ELISA

Introduction

The enzyme-linked immunosorbent assay, or ELISA, test is used to screen patients for various diseases or allergies. It is one of the most prevalent diagnostic tools available for doctors today. Guide your students to take on the role of medical diagnostic technicians by determining the results of comprehensive allergy testing for a patient.

Concepts

• Antigen

• ELISA
• Antibody
• Allergen

Materials Included In Kit

Additional Materials Required

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. The indicator solution is a dye solution and will stain skin and clothing. Avoid contact of all chemicals with skin and eyes. Dilute (0.01 M or less) solutions of hydrochloric acid and sodium hydroxide are body tissue irritants. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron when handling these chemicals. Avoid exposure to eyes and skin and clean up all spills promptly. Wash hands thoroughly with soap and water before leaving the laboratory. 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 final solutions from this laboratory exercise can be disposed of down the drain according to Flinn Suggested Disposal Method #26b. The ELISA test plates and excess agar may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a.

Prelab Preparation

1. Prepare simulated solution bottles.
1. Fill the following bottles with deionized water—2° antibody solution, negative control solution, positive control solution, sera solution and wash solution. Note: Bottles contain only water and are not actual solutions.
2. Dry the outside of each bottle, if necessary.
3. Flinn labels provided to each bottle. Adhere the labels to the bottles.
2. Prepare the acidic agar. May be prepared several days in advance and re-melted for step 4.
1. Label one of the borosilicate glass beakers “acidic agar.”
2. Add 250 mL of 0.01 M hydrochloric acid solution to the beaker.
3. Place the beaker on a hot plate or magnetic stirrer/hot plate and heat slowly to warm the acid solution. Use a stirring rod or magnetic stir bar to slowly stir the solution.
4. Slowly stir 7 g of agar into the warm acid. Use the stirring rod to break up any clumps of agar.
5. Allow the agar to dissolve into the dilute acid solution.
6. Bring the agar up to a slow boil. Boil for 5 minutes.
7. Allow the agar to cool to 55 °C before preparing the ELISA plate (step 4).
3. Prepare the basic agar. May be prepared several days in advance and re-melted for step 4.
1. Label the second borosilicate beaker “basic agar.”
2. Add the 250 mL of 0.005 M sodium hydroxide solution to the beaker.
3. Place the beaker on a hot plate or magnetic stirrer/hot plate and heat slowly to warm the base solution. Use a stirring rod or magnetic stir bar to slowly stir the solution.
4. Slowly stir 7 g of agar into the warm base. Use the stirring rod to break up any clumps of agar.
5. Allow the agar to dissolve into the dilute base solution.
6. Bring the agar up to a slow boil. Boil for 5 minutes.
7. Allow the agar to cool to 55 °C before preparing the ELISA plate (step 4).
4. Prepare one ELISA plate for each demonstration. Best if made a day or two before the day of the demo.
1. About 35 mL of each agar is needed for each ELISA plate that is being prepared. If all 7 ELISA plates are being prepared, all of the agar will be used. If proceeding directly from steps 1 and 2, skip to step c.
2. If the agar has set, melt the agar by one of the two following methods.
1. Use a hot plate set at a low to medium setting. Do not overheat the agar as it may burn. Stir frequently with a stirring rod or use a magnetic stir bar on low speed.
2. Use a laboratory microwave; do not microwave for more than 30 seconds at a time. Stir the agar with a stirring rod between each cycle in the microwave.
3. Use a graduated pipet to add about 1.5 mL of the acidic agar into each well in columns 3 and 6 as well as two wells in column 2 and two wells in column 5.
4. Use a clean, graduated pipet to add 1.5 mL of the basic agar into each well in columns 1 and 4 as well as the remaining wells in columns 2 and 5.
5. Allow the agar to solidify in the wells. This will take at least 30 minutes but it is best if done at least 12 hours before.

Procedure

1. Explain to the students what each solution represents and its purpose in an ELISA test (see Discussion section).
2. Place three drops of the negative control solution into each well in columns 1 and 4 (see Figure 1).

3. Place three drops of the positive control solution into each well in columns 3 and 6.
4. Place three drops of the sera solution into each well in columns 2 and 5.
5. Fold a paper towel until it fits just over the top of the ELISA test plate. Holding the paper towel in place on top of the ELISA test plate, quickly flip the ELISA test plate upside down. This should cause the solutions to run into the paper towel. Flip the ELISA test plate right-side up.
6. Place five drops of the wash solution into each well.
7. Place a clean, folded piece of paper towel on top of the ELISA test plate; quickly flip the ELISA test plate upside down. Flip the ELISA test plate right-side up.
8. Place three drops of the 2° antibodies into each well.
9. Place a clean, folded piece of paper towel on top of the ELISA test plate; quickly flip the ELISA test plate upside down. Flip the ELISA test plate right-side up.
10. Place five drops of the wash solution into each well.
11. Place a clean, folded piece of paper towel on top of the ELISA test plate; quickly flip the ELISA test plate upside down. Flip the ELISA test plate right-side up.
12. Finally, place 3–5 drops of indicator solution into each well.
13. Have the students record observations (color changes and the test results) on the ELISA Test Worksheet. A color change of the indicator is evidence of a positive reaction. Answer the questions on the worksheet.

