Using the ELISA Assay for Disease Detection-Teacher Guide

Using the ELISA Assay for Disease Detection-Teacher Guide

Using the ELISA Assay for Disease Detection- Teacher Guide

This laboratory is a simulation of the ELISA test (Enzyme Linked ImmunoSorbent Assay) for detecting the presence of antigen, such as a disease-related agent, from a sample of body fluid. The "antigen" used in this experiment is an innocuous protein (biotin-bovine albumin) present in the "body fluid," which is a buffer. The "antibody" is represented by another protein (streptavidin) which is linked to the enzyme peroxidase. The protein used as the antibody binds with high affinity and specificity to the antigen. A color substrate (TMB) is then added to visualize the presence streptavidin- peroxidase (antibody) binding to biotin-bovine albumin (antigen).

Each student is given a solution representing his/her body fluid. Students then exchange body fluid with three other individuals. The ELISA is used to detect the simulated disease agent in the mixed body fluids. Finally, the class can trace back through the transmission routes to determine identity of the original carrier(s).

This assay is similar to that used for the AIDS test, in which an ELISA is used to detect individuals infected with the virus that causes AIDS (HIV) by screening for virus-specific antibody.

Classroom time needed for this lab:

* two class periods (~50 minutes each) or one block period.
Day 1 for performing first ELISA test on shared samples
Day 2 for analysis of classroom data and performing a second ELISA test

You will need to prepare the following materials:

* Washing Solution
* 1X Na2CO3 Buffer Solution
* Positive Antigen Solution
* Antibody Solution
* Color Reagent Solution

Setting up the classroom for this lab:

Each student will need a microcentrifuge tube of "body fluid," either 1X Na2CO3 buffer solution (non-infected) or positive antigen solution (infected). We distribute one infected tube for up to 20 students and two infected tubes for 21- 40 students.

Each student will also need a clean transfer pipet to be used for the exchanges of solution.

During the lab:

After the exchanges, each group of four students receives:

* ELISA plate (contains 8x3 wells or a quarter section of a full ELISA tray)
* positive control solution (in dropper bottle)
* negative control solution (in dropper bottle)
* Paper towels
* Antibody solution (in dropper bottle)
* Washing solution (~ 100 ml in wash bottle)
* TMB Color reagent solution (in dropper bottle)

Be sure that students dispose of the transfer pipets immediately after they use them with a reagent. Contaminating the transfer pipets with another reagent will confound the results!
Recipes:

Washing Solution: 1X PBS with 0.1% Tween 20

1. Dissolve 160 g NaCl, 4g KCl, 22.4 g Na2HPO4, and 4g KH2PO4 in deionized or distilled water to give final volume of 1000 ml buffer. This is 20X PBS.

2. Dilute 50 ml of 20X PBS to 950 mL with deioinized or distilled water for each class. Add 10 ml 10% Tween 20 to buffer. This buffer (1X PBS, 0.1% Tween 20) can be stored indefinitely at room temperature.

This buffer is used to wash plates after antigen addition and after antibody addition.

Sodium Carbonate: 1X Na2CO3 Buffer Solution

1. Dissolve 3.2g Na2CO3 and 5.86 g NaHCO3 in deionized or distilled water to final volume of 200 mL. This is a 10X Na2CO3 concentrate. This solution can be stored indefinitely in the refrigerator.

2. To make a 1X Na2CO3 buffer solution for classroom use, add 20 ml 10X concentrate to 180 ml deionized or distilled water. This solution can be stored indefinitely in the refrigerator.

3. Give 1 ml of the 1X Na2CO3 buffer solution to each student as simulated body fluid. Distribute 1 ml aliquots in microcentrifuge tubes as a negative control for the ELISA test.

Positive Antigen Solution (biotinylated albumin)

1. Dissolve 10 mg biotinylated bovine albumin in 20 mL 1X Na2CO3 buffer for a final concentration of 0.5 mg/mL. Store as 1 mL aliquots in freezer.

2. The positive antigen solution contains two types of bovine albumin, biotinylated bovine albumin and normal, non-biotinylated bovine albumin. To prepare positive antigen solution, mix 0.1 mL of the 0.5 mg/mL biotinylated bovine albumin solution with 9.85 mL 1X Na2CO3 solution and 50 ul of 10 mg/ml normal, non-biotinylated bovine albumin (for recipe, see "Antibody Solution"). This solution can be stored for up to one week in the refrigerator.

3. This solution is given to 1-2 students in a class as the infected samples. These students would be the original infected carriers in the class.

4. Use the remaining solution as positive control samples for the ELISA test. Put 1 ml of positive antigen solution into a centrifuge tube (1.5 ml size) for each lab group.

Antibody Solution (Streptavidin peroxidase)

1. Add 1 mL of a 50% glycerol solution to the 0.5 mg of streptavidin peroxidase in the container. Store in refrigerator; this concentrate should be stable for several years.

2. Prepare a 10 mg/ml bovine albumin solution by mixing 0.5 g bovine albumin (this is normal, non-biotinylated albumin) in 50 ml deionized or distilled water. The 10 mg/ml solution can be stored in 10 ml aliquots in the freezer.

3. To prepare antibody solution, mix 0.5 mL 20X PBS, 50 ul of 10 mg/ml normal bovine albumin and 9.45 mL deionized or distilled water. To this mixture, add 1 uL streptavidin peroxidase solution. Store in refrigerator and use within one week after dilution. Distribute in 3 ml aliquots to each student group.

Color Reagent Solution (TMB)

1. Dissolve 1.46 Na2HPO4 and 1.02 g citric acid in water to final volume of 200 mL. This is citrate phosphate solution (0.05 M). This solution can be stored indefinitely in refrigerator.

2. To prepare color reagent solution, add 1 mg TMB or 1 TMB tablet to 10 mL citrate phosphate solution. Next, add 2 uL of hydrogen peroxide (30%) to this solution.

3. Use this solution on the same day and store in the refrigerator.