Teacher Tips Sheet:  Immunoassay Experiment

Immunoassay is a technique that has been used since the 1940s. It is basically an antigen-antibody test. It relies on the fact that each antibody is specific for a particular antigen (in this case human blood) due to the unique features of an antigen’s charge and 3-D shape. In this technique when the antibody binds to its specific antigen, a visible band of precipitate forms. In this experiment, the term “antiserum” refers to the antibody – cat antiserum is antibody that reacts to cat serum; human antiserum is antibody that reacts with human serum. These antisera would come from commercial sources to be used for testing purposes.

The experimental setup uses an agar plate as a diffusion medium and the agar contains holes in it (wells) for the reagents. You are going to carefully place the test substances into the marked wells, allow diffusion of substances to take place, and look for a line of precipitation to link the unknown substance to its specific antibody. The experiment also contains controls in section A to show that diffusion will take place and in section B to show that human serum reacts with human antiserum but NOT with cat antiserum – i.e. the reaction is specific.

Credits:  This laboratory is adapted from the Shoestring Biotechnology project of NABT. 

Further Explorations:

You can do a sensitivity experiment to test how far you can dilute the Human serum and still get a positive reaction with Human antiserum. Start by diluting HS 1:2 (1 ml HS in 1 ml water), (or 1:4, or 1:10) then dilute again, in a series, and determine at what dilution can the HS still be detected by Human antiserum. This type of testing is important in diagnostics to quantify antigens or antibodies against a known standard.

You could measure diffusion rates by making precise measurements of distances between wells and time till precipitation. You could also test rates of diffusion at different temperatures

Suggestions by Jana Jean, Sante Fe South HS

• Cut straws down into thirds.  This will provide more suction for removal of the agar plugs.
• Place Petri dishes on overhead projector to show students how their plate should look.
• If you are pressed for time and would like to get results more quickly, place the loaded Petri dishes in an incubator (if you have one).

SETUP

Agar Plates (makes about 65 plates of 2% agar)

Add 20 g. agar to 1 liter distilled water, stir while bringing to boil till completely dissolved. Pour about 15 mL of agar solution into bottom half of Petri plate. Let plates solidify at room temperature. May be placed in sealed bag and stored in refrigerator for several weeks. Do not freeze.

2 M saturated Calcium chloride: 58.8 g calcium chloride in 200 mL distilled water- mix well, store at room temperature.

2M saturated potassium monohydrogen phosphate: 69.6 g. potassium monohydrogen phosphate in 200 mL distilled water – mix well, store at room temperature.

To prepare Simulated Sera and Antisera, use dropper bottles as follows:

· Human Serum (HS): fill with 2M saturated calcium chloride

· Unknown Serum (?S): fill with 2M saturated calcium chloride

· Dog Antiserum (DA): fill with distilled water

· Cat Antiserum (CatA): fill with distilled water

· Rooster Antiserum (RA): fill with distilled water

· Human Antiserum (HA): fill with 2 M saturated potassium monohydrogen phosphate

Also need yellow and blue food coloring.