Immunology Field Trip-Background

K-12 Programs

Immunology Field Trip Background

Immunology is the study of the immune system. The immune system is a network of cells and organs that produce substances, such as antibodies (Ab), which defend the body against attack by "foreign" substances called antigens (Ag). Antigens can be viruses, bacteria, proteins or other foreign compounds. When the immune system works properly, it protects your body against infections. If the immune system malfunctions, it can produce undesirable effects, such as minor allergies, or, occasionally it can result in serious, autoimmune diseases. In autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis (MS), the antibodies attack the body's own organs.

Antibodies, also called immunoglobulins, are protein molecules produced by the B-lymphocytes (white blood cells) that bind very tightly and specifically to the antigens they recognize. Medical tests have been developed that use this specific binding of antibody and antigen (Ab:Ag) to diagnose infectious diseases by detecting the presence of the relevant antibody or antigen in the patient's blood. Commercial antibody production involves injecting the specific antigen into an animal such as mice, rabbits, goats, etc. After several weeks, the animal should be producing antibodies against the antigen and blood is drawn from the animal. The serum containing the antibodies is separated from the blood cells and the specific antibodies are further purified. This process produces only the antibodies against a specific antigen, which can then be used in a diagnostic test to detect the presence of that antigen.

The BTCI immunology field trip uses a dot blot. Dot blots are crude assays to quickly determine the presence or absence of a protein. In this experiment, IgYs are isolated from egg yolk. The IgYs are deposited in the yolk by the hen to provide the developing chick with a passive immunity to pathogens to which the hen has been exposed and which therefore are likely to be present in the chick’s environment. In this case, E.coli and Salmonella antigens have been spotted on a filter to create a “dot blot”. E.coli and Salmonella are two common antigens found in many environments, especially chicken coops. The isolated IgYs are incubated with the dot blot. If the hen was exposed to E.coli or Salmonella, IgYs should bind the antigens. Because the IgYs are not labeled a second antibody is used to trace them. The secondary antibody was raised against IgYs by injecting IgYs into a goat. The secondary antibody is labeled with a colorimetric reagent. The secondary antibody will bind any IgYs bound to E.coli or Salmonella antigen. Presence or absence of IgYs recognizing E.coli or Salmonella antigen will be determined when the colorimetric label is activated.

In addition, we will discuss how clinical laboratories use antibodies to detect various infectious diseases. If you have any questions about this field trip, please give us a call, or bring your questions along and we can discuss them during the lab. Thank you for your interest in BTCI's Biotechnology Field Trips program.

References for Antibodies:

Antibody Diversity (http://www.aw-bc.com/mathews/ch25/c25gad.htm)

Everything and then some (http://antibodyresource.com/educational.html)

Computer graphics of Antibody structure (http://www.path.cam.ac.uk/~mrc7/mikeimages.html)

Laboratory Activity:

In this lab, students will purify IgY antibodies from chicken egg yolks. The antibodies in the egg yolk are made naturally by the chicken against bacteria in her environment, such as E. coli and Salmonella. The hen passes on antibodies to these bacteria in her eggs. The isolated IgY antibodies will be used to test whether the hen was exposed to E. coli or Salmonella by using a dot blot assay.

Part 1: Purify Antibodies from the Chicken Egg Yolk

A. Remove large proteins and lipids from the yolk. Do this using low concentration Polyethylene Glycol (PEG).

B. Use a higher concentration of PEG to precipitate the smaller IgY proteins from the rest of the solution.

Part 2: Detect the Antigens.

A. E. coli, Salmonella, and a positive control protein have been blotted onto a membrane and permanently bound to that membrane.

B. Using two antibodies, one that students purify and one that was made specifically to allow us to "see" the proteins, students will detect the antigens on the membrane. This immunodetection technique is referred to as a “dot blot”.

Dot Blot Filter Secondary Antibody Binding

FOR MORE INFORMATION

Contact: Barbara Bielec, 608-273-9737