antibodies is stimulated by the presence of foreign agents such as viruses and bacteria. White blood cells can identify molecules located on the foreign agents. These molecules are also proteins and are referred to as antigens. The antibodies produced in response to the antigens bind specifically to the antigens and render them harmless. Under normal circumstances, your body does this any time a foreign protein is identified in your body fluids and the response is the destruction of the foreign substance. It is important to remember that a specific antibody will bind only to the antigen that it was manufactured for. This phenomenon is the basis of the body’s defense against disease.

There are many laboratory procedures that utilize the antibody-antigen reaction. One such test is called ELISA (Enzyme Linked Immunosorbent Assay). This technique is used to detect and quantify specific antibodies found in blood serum. In ELISA, serum to be tested is allowed to react with specific antigens. Serum antibodies that combine with the antigens are detected by treating the test with a second antibody which has an enzyme attached to it. The second antibody is called a conjugate and it binds to the antibody-antigen complex and serves as a marker. Later in the test a substrate for the enzyme is added. The substrate will react with the enzyme and produce a color change. If no serum antibodies were present to bind with the antigen and the conjugate, no enzyme-substrate complex will form and therefore no color change will be observed. This test is done in polyvinyl well plates under controlled conditions. ELISA is done routinely to screen humans for antibodies to the HIV virus.

Definitions:

Antibody: A protein produced by white blood cells (B-cells) in response to a foreign chemical or disease organism in the body. Antibodies bond to the foreign substance, aiding the body’s ability to recognize and remove the foreign material.

Antigen: A term for a foreign material in the body. Antigens could be carbohydrates, lipids, nucleic acids, proteins, dust, bacteria, viruses, etc. They stimulate an immune response in animals with an immune system.

ELISA: (Enzyme Linked Immunosorbent Assay). This technique is used to detect and quantify specific antibodies found in blood serum.

Guiding Questions:

1. 
   Distinguish antigens from antibodies.
   
2. 
   Describe the antibody/antigen reaction.
   
3. 
   Describe the ELISA test.
   
4. 
   Why is this test important clinically?
   

Materials:

safety glasses HIV antigens

gloves positive control (primary antibody)

paper towels negative control

HIV antibody test well plate donor 1 serum

stopwatch donor 2 serum

transfer pipettes secondary antibody

substrate

Safety Notes:

1. Although no human products are used, the chemicals you will be working with are irritants. Avoid eye/skin contact and do not ingest. Flush spills with water for 15 minutes. Notify your instructor.

2. Wear safety glasses and gloves at all times.

3. Wash your hands thoroughly at the end of each lab session.

Procedure:

1. 
   Place your initials on the HIV antibody test well plate and put it on a paper towel.
   

2. Using a transfer pipette, place one drop of HIV antigen in the center of each well on the well plate.

3. 
   Incubate at room temperature for 2 minutes.
   

4. 
   Using a new transfer pipette, place one drop of the negative control to the wells in row 1.
   

5. 
   Using a new transfer pipette, place one drop of the positive control to the well in row 2.
   

6. 
   Using a new transfer pipette, place one drop of donor 1 serum to the well in row 3.
   

7. 
   Using a new transfer pipette, place one drop of donor 2 serum to the well in row 4.
   

8. 
   Gently shake the well plate then incubate at room temperature for 2 minutes.
   

9. Using a new transfer pipette, place one drop of secondary antibody to all wells.

10. Gently shake the well plate then incubate at room temperature for 2 minutes.
11. Using a new transfer pipette place one drop of substrate to all wells.
12. A pink color change indicates a positive test result. Record the color changes on the data sheet.
Well Plate – only one row will be used per lab group


Steps in ELISA

1. Add antigens to the wells on the plate.

HIV Antigen

2. Serum containing a specific antibody is added to the wells; this antibody will bind with a specific antigen if the antigen is present.

Antigen-Antibody

3. A second antibody, which specifically binds with the first antibody, is added. This is called an anti-antibody and it has a specific enzyme attached to it.

Antigen—Antibody - Anti-Antibody—Enzyme

4. A substrate for the enzyme is added. If an enzyme - substrate complex forms, a pink color develops in the well.

Antigen—Antibody - Anti-Antibody—Enzyme---Substrate

(color reaction)

References
Carnegie Mellon Website. {Retrieved 1 June 2011:

www.andrew.cmu.edu/user/berget/Education/AIDS/html.AIDS_gifs/ELISA.gif } introductory diagram.

Student Data Sheet

Name_____________________________ Date ________________

Observations:

Record the color changes observed in the wells.

Row 1_________________________
Row 2_________________________
Row 3_________________________
Row 4_________________________

Analysis/Conclusions:

1. 
   Which donor/donors has been exposed to the HIV virus? How do you know?
   

2. 
   Which control will show that a donor has been exposed to HIV?
   

2. 
   Why is the substrate added to the wells?
   

3. 
   Does a positive ELISA test for antibodies against HIV indicate that the individual has AIDS? Explain.
   

5. 
   Summarize the reactions occurring during the ELISA test.
   

Teacher Notes:

Approximate Time to complete lab:

Introduction, procedure, and results: 40 minute period

Expected Results:

Row 1 – no color change

Row 2 – pink color change

Row 3 – no color change

Row 4 – pink color change

Sample answers to Evaluation questions:

1. 
   Donor 2 has been exposed to the HIV virus. There is a pink color change.
   
2. 
   Row 2 is the positive control.
   
3. 
   Without the substrate-enzyme reaction, there is no color change.
   
4. 
   No, because a person could be HIV positive and not have AIDS.
   
5. 
   Refer to Figure 1.