Student Worksheet PDF

10753_Student1.pdf

Teacher Tips

• This kit contains enough chemicals to perform the demonstration seven times: 50 mL of phenol red indicator solution, 250 mL of 0.01 M hydrochloric acid solution, 250 mL of 0.005 M sodium hydroxide solution, 14 g of agar, pipets, seven 24-well test plates, five 60-mL dropping bottles and five Flinn labels.
• If the agar does not set properly, reheat to a faster boil for several minutes. The agar should be transparent.
• The demonstration is designed to “test” for eight items. The following scenarios may be presented to the students:
  —The eight most common food allergies are dairy, soy, wheat, shellfish, fish, peanuts, tree nuts and eggs.
  —The eight most common environmental allergies are dust mites, rye pollen, ragweed pollen, cockroach dust, latex, penicillin, bees and other stinging insects.
  —Test eight different patients for the same virus, such as HIV or H1N1, or other condition, such as pregnancy or drug screening.
• Phenol red is the indicator used in this kit. Phenol red is yellow at pH 6.8 and red at pH 7.8.
• Although this cost-effective demonstration is based on an acid–base idea, the steps involved in the procedure are the same steps as those used in a real ELISA test.

Sample Data

Observations

Answers to Questions

1. The initial color of the indicator used in this demonstration is pink. If the indicator remains pink after it is added to the ELISA test plate, does the patient’s serum contain the antibody for that particular allergy?

A pink well indicates a negative test—the patient’s serum does not contain the antibody and he or she is not allergic to that allergen.

2. Draw a sample ELISA test plate. Indicate which wells are positive for the antibody of interest.

3. In this demonstration the ELISA test plate was set up to simulate an allergy test. Answer the following questions to define the specific substance that would be used for an HIV ELISA test.

a. What is coated on the ELISA plate?

The ELISA plate is coated with inactive HIV.

b. What is an appropriate negative control?

Human serum that is known to be HIV negative should be used as the negative control.

c. What is an appropriate positive control?

HIV antibodies should be used as the positive control.

d. What does the 2° antibody bind to?

The 2° antibody binds to HIV antibodies.

e. What causes the indicator to change color?

The indicator binds to the active site on the 2° antibody which is bound to the HIV antibody and the underlying inactive HIV.

Discussion

The ELISA test is based upon the specific response of the immune system. Antibodies are protein products of the immune system and are found in the blood and other body fluids of vertebrates. The main function of antibodies is to recognize and initiate the removal of foreign objects, such as bacteria, viruses or allergens. Antigens are foreign molecules such as bacteria, viruses or pollen that do not belong to the host organism. Antibodies recognize and bind to a specific region of the antigen much like a lock and key. Once the antibody binds to the antigen, the antigen is tagged to be destroyed by the immune system. The fit between the antigen-binding site of the antibody and the antigen is highly specific. This specificity allows antibodies to bind only with their matching antigen so other molecules are not tagged for destruction. Using the idea of a tagging system, scientists have developed numerous biochemical antigens that selectively bind to desired antibodies for a wide variety of diseases or allergies. Once the antigens have been tagged, a colorizing agent or indicator is added. A color change indicates for a “positive” result while the solution remains colorless if no antibody/antigen complex is present. Common ELISA tests include pregnancy tests, viral disease tests (including HIV), screening for illicit drugs and allergy testing.

Allergies occur when the immune system mistakenly identifies a “harmless” substance as a “non-self” harmful substance. The immune system attacks the allergen in the same way it would a virus. It is this attack that causes typical allergy symptoms such as puffy eyes, sneezing and nasal drip, or even the severe reaction of anaphylactic shock. A typical ELISA allergen test can screen for up to 140 different allergens from a single sample of blood serum. Blood serum is the part of blood that is left when the red and white blood cells have been removed.

The ELISA test is done in a plastic well plate containing rows of very small wells that hold small volumes of chemicals. The desired antigen (allergen) is bound to the bottom of each well (see Figure 4a). A sample of the patient’s blood serum is added to each well. If allergen antibodies are present in the blood sera, they will bind to the antigen in the well (see Figure 4b). Only individuals that have developed an allergy will have antibodies, called primary antibodies, which recognize the allergen (antigen). In between each step of the test procedure, the well is rinsed to remove any excess reagents. Only the bound antibodies remain after rinsing.

To detect whether any primary antibodies are bound to the antigen in the well, a second biochemical antibody is added to the well (see Figure 4c). This second antibody, called the secondary or 2° antibody, only recognizes the primary antibodies that remained in the well after rinsing. If any primary antibodies are present, the secondary antibodies will bind to them. An enzyme attached to the “tail” of the secondary antibody reacts with an indicator solution added to the well in the final step of the procedure. If antibodies are present in the patient’s blood sera, the indicator will change to its colored form (see Figure 4d). If no antibodies are present, the indicator remains colorless. In a clinical setting ELISA tests are quantitative.
The color intensity of a positive test is directly proportional to the amount of antibodies present in the patient’s blood sera. Figure 5 is a schematic representation of positive and negative ELISA reactions: