Epidemiology, ELISA and HIV

Scott MacClintic The Loomis Chafee School Windsor, CT Scott_MacClintic@loomis.org Genevieve Nelson Germantown Friends School Philadelphia, PA Gen_Nelson@gfsnet.org NSTA National Convention Philadelphia, PA March 21, 2010

Epidemiology, ELISA and HIV

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Introduction:

Epidemiology is the study of the incidence,

distribution and control of disease within a population. Exploring this topic offers numerous opportunities to study the relationships between humans and microbes, diagnostic techniques, antigenantibody interactions and other important biological concepts. This workshop will be divided into three parts: a simulated outbreak of an infectious disease, and Enzyme linked

Immunosorbent Assay (ELISA) and a discussion of how the ELISA is used to diagnose HIV infection.

I. Outbreak! Identifying the source of an epidemic is critical to controlling the disease and protecting public health. the following simulation can be used to give students direct experience with the problems inherent in finding the index case of a propagated epidemic. In order to make this activity meaningful to my students, I place it in the context of HIV, but it could work as a simulation of the spread of any infectious disease through a population. How much or how
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little background information you reveal to your students before doing this activity will depend on what you want them to get out of it. In its simplest form, it is merely a puzzle to be solved. Framed by a more specific storyline, it becomes a public health problem that students must work through in order to find patient 0. It can also be modified to include behavioral risk factors for contracting HIV. Procedure: Collect enough CLEAN test tubes and droppers so that you have one test tube and dropper for each student in the class. Place 2-5 mL 0.2M NaOH in one test tube and place an equal volume of water in all the others. Distribute (or let students pick) one tube and dropper to each student. Keep your eye on the NaOH tube and make a mental note of which student picks it up. Explain that one of the tubes is infected with a pathogen (I use HIV) and that the class is going to simulate an epidemic, and then try to determine who the initial case was. This pathogen is transmitted through bodily fluids. Instruct students to perform reciprocal fluid exchanges with 3 people. (If you have more than 25 students per class, you may want to increase the number of
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exchanges to 4 or 5). A reciprocal exchange is accomplished by student A transferring 1 dropperful (at least 1 mL) of fluid from their tube into student Bs tube, and then student B transfers 1 dropperful of fluid from their tube into student As tube. Students might ask if this exchange must occur sequentially or

simultaneously, and it is well worth having them discuss as a class whether or not that will make a difference to their ability to find the source of the epidemic. You might also wish to assign some students specific roles to play, such as a monogamous couple, one or 2 promiscuous people, one or two people who practice safe sex (i.e., never actually transfer their fluid to their partners tube), and one or two people who practice abstinence. Answer any questions about the procedure and instruct students to return to their seats after completing their exchanges. How much advice you give them at this point is up to you. For example, you might want to suggest that it would be worthwhile for them to remember with whom they exchange fluids and in what order those exchanges occur, or you might choose not to offer any suggestions at all. When the exchanges are complete, circulate
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around the room and add 1 or 2 drops of phenolthalein solution to every test tube. Those tubes that turn purple are positive for the pathogen, those that remain clear are negative. Ask students to determine who the source of the epidemic was and then step back and let them work it out. Try not to guide their deliberations in any way. Just say that once they think they have identified the index case they will have to explain their conclusions to you and provide relevant evidence to support their opinion. Once the students have made their case, you may tell them whether or not they are correct (but you dont have to!). Follow Up Questions: Depending on how much time you want to spend on this, there are several questions worth exploring, and the simulation can be repeated several times to investigate different variables such as... How does the number of exchanges each person participates in affect the final number (or percentage) of people infected? How does the number of initial infected people affect the final number (or percentage) of people infected? How does the mode of transmission affect the spread of the
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disease? What if this pathogen were airborne instead of fluid borne? How might knowledge of ones infection status alter ones behavior? In this scenario, no one knows who is infected until the end, but what if people could go get tested after one or 2 exchanges? This discussion can be expanded to include ethical issues related to testing and disclosure of test results. How does anonymous testing and reporting differ from name-based reporting? Would name-based reporting discourage people from getting tested? Besides the patient, who has a right to know a persons status after a test has been performed?

