Investigating the Antibody:
An ELISA Laboratory Investigation

Developed by: Peter Seymour
© American Association of Immunologists 1999

Overview
This curriculum is an immunology lab that has several purposes. Primarily it will serve as an effective way in which to introduce students to the structure and function of the immune system. The lab procedure investigates the specific binding of antibodies to their targets, and exploits our knowledge of other biological systems to gain insight into this elusive event. Because of the complex nature of the interactions of the many parts of the immune system, it will require that student shave had exposure to a significant amount of basic biology prior to taking on this lab. Specific areas of knowledge that will be required are talked about in a later section. Since this curriculum draws on many different areas of knowledge, it will serve effectively as a capstone event for a biology class. It will require that a student recall, integrate, and apply knowledge gained over the course of the year, in order to understand fully the interactions of the many parts of the immune system.

The lab section of this curriculum uses materials and protocols similar to those found in modern immunology research facilities. It will emphasize the structure of a well-designed experiment, using positive and negative controls and will reward the student for paying close attention to detail. The lab is a derivative of an ELISA Protocol, which is a powerful tool for clinical diagnostic work, as well as scientific research. Because this is a protocol that is regularly used in a number of different areas of medical science, students will be able to be involved with an activity that is currently being used in laboratories. In addition to the physiological information that will be learned, any discussion of the immune system immediately brings to light issues that are prevalent in today's society. Topics such as AIDS and hepatitis, bacterial infections, antibiotics, antibiotic resistance and vaccines are all issues that can tie the investigation of the immune system directly to the students' lives.

Objectives
The lab experience will offer several things to the student. First, it will provide for them a first hand demonstration of the specific nature with which antibodies bind to proteins, including other antibodies. But this lab will also give students several specific skills. Each student will be able to:

1. determine volumes;
2. accurately dispense small volumes of fluid using a micropipette or other instrument;
3. observe and interpret complex interactions between antibodies;
4. map concepts of the immune response and of the lab procedure to show integration of knowledge;
5. identify the function of positive and negative controls in a controlled experiment.

Suggested Time
Students should be given time to read about the immune system. A cursory understanding of what an antibody is and what it does will be necessary for the students to understand what he/she is doing on the first day. Reading assignments can augment discussion/question over the two to three days in which the students perform the actual lab.

There are two ways in which to perform this lab and the way in which you choose will depend upon your class time constraints. There are three distinct areas on the lab, each of which are separated by a rather lengthy incubation periods (typically overnight).  

Day One: 

1. coating the plate with primary antibodies (need to incubate overnight)

Day Two: 

1. blocking with bovine serum albumin (BSA)
2. adding secondary antibodies

Day Three: (this will require a double period)

1. add third antibody and an hour-long incubation followed by addition of the substrate, which is transformed into product in 20 minutes.