Antibody Capture Simulation

Developed by: Eric Burtson
© American Association of Immunologists 1995

Background
When we get sick from an infection, our antibodies respond by attaching themselves to the proteins that come in with the invading viruses or bacteria. Immunologists call the bonded proteins antigens. Since antigens can have so many chemical forms, our bodies must provide many thousands of different kinds of antibodies. As a result, it takes a while for the right antibodies to find a particular unwelcome protein. When they do, our immune system begins reproducing many of those antibodies.

At times, immunologists need to test certain fluids to see if they contain the right kind of antibody and how much of that antibody is present. One such test is called an Antibody Capture Assay . To do the assay, the scientist pours an antigen solution into special plastic plates containing wells that can bind the antigen to their floors. Since the scientist knows the concentration of antigen in the fluid, he or she then knows how many antigens bind to the floor of each well.

Next, the immunologist pours certain volumes of the antibody solution into the wells. After letting the tray incubate and the antibodies bind, the scientist rinses out the wells. Then the immunologist pours a labeled reagent that will bind with any captured antibody and will also be detected and counted by special lab equipment. In this way, not only can scientists verify the presence of the antibody, but they can know exactly how much is in the solution.

Finding out how much antibody a test solution contains is called a titration . To titrate a solution, a scientist will analyze the machine's readings to determine what percentage of the antigen on the plate is bonded. This information will then give the scientist the number of antibodies in the volume of antibody solution used.

In this lab, you will simulate an antibody capture assay. Using the tools provided, you will find out what percentage of "antigen" is bonded by various volumes of "antibody test solution." From these numbers, you will make a graph to titrate the test solution.

In addition, you will begin to familiarize yourself with the unit mole. To compensate for the extreme smallness of molecules, we like to use a large number, the mole, when we discuss how much of a measurable amount of molecules we have. Therefore, just as it is convenient for bakers to sell things in units of a dozen, scientists often find it convenient to work with moles of molecules.

Procedure

1. Count how many velcro antigens are in your shoe box.
2. Record which container of antibody test solution you are assigned: "A," or "B."
3. Scoop out one liter of the assigned styrofoam antibody test solution and dump it into your box.
4. Allow your antibodies to incubate by gently shaking the box for a few seconds. Open the lid and dump out the unbound contents.
5. Count the number of antibodies that are bound to antigens. Record this and the volume of test solution you used in a table with column headings "Percentage of Antigens Bound," "Number of Antibodies Bound," and "Volume of Test Solution (Liters)." Return all antibodies to the assigned container and gently mix them back into solution.
6. Repeat steps three through five to make a total of twenty trials. Use an array of different volumes, adjusting the amount taken so that most of your measurements will result in less than all the antigens sites being bonded.

Processing the Data

1. Calculate and record the percentage of antigen sites that are bound for each trial.
2. Graph the percentage of antigens bound versus the volume of test solution.
3. Titrate by reading from your graph the volume of antibody test solution at which one hundred percent of the antigen is bound.
4. How many of the antibodies that you are testing for should be in that volume?
5. How many liters would contain a dozen of the desired antibodies?
6. How many would contain one hundred?
7. How many would contain a mole?
8. How many shoe boxes would we need to capture a mole of the antibodies?
9. Estimate how many rooms this size would be needed to hold all those shoe boxes.
10. Separate a liter of antibody test solution into desired antibody (with velcro) and undesired antibody (without velcro). What fraction of your solution contains desired antibody?
11. If you had one mole of antibodies in the proportions of those in your solution, how many desired antibodies would you have?
12. Do a quick check of the other test solution. About how many liters of that solution do you need to bind all the antigen? Using that volume and your previous titration, what would you say is the proportion of desired antibody in this solution?
13. Compare this ratio with a lab group that was assigned this solution. How close is your estimation?
14. Write a short paragraph on why we often use moles when we refer to quantities of chemicals.

Teachers Notes

Chemistry Concepts: The mole, Math review
To simplify the graphing of percentages, glue ten velcro antigens onto shoe box bottoms.

1. Each liter of antigen will hold more than thirty-five one inch styrofoam spheres.
2. I recommend preparing test solutions A and B in the ratios of 1:3 and 2:3 binding to total antibodies.
3. To illustrate binding with labeled secondary agents, consider marking each binding antibody with a yellow dot on the side opposite its velcro patch.