Epidemiology, ELISA and HIV

Epidemiology, ELISA and HIV

Scott MacClinticThe Loomis Chafee School

Windsor, [email protected]

Genevieve NelsonGermantown Friends School

Philadelphia, [email protected]

NSTA National ConventionPhiladelphia, PAMarch 21, 2010

mailto:[email protected]

Epidemiology, ELISA and HIV MacClintic and Nelson

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, antigen-

antibody 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 B’s tube, and then student B transfers 1

dropperful of fluid from their tube into student A’s 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 partner’s 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 don’t 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 one’s infection status alter one’s

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 person’s 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 6


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 (Ward’s 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 (µL) 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 µL of TBS into

wells A2-6, B2-6, C2-6 and D2-6 of the microtitration plate. Place

55µL of chicken serum into well A-1, 55µL of cow serum into well

B-1, 55µL of horse serum into well C-1 and 55µL 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 5µL 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 µL from well A2 into A3 and

mix as above. Transfer 5 µL from well A3 into A4 and mix, then

transfer 5 µL 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 kit’s

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 µL 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 kit’s Instructor Guide.

Dispense about 1.5 mL of this solution in a small test tube to each

group. ***

Add 50 µL 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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Epidemiology, ELISA and HIV