PROTOCOL I
TRACKING DISEASE OUTBREAKS
Protocol I: ELISA for Tracking Disease Outbreaks
Instructor’s Guide
This interactive procedure provides a context for introducing a real-world, topical application
of ELISA. Students first model the spread of disease in a population by the sharing of
simulated “body fluids”. Each student is given a sample to share, one or two of which are
positive for the “disease agent”. After students share their “body fluids”, they assay their
shared samples using ELISA.
Students’ ELISA results reveal that a large portion of the class now tests positive for the
disease! This leads to a guided, inquiry-based activity about how the disease has spread
through the population. Because students have a personal connection to the results, this
activity tends to captivate students’ imaginations and is particularly relevant in light of the
recently emerged contagious disease SARS.
Many other diseases also work with this protocol, including West Nile virus, HIV, colds,
influenza, and STDs, to name a few. A review of Appendix C will provide useful information
about appropriate diseases and scenarios for implementation in the classroom. A simple
approach may be to leave the actual disease unspecified for a generic lesson.
Implementation Timeline
Lesson 1 Set the stage Lecture and discussion
Lesson 2 Sharing of simulated body fluids ELISA lab
Lesson 3 Analyze ELISA results Tracking exercise
12
, TRACKING DISEASE OUTBREAKS
Instructor’s Laboratory Overview
Step 1: Students share “body fluids” by mixing their sample with those of other students.
Within each pair of students who share, each student takes back half of the combined sample.
Each student repeats the sharing process with a different student either 1 or 2 more times
(depending on class size) and records sharing partners. Note: To ensure dissemination of the
“disease”, the sharing must be performed in two or three separate rounds.
PROTOCOL I
750 µl
Student A Students A+B Students A+B+C
Student B Student C Student D
Step 2: Using a pipet, 50 µl of each student’s sample (unknowns)
along with positive and negative controls are added to the wells of
the microplate strip and incubated for 5 minutes, allowing proteins in
the sample to bind to the wells. The wells are rinsed with wash
buffer (PBST: phosphate buffered saline containing 0.05%
Tween 20) that also blocks the unoccupied protein binding sites in
the wells.
Step 3: Primary antibody (50 µl) is added to each well of the
microplate strip and incubated for 5 minutes at room temperature.
The primary antibody is an antibody that recognizes and binds to
the “disease agent”/antigen. The wells are rinsed with wash buffer
to remove unbound antibody.
Step 4: Horseradish peroxidase (HRP)-labeled secondary antibody
(50 µl) is added to each well and incubated for 5 minutes at room
temperature. The secondary antibody is antibody that recognizes
and binds to the primary antibody. HRP is an enzyme that will
oxidize a color-producing substrate. Wells are rinsed with wash
buffer to remove unbound secondary antibody.
Step 5: The enzyme substrate (50 µl) is added to each well and
students watch color development. If HRP is present (meaning that
the antigen was present in the sample), the solution in the wells will
turn blue within 5 minutes. If the antigen was not present in the
sample, the wells will remain colorless.
13
,PROTOCOL I
TRACKING DISEASE OUTBREAKS
Typical ELISA results.
Using Students’ ELISA Results to Track the “Disease”
The number of positive tests in the class results will depend on how many positive
samples you released at the beginning. You can now track the progress of the disease
through your class.
You may want the students to work out a method to track the disease to its source by
themselves. For a more guided inquiry approach, we recommend using the class results
table on page 32 in the student manual. Make a transparency from the page with the
students' names listed and place it on an overhead projector. Ask each student to come up
and write a plus (+) or a minus (-) in the second column to indicate if their ELISA tested
positive or negative. Then, depending on whether their ELISA tested positive or negative,
they should also write a plus (+) or a minus (-) respectively next to the names of the students
with whom they shared their sample.
For example, if Kiko tested positive and shared with Alexander, Florence, and Mustafa, she
would write a “+” by Alexander, Florence’s, and Mustafa’s names.
The students with all pluses against their names will be revealed as early sources of the
infection.
Question: Why will the class not be able to track the infection to a single student?
Answer: When a single student who is the primary source of infection first shares his or
her sample with a second student, the second student will also have all pluses. This is
representative of the kind of problem that epidemiologists face in the real world. You may
turn this occurrence to your advantage by discussing why epidemiologists investigate many
factors when tracking diseases, such as patients’ locations, histories, and behaviors, in
addition to testing for the infection. You may also have your students perform a more
detailed analysis involving tracking the order in which the samples were shared and deducing
if some of the students can be eliminated from the pool of students suspected as being the
original source.
Epidemiologists rarely have patient samples prior to the outbreak of infection, and rarely
are they able to track an outbreak to a single source. However, you have the advantage of
keeping a record of which students received the infected samples, which for the sake of
this exercise may prove helpful. Alternatively, for a more anonymous approach, you may
sequentially number all the student samples and record the numbers of tubes that are
“infected”. The source can be revealed at the end of the activity to see if it matches your
students’ data analysis.
14
, TRACKING DISEASE OUTBREAKS
Instructor’s Advance Laboratory Preparation
This section is designed to help you prepare for the laboratory efficiently. We recommend that
you read this section of the manual (Protocol I: Tracking Disease Outbreaks) in its entirety
before beginning your preparation. In addition, if you are choosing to perform a scenario-based
activity (for example, HIV testing), we recommend using the information given in Appendix C
to help plan your lesson.
The most important thing for the students to do is to put the correct components in the
PROTOCOL I
assay wells in the correct order, so having the tubes clearly labeled and properly color-coded
is crucial to a successful outcome.
Objectives
Step 1. Prepare buffers
Step 2. Rehydrate the freeze-dried antigen, primary antibody, and secondary
antibody to make 50x stocks
Step 3. Dilute 50x stock solutions
Step 4. Dispense reagents for student workstations
Step 5. Set out student workstations
Time Required 1–3 hours
Preparation Timeframe
We recommend rehydrating and diluting the antigen and primary antibody no more than
3 days before the lesson, and the secondary antibody less than 24 hours before the lesson.
We also suggest using sterile distilled water to prepare the 1x PBS to avoid contaminating
rehydrated reagents. These reagents must be kept on ice or in the refrigerator if prepared
more than 4 hours before the lesson.
Note: If you are planning to use this kit for multiple lab sessions over a 1- or 2-week
period, we strongly suggest using sterile water to prepare the PBS buffer in order to
avoid contaminating reagents. (Water can be sterilized by boiling it in a microwave oven
for 5 minutes in a loosely capped bottle; after you remove the bottle from the microwave
oven, let it cool, then secure the cap.) Dilute only as much concentrated antibody and antigen
as required for each lab session. The rehydrated antibodies are 50x concentrates. Store
the remaining concentrated antigen and antibodies in the refrigerator at 4°C. We do not
recommend storing the concentrated antibody and antigen for more than 2 weeks, even at
4°C. Do not freeze the solutions.
Volume Measurements
This kit contains graduated disposable plastic transfer pipets (DPTPs) to use for preparing
some of the reagents where volumes between 250 microliters (µl) and 5 milliliters (ml) are
required. In addition, adjustable- or fixed-volume micropipets are needed to measure 50 µl
volumes. The illustration shows the marks on the DPTP corresponding to the volumes to
be measured. Volumes over 1 ml will require multiple additions. For each step of the laboratory
preparation, use a fresh DPTP or a fresh pipet tip.
1 ml
750 µl
500 µl
250 µl
100 µl
Measuring liquids that contain detergents that foam (e.g., the wash buffer) requires that you
read the volume at the interface of the liquid and the bubbles.
15
,PROTOCOL I
TRACKING DISEASE OUTBREAKS
PROTOCOL I: Step-by-Step Instructor’s Advance Preparation Guide
These instructions are for the setup of 12 student workstations of 4 students each.
Supplied Reagents Quantity
Antigen, chicken gamma globulin, freeze-dried 1 vial
Primary antibody, rabbit anti-chicken polyclonal antibody, freeze-dried 1 vial
Secondary antibody, goat anti-rabbit antibody conjugated to (HRP),
freeze-dried 1 vial
HRP enzyme substrate (TMB) 1 bottle
10x phosphate buffered saline (PBS) 1 bottle
10% Tween 20 1 bottle
Required Reagent
Distilled water, sterile is recommended, see note on page 15 1L
Step 1. Prepare buffers.
We recommend you use a 100 ml and a 1 liter (L) graduated cylinder for preparing the
buffer solutions. You will also need 1 L of distilled water.