II. Enzyme Linked Immunosorbent Assay I use the simulation above as an introduction to a unit on infectious disease. Later in this unit, my students do this lab in which they use a labeled antibody to probe for the presence of specific serum proteins. ELISA is a common diagnostic technique which has a wide variety of applications, including home
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pregnancy tests and HIV testing. This lab would be equally appropriate as part of a unit on immunology, since the central concept involved is the specificity of antigen-antibody

interactions. Background: An antigen is a molecule or part of a molecule that is capable of eliciting an immune response. An antibody is an immunoglobulin, or immune system protein that is produced in response to a specific antigen. Although all antibodies have some structural similarities (which are used to classify them into isotypes, such as IgG, IgA, IgM, IgD and IgE), the parts of these molecules that interact with antigens are highly variable, and this variability enables them to detect different antigens. Since antibodies are themselves proteins, they can also serve as antigens. In other words, an antibody from one species will act as an antigen in another species. For example, injecting antibodies isolated from rabbit serum into a horse will cause the horse to produce antibodies of its own which will bind to the rabbit antibodies. Furthermore, antibodies can be connected to (or conjugated with) other compounds such as enzymes that, when
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exposed to the proper substrate, form a colored product, thus enabling one to detect the presence (or absence) of a certain antibody and its corresponding antigen. This particular procedure uses serum proteins from four different animals (chicken, cow, horse and rabbit) as the primary antigen. These antigens are serially diluted in a polyvinyl chloride (PVC) plate so that the sensitivity of the assay can be quantified. The antibody used to detect these antigens is goat anti-rabbit IgG conjugated to horseradish peroxidase (GAR-HRP). This antibody was produced by injecting rabbit IgG into a goat, isolating the antibodies the goat made in response to the rabbit IgG and then conjugating those antibodies to the enzyme horseradish

peroxidase (HRP). As previously stated, all antibodies have some degree of structural similarity. The central question in this lab is: How structurally similar are antibodies isolated from different species? It is reasonable to suggest that the more closely related two species are, the more structurally similar their antibodies will be. If that is true, then it is possible that a secondary antibody (such as
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GAR-HRP) made to one of them will also cross-react with the structurally similar antibody from the other species. On the other hand, lack of cross-reaction indicates that antibodies are

extremely specific and can distinguish between two similar molecules. This specificity is the basis of many of the therapeutic applications on antibodies. For example, antigens that are unique to cancer cells can be used as targets for antibodies that have been conjugated to toxic compounds. In this situation, the antibody-toxin complex acts as a magic bullet by delivering the toxin only to cancer cells. This immunotherapy has considerably fewer side effects than other chemotherapies which target all rapidly dividing cells, not just cancer cells. Procedure: All materials for this activity can be purchased from Modern Biology of West Lafayette, Indiana (1-800-733-6544); The kit is Catalog number IND-3 (about $65), and includes sufficient materials for 16 groups of students working in pairs. The entire activity takes about 2 hours, but there are places where the procedure may be stopped overnight without compromising the results. the kit includes background information about ELISA and
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other immunoassays as well as study questions. The only necessary materials that are NOT provided with the kit are microliter dispensers. Clean 1 mL beral pipets (Wards Catalog #18W2971. $17.75 for a case of 500) work fine for most of the procedure, but a microliter dispenser capable of measuring volumes of 5 microliters (L) is necessary for the first step. Modern Biology sells inexpensive graduated capillary tubes and plungers that are perfect for this (catalog # 6-7-4, $43.46). Pre-Lab Preparation: Cut the 96-well microtitration plates into quarters using a sharp scissors. Dilute the Tris Buffered Saline

