Immunology is the study of the immune system and how the body protects itself against disease. Over 100 years ago, biologists found that animals' internal immune systems respond to invasion by

foreign entities or antigens. When an invader enters the body, it provokes an immune response that begins with the production of proteins called antibodies. Like magic bullets, antibodies seek out and attach themselves to invading entities (antigens), flagging the invaders for destruction by other cells of the immune system. The antigenic invaders may be any molecules foreign to the body, including components of infectious agents like bacteria, viruses, and fungi. Today, antibodies have become vital scientific tools, used in biotechnology research and to diagnose and treat disease. The number of different antibodies circulating in the blood has been estimated to be between 10 and 10¹1, so there is usually an antibody ready to deal with any antigen. In fact, antibodies make up to 15% of your total blood serum protein. Antibodies are very specific; each antibody recognizes only a single antigen. You are about to perform an ELISA (enzyme-linked immunosorbent assay). The ELISA relies on antibodies to detect the presence of antigens in liquid samples. Because they are antibody-based, ELISAS are called immunoassays. ELISAS can detect minute amounts of disease agents in samples such as body fluids (before the body has had a chance to mount an immune response). Smallpox virus is an example of a disease agent that can now be detected using an ELISA. If exposure is detected and treated with vaccine within 2-3 days, patients do not develop smallpox. Other applications for ELISA include testing for West Nile virus, HIV coat protein p24, SARS virus, anthrax spores, hormones such as hCG in pregnancy tests, illegal steroids in drug tests, bacteria in food safety tests, and the presence of genetically modified organisms contaminating non- GMO food. Where Is ELISA Used in the Real World? With its rapid test results, the ELISA has had a major impact on many aspects of medicine and agriculture. ELISA is used for such diverse purposes as home pregnancy tests, disease detection in people, animals, and plants, detecting illegal drug use, testing indoor air quality, and determining if food is labeled accurately. For new and emerging diseases like severe acute respiratory syndrome (SARS), one of the highest priorities of the US Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) has been to develop an ELISA that can quickly and easily verify whether patients have been exposed to the virus. Over-the-counter kits that are based on the same principles as this ELISA activity include home pregnancy and ovulation tests, and tests for the presence of illegal drugs like marijuana and cocaine. PROTOCOL II Antigen Detection ELISA/nSome tests give positive or negative results in a matter of minutes. For example, home pregnancy dipstick tests detect levels of human chorionic gonadotropin (hCG), a hormone that appears in the blood and urine of pregnant women within days of Step 1. fertilization. The wick area of the dipstick is coated with anti-hCG antibody labeled with a pink compound (step 1). When the strip is dipped in urine, if hCG is present it will bind to the pink antibody, and the pink hCG-anti- Step 2. body complex will migrate up the strip via capillary action (step 2). When the pink complex reaches the first test zone, a narrow strip containing an unlabeled fixed anti- hCG antibody, the complex will bind and Step 3. concentrate there, making a pink stripe (step 3). The dipsticks have a built-in control zone containing an unlabeled secondary antibody that binds unbound pink complex (present in both positive and Step 4. negative results) in the second stripe (step 4). Thus, every valid test will give a second pink stripe, but only a positive pregnancy test will give two pink stripes. How Are Antibodies Made? Wick TRA 182 Capillary action Test XXX YYY YYY Control AAA AAA 74 YYY Antigen (hCG) Y Colored anti-hCG antibody Y Fixed anti-hCG antibody Fixed secondary antibody When exposed to antigens, all mammals generate an immune response and produce antibodies, proteins that recognize and bind tightly to the specific antigens. Each antibody recognizes only a single antigen. Animals such as goats, rabbits, and mice can be injected with an antigen and, after a period of time, their serum will contain antibodies that specifically recognize that antigen. If the antigen was a disease-causing agent, the antibodies can be used to develop diagnostic tests for the disease. In an immunoassay, the antibodies used to recognize antigens like disease agents are called primary antibodies. PROTOCOL II Antigen Detection ELISA/nAntigen Detection ELISA PROTOCOL II Secondary antibodies recognize nary antibodies noassay. They are prepared by injecting antibodies produced by one species of animal into another species. This works because the antibodies produced by different species are different enough from each other that they will provoke an immune response. For example, if you want a secondary antibody that will recognize a human primary antibody, inject human antibodies into an animal like a rabbit. After the rabbit immune response, the rabbit serum will contain antibodies that recognize and bind to human antibodies. Secondary antibodies are frequently labeled to make them visible. In this experiment, the secondary antibodies you will be working with are conjugated to an enzyme named horseradish peroxidase (HRP); HRP in the presence of its substrate, TMB, produces a blue color. Controls in Immunoassays For any immunoassay to be valid, it must include both positive and negative controls, i.e., samples that will give known results. Controls are always run side by side with experimental samples. If you do not run a positive control and the experiment gives negative results, how can you be sure the results are truly negative? What if the assay simply did not work? If a positive sample gives a negative assay result, it is called a false negative. Conversely, if you do not run a negative control and the experiment gives all positive results, how can you be sure the results are truly positive? What if the assay was contaminated with antigen? If a negative sample gives a positive assay result, it is called a false positive. Controls are also needed to guard against experimental error and to ensure that the assay is working correctly. There can be problems with reagents, which can degrade due to age or poor storage conditions. Operators can make mistakes by choosing the wrong reagents, making errors in dilutions or in pipetting, or failing to remove unbound reagents. Poor record keeping is another source of false assay results. Most of these possibilities can be checked for within the assay with the appropriate controls. Now let's put this all together. The main steps in this antigen detection ELISA are: 1. Add your sample and control samples to the wells in a microplate strip. Your samples contain many proteins and may or may not contain the antigen. Incubate for 5 minutes to allow all the proteins in the samples to bind to the plastic wells via hydrophobic interaction. This is called an immunosorbent assay because proteins adsorb (bind) to the plastic wells./n2. Add primary antibody to the wells and incubate. The antibodies will seek out the antigen from the many proteins bound to the well. If your sample contains the antigen, the antibodies will bind it tightly and remain in the well. 3. Detect the bound antibodies with HRP-labeled secondary antibody. If the primary antibodies have bound to the antigen, the secondary antibodies will bind tightly to the primary antibodies. 4. Add enzyme substrate to the wells, wait 5 minutes, and evaluate the assay results. If the antigen was present in your sample, the wells will turn blue. This is a positive diagnosis. If the wells remain colorless, the antigen was not present in your sample and the diagnosis is negative.