Buffer Volume Reagent Used for
1x PBS, 100 ml 90 ml Distilled water • Rehydrating antigen,
10 ml 10x PBS Primary and secondary
antibodies to make 50x
reagent stock solutions
• Diluting 50x antigen to make
positive control and “infected”
student samples
• Negative control
• Negative student samples
Wash Buffer, 900 ml 805 ml Distilled water • Dilution of 50x antibody
stocks
90 ml 10x PBS • Plate washing
4.5 ml 10% Tween 20
Step 2. Rehydrate the freeze-dried antigen, primary antibody, and secondary
antibody.
Carefully remove the stoppers from the three freeze-dried reagents and use a fresh DPTP to
add 0.5 ml 1x PBS to each. Close the stoppers and shake to mix. These solutions are 50x
concentrates, or stock solutions. NOTE: You must not use wash buffer in this step.
Freeze-Dried Reagent Protocol for 50x Stock Solution Used for
Antigen Add 0.5 ml of 1x PBS to vial • Positive control
• “Infected” student samples
Primary antibody Add 0.5 ml of 1x PBS to vial • Primary antibody
Secondary antibody Add 0.5 ml of 1x PBS to vial • Secondary antibody
16
, TRACKING DISEASE OUTBREAKS
Step 3. Dilute 50x stock reagents.
Label one 30 ml bottle for each of the diluted solutions below. Use a fresh DPTP to add the
contents of the appropriate 50x concentrated stock to the corresponding 30 ml bottle.
Diluted solution Volume Reagent Used for
Positive control (1x antigen), 7.5 ml 1x PBS Positive control
label one 30 ml bottle 150 µl 50x antigen
stock
PROTOCOL I
NOTE: you must not add any buffer containing Tween 20 to
the antigen, or the experiment will not work.
1x primary antibody, 24.5 ml Wash buffer Primary antibody
label one 30 ml bottle 0.5 ml 50x primary
antibody stock
• Use the DPTP to rinse out the vial with some of the diluted
reagent to ensure that all of the stock solution is used.
• Close the cap and shake to mix.
1x secondary antibody, 24.5 ml Wash buffer Secondary antibody
label one 30 ml bottle 0.5 ml 50x secondary
antibody stock
• Dilute the secondary antibody less than 24 hours before the start
of the lesson. Use the DPTP to rinse out the vial with some of the
diluted reagent to ensure that all of the stock solution is used.
• Close the cap and shake to mix.
Step 4. Dispense reagents for student workstations.
Tubes Description Label Contents (Each Tube)
Violet tubes, 12 Positive control “+” 0.5 ml positive control solution
(1x antigen)
Blue tubes, 12 Negative control “–” 0.5 ml 1x PBS
Green tubes, 12 Primary antibody “PA” 1.5 ml 1x primary antibody solution
Orange tubes, 12 Secondary antibody “SA” 1.5 ml 1x secondary antibody solution
Brown tubes, 12 Enzyme substrate “SUB” 1.5 ml HRP enzyme substrate (TMB)
Note: TMB is light sensitive, so it is important to use the dark tubes to store this
reagent.
Yellow tubes, # “Infected” student Determined 100 µl 50x antigen stock solution
depends on # of sample(s) (6.6x antigen) by 650 µl 1x PBS
students (1–3 instructor
tubes)
The number of students in the class will determine the number of “infected” samples
you mix with the blanks. For a result where about half the students become infected,
we recommend making one infected sample per 16 students. (Note: if your class is
less than 10 students, use a single sample and perform just 2 rounds of sharing.)
Note: You must not add any buffer containing Tween 20 to the antigen, or the
experiment will not work. You may want to keep the infected samples separate
from the blank samples until the lesson to keep track of who receives them.
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,PROTOCOL I
TRACKING DISEASE OUTBREAKS
Tubes Description Label Contents (Each Tube)
Yellow tubes, # Blank (uninfected) Determined 750 µl 1x PBS
depends on # of student samples by
students instructor*
Make enough blank yellow tubes for your student number minus the "infected"
samples. Note: You must use 1x PBS and not wash buffer for the blank
students samples, or the experiment will not work.
* (Optional) For your own information, you may wish to number each tube of student sample (infected
and blank) from 1 to 48 and record which numbered tubes contain the infected samples.
Step 5. Set out student workstations.
Student Workstation Checklist
One workstation serves 4 students.
