Immunology: The ELISA assay

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1 Biology 2180 Laboratory # 10 BACKGROUND INFORMATION Name Immunology: The ELISA assay Antibodies are specific animal proteins that are produced by white blood cells in response to foreign material. Examples of such foreign material, known as antigens, include infectious agents and various environmental "non-self" materials. Biological antigens are high molecular weight biomolecules such as proteins, carbohydrates and nucleic acids which can be circulating freely or as part of a complex such as a virus coat protein or bacterial cell surface. Antibodies are made in response to antigens. They bind to antigens and play a significant role in the subsequent removal of such materials from circulation. For example, exposure to an infectious agent causes the individual to mount an antibody response which eventually results in plasma antibody molecules that bind to different viral proteins (and/or different areas of the same polypeptide). When an antibody binds to a specific biological antigen, it can recognize specific chemical charges, sequences or structural conformational elements. These structural binding characteristics make up the specific fingerprint of an antigen. Each antibody molecule can bind two antigen molecules. This recognition and binding is highly specific and makes possible the differentiation between two circulating viruses that may be very closely related, as in the case of two strains of the same virus. When an antigen and its antibodies form insoluble complexes, this highly specific binding reaction is known as immunoprecipitation. Precipitation of the complex is the result of various polyclonal antibodies binding to the antigens to form a network. In the traditional immunoprecipitation assay, antibodies are obtained from the serum of an animal exposed to the specific antigen. The serum, also known as plasma, is prepared by the removal of red blood cells. It contains the specific proteins for that particular animal and antibodies against a "non-self" antigen that is introduced in the animal by either design or an infection. Antibodies are purified from animal sera samples and can be used to detect particular antigens, such as human infectious agents. Immunological Screening Test Enzyme linked immunosorbent assay (ELISA) tests were originally developed for antibody measurement. These immunoassays have also been adapted to successfully detect samples that contain antigens. ELISAs are done in microtiter plates which are generally made of polystyrene or polyvinyl chloride. The plates are somewhat transparent and contain many small wells, in which liquid samples are deposited. First, the antigens are added to the wells where some remain adsorbed by hydrophobic association to the walls after washing away the excess. The antigens can be the whole infecting agent, such as a virus or lysate, specific proteins, or a mixture of the two. There is no specificity involved with the adsorption process although some substances

2 may exhibit low binding to the walls. In certain cases the antigens can be covalently cross-linked to the plastic using UV light. After washing away unadsorbed material, the unoccupied sites on the walls of the plastic wells are blocked with a blocking agent such as gelatin, milk proteins or bovine serum albumin. Serum samples from individual patients are then added to each microtiter well. If a particular patient s serum is positive, it will have antibodies that bind to the adsorbed antigens in the wells (see below). These antibodies are called the primary antibodies. If the primary antibody remains in the well when the serum is removed, it can then be visualized using a secondary antibody with an attached enzyme. These secondary antibodies are usually raised in rabbits and goats immunized with the equivalent of the primary antibodies. The secondary antibodies are purified and covalently cross-linked to the enzyme horseradish peroxidase (HRP). This modification (called a conjugate) does not significantly affect the binding specificity and affinity of the antibody or the enzymatic activity of the peroxidase. After washing, a solution containing hydrogen peroxide and an enzyme substrate called azino-di-ethylbenz-thiazoline sulfonate (ABTS) is added to each well. The substrate solution added is nearly colorless. Peroxidase converts the peroxide to H 2 O + O 2 using the ABTS as the hydrogen donor. The oxidized ABTS is green and can be easily observed in positive wells. Colorless Green Enzyme Enzyme Enzyme Enzyme Secondary Antibody Primary Antibody Adsorbed Antigen Elisa Assay