(TBS) and TBS+Nonidet-40 (TBS+NP-40) as directed in the Instructor Guide. Prepare the TBS-Gelatin solution as directed by adding the 6g of gelatin to 300 mL boiling TBS and stirring until the gelatin dissolves. Do not prepare the Color Development solution and the Goat anti-Rabbit IgG-Peroxidase until just before they are needed. Each pair of students will need: 1/4 section of a microtitration plate, 3-4 pipets, small test tubes containing 5 mL TBS, 13 mL TBS-Gelatin, 20 mL TBS+NP-40, 10 mL distilled water, 1 250 mL
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beaker for discarding liquid, 1 250 mL beaker containing water for rinsing pipets. Using a pipet or microliter dispenser, place 50 L of TBS into wells A2-6, B2-6, C2-6 and D2-6 of the microtitration plate. Place 55L of chicken serum into well A-1, 55L of cow serum into well B-1, 55L of horse serum into well C-1 and 55L of rabbit serum into well D-1. Perform serial ten fold dilutions of the sera so that the final concentrations are 1% in A1, B1, C1 and D1, 0.1% in A2, B2, C2 and D2, 0.01% in A3, B3, C3 and D3, 0.001% in A4, B4, C4 and D4, 0.0001% in A5, B5, C5 and D5, and 0.0% in A6, B6, C6 and D6. To perform these dilutions, transfer 5L of the serum from well A1 into well A-2. Mix the contents of well A-2 by drawing it into the pipet and expelling it back into the well a few times or by shaking the plate gently. Transfer 5 L from well A2 into A3 and mix as above. Transfer 5 L from well A3 into A4 and mix, then transfer 5 L from well A4 into A5 and mix. Do NOT place any serum in A-6 (this is a negative control). Repeat this procedure to dilute the sera in rows B, C and D. Let the plate sit undisturbed at room temperature for about 20 minutes to allow the antigens to
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adsorb onto the plastic of the plate. ***This dilution procedure can be fairly time consuming. If you feel it is important for your students to perform this part of the procedure, it will take about 30-40 minutes (a whole class period). Cover the plates with plastic and refrigerate them overnight. I prefer to perform the dilutions and the 20 minute incubation BEFORE my students arrive so that they can continue from here.*** Add 2 drops of TBS-gelatin to each well using a pipet. Keep this pipet in the TBS-gelatin solution. The gelatin blocks the sites on the plastic that do not have serum proteins bound to them. In other words, it prevents non-specific binding of the secondary antibody (goat anti-rabbit IgG). ***While your students are doing this step, prepare the Goat anti-Rabbit IgG conjugated to peroxidase as directed in the kits Instructor Guide. Dispense about 1.5 mL of this solution in a small test tube to each group. *** Remove all the liquid from the wells in reverse order: from A6 to A1, B6 to B1 and so on. Place this discarded liquid in an empty
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beaker. Discard this pipet. Use the TBS-gelatin pipet to add 3-4 drops of TBS-gelatin to each well. Let sit for about 3 minutes, then discard this solution by flipping the plate over on a paper towel and tapping it gently. This step washes any unbound serum proteins out of the well. Add 50 L of Goat anti-Rabbit IgG conjugated to peroxidase to each well of the plate and shake the plate gently to ensure that all surfaces are in contact with the antibody solution. Allow 15-20 minutes for the antibody to bind to the immobilized antigens. ***If necessary, the plates may be covered with plastic and refrigerated overnight at this time.*** Add two drops of TBS-gelatin to each well and then empty the plate by flipping it over on a paper towel and tapping it gently. Immediately wash the plate with 3-4 drops of TBS-gelatin and then empty the wells again. Wash the plate THREE times with 3-4 drops of TBS+NP-40 solution and then wash once with 3-4 drops of water. These washes remove any excess unbound antibody from the plate. If the plate is not adequately washed, false positive results will occur because of residual goat anti-rabbit IgG in the
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plate. ***While your students are doing this step, prepare the Color Development Solution as directed in the kits Instructor Guide. Dispense about 1.5 mL of this solution in a small test tube to each group. *** Add 50 L Color Development Solution to each well. Wait for 10-15 minutes and then observe the intensity of the blue color, which is produced by an insoluble product of the peroxidase reaction. Record the relative intensity of the blue color in each well in your data chart. Use + for the lightest wells and +++ for the darkest wells. If you would like to collect quantitative data, add 1 drop of 0.1N HCl to each well. This turns the blue product yellow. Transfer the contents of each well to a spectrophotometer cuvette containing 2 mL of water. Set the wavelength to 450 nm and adjust the spectrophotometer to 0% transmittance (100%

absorbance). Insert a cuvette of water into the spectrophotometer and adjust it to 100% transmittance (0% absorbance). Using this solution as a blank, read and record the absorbance of the
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solutions from the plate. Discussion Questions: 1. What do your results suggest about the degree of structural similarity between serum proteins from different species? 2. Does this evidence suggest a close evolutionary

relationship between rabbits and horses, cows and chickens? Explain. 3. Knowing that the concentration of IgG is 10 mg/mL, estimate the lowest concentration of IgG that is detected in this assay. This is a measure of the sensitivity of this assay. 4. How might this technique be used to diagnose Human Immunodificiency Virus (HIV) infection? Explain how a false

negative test might result. Why are false negatives more common than false positives when using ELISA to diagnose HIV?

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