3 Using the ELISA Assay for Disease Detection In our class, at least one person has been infected by a newly discovered strain of the influenza virus called Salt Lake Flu. This is a particularly devastating virus which causes infected individuals to cheer for a hideous football team located about an hour south of Salt Lake City (hence the name). Other people in the lab have similar symptoms but have actually been infected by a much less virulent strain of influenza called 49er Flu. Your task is to determine who in the lab has been infected by these two viruses. Antigen Adsorption 1) Each group should obtain one microtiter plate and samples of antigen from Salt Lake Flu (A1) and from 49er Flu (A2). 2) Add 1 drop from a small transfer pipette of A1 to each of the wells in rows A and B then use a new pipette to add 1 drop of A2 to each of the wells in rows E and F. 3) Incubate the plate at room temperature for 5 minutes. 4) Using a transfer pipette, remove the A1 solution from each well in rows A and B. Using a new pipette, remove the A2 solution from each well in rows E and F. 5) Wash the wells by adding 4 drops Phosphate Buffered Saline (PBS) to each well. You may use the same pipette for all the wells if you don t touch the plate. If you touch the well, change the pipette so you don t contaminate the PBS. 6) Using a single transfer pipette, remove the PBS solution from each A1 well then use a new pipette to remove the PBS solution from each A2 well. 7) With a clean pipette, add 1 drop blocking agent to each of the wells. 8) Incubate 10 minutes at 37 C. 9) Using a transfer pipette, remove the blocking solution from each A1 well. With a new pipette, remove the blocking solution from each A2 well. 10) Wash the wells again by repeating steps 5 and 6. Addition of Body Fluid Sample (Primary Antibody) 1) Each person in the lab should obtain a body fluid sample and add 1 drop to the appropriate microtiter wells for each group. Look on the board to see which wells you should put your body fluid sample into. 2) After everyone in the lab has added their sample to the microtiter wells for your group, add 1 drop positive control to the appropriate well and 1 drop PBS to any leftover wells. Incubate the plate 30 minutes at 37 C.

4 3) Using a single transfer pipette, remove the body fluid samples from each A1 well. Change pipettes and remove the samples from each A2 well. 4) Wash the wells by adding 4 drops Phosphate Buffered Saline (PBS) to each well. You may use the same pipette for all the wells if you don t touch the plate. If you touch the well, change the pipette so you don t contaminate the PBS. 5) Using a single transfer pipette, remove the PBS solution from each A1 well then use a new pipette to remove the PBS solution from each A2 well. Addition of Secondary Antibody 1) Add 1 drop of secondary antibody to each well and incubate 15 min at 37 C. 2) Using a single transfer pipette, remove the secondary antibody solution from each A1 well. Change the pipette and remove the samples from each A2 well. 3) Wash the wells by adding 4 drops Phosphate Buffered Saline (PBS) to each well. You may use the same pipette for all the wells if you don t touch the plate. If you touch the well, change the pipette so you don t contaminate the PBS. 4) Using a single transfer pipette, remove the PBS solution from each A1 well then use a new pipette to remove the PBS solution from each A2 well. Addition of substrate Add 1 drop substrate to each well and watch for color changes over the next few minutes. When finished, place the tray on the white light box to see the color. Questions 1) Make a diagram of your microtiter plate showing what was placed into each well and whether or not the assay gave a positive or negative result.

5 2) Who in the lab has been infected by the Salt Lake Flu? 3) Who in the lab has been infected by the 49er Flu? 4) Briefly describe the role each of the following plays in the ELISA assay. Horseradish Peroxidase ABTS Antigen Secondary Antibody PBS 5) Can an antibody act as an antigen? Explain. 6) What were the positive and negative controls for this experiment? What did they control for?

6 7) Describe at least two ways that the ELISA might give an incorrect result and what you might do to see that they don t happen. References: EDVOTEC experiment #278 Quantitative Elisa Assay THE ELISA ASSAY AN IMMUNOLOGY EXPERIMENT June 2001 Kristi DeCourcy Fralin Biotechnology Center Virginia Tech