Item (Label) Contents Number ✔)
(✔
Yellow tubes Student test samples (0.75 ml) 4 (1 per student) ❑
Violet tube (+) Positive control (0.5 ml) 1 ❑
Blue tube (–) Negative control (0.5 ml) 1 ❑
Green tube (PA) Primary antibody (1.5 ml) 1 ❑
Orange tube (SA) Secondary antibody (1.5 ml) 1 ❑
Brown tube (SUB) Enzyme substrate (1.5 ml) 1 ❑
12-well microplate strips 2 ❑
50 µl fixed-volume micropipet 1 ❑
or 20–200 µl adjustable micropipet
Yellow tips 10–20 ❑
Disposable plastic transfer pipets 5 ❑
70–80 ml wash buffer in beaker Phosphate buffered saline 1 ❑
with 0.05% Tween 20
Large stack of paper towels 2 ❑
Black marking pen 1 ❑
Note on sharing protocol: Make sure that the students share “body fluids” with students
from other parts of the room, not just with their near neighbors. The best way to do this is to
have orderly sharing: Tell the students to share with one other person, then return to their
lab station. After all students are finished with the first sharing and are back in place, then
tell them to share with a different person. The degree of sharing will depend on your class
size. We recommend one "infected" sample per 16 students. If your class size is less than
10, use a single "infected" sample and perform just two rounds of sharing.
Stopping points: Although this procedure is designed to fit into a single lesson period, you
may stop the laboratory activity after sharing the “body fluids” and place all the reagents in
the refrigerator at 4°C overnight. Alternatively, if you wish to stop during the ELISA you may
add wash buffer to the microplate wells at any stage after the addition of antigen and prior
to the addition of enzyme substrate. Place the microplate strips and all the reagents in the
refrigerator at 4°C overnight.
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, TRACKING DISEASE OUTBREAKS
Setting Up The Activity to Test for a Specific Disease (e.g., HIV)
Appendix C provides information on a variety of diseases that can be diagnosed using
ELISA. In addition, for each disease, we provide a table describing what the reagents for
the activity represent in a real-world diagnostic ELISA. Below is an example of a diagnostic
test to detect HIV viral proteins in a patient's blood sample.
Detecting p24 HIV-1 Capsid Protein.
PROTOCOL I
Tube Actual Tube
Tube Description Color Contents Simulated Tube Contents
Student samples Yellow 1x antigen or Sample derived from patient's blood
(unknowns) 1x PBS
Primary antibody Green 1x primary Anti-p24 capsid protein antibody from
antibody mouse
Secondary Orange 1x secondary Anti-mouse immunoglobulin antibody
antibody antibody conjugated to HRP
Positive control Violet 1x antigen Heat-inactivated viral antigen (p24 protein)
Negative control Blue 1x PBS HIV negative human serum
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TRACKING DISEASE OUTBREAKS
Instructor’s Answer Key and Discussion Points
Pre-Lab Focus Questions
1. How does the immune system protect us from disease?
The immune system includes physical barriers, such as the skin and mucous membranes
that prevent pathogens from entering the body, and cellular responses, such as
circulating macrophages that respond to foreign invaders. Our acquired immune system
mounts a specific antibody response when the body is exposed to a foreign invader,
and our immune cells attack the invader.
2. How do doctors use the immune response to protect you from disease?
Doctors use the immune response when we are vaccinated against diseases. Our
immune system remembers the pathogens to which we have been exposed, and the
next time we are exposed to the pathogens our immune system attacks them more
quickly and efficiently. Doctors take advantage of this priming effect by exposing us to
inactivated pathogens (killed or weakened organisms that cannot make us sick) so that if
we are later exposed to the live pathogen, our body will mount a strong and immediate
antibody response, reducing or eliminating the chance that it will make us sick.
3. What are some ways that diseases spread?
Diseases Can Spread Through: Examples:
Exchange of bodily fluids • HIV, SARS, Epstein-Barr virus (cause of
mononucleosis), STDs
Ingestion of contaminated • E. coli O157:H7, prions that cause
food or water Creutzfeldt-Jakob and mad cow diseases,
protozoa that cause giardiasis, nematodes
that cause trichinosis
Inhalation • Viruses that cause the flu, bacteria that
cause tuberculosis
Vector transfer • Mosquito-borne diseases (malaria, West Nile
virus, dengue fever, yellow fever), tick-borne
diseases (Lyme disease, Rocky Mountain
spotted fever)
4. What is an example of a disease that attacks the human immune system?
Diseases that attack the immune system include autoimmune diseases (e.g.,
rheumatoid arthritis, lupus, asthma, eczema, SCID) and AIDS. An extensive list can be
found in Appendix A.
5. What problems can prevent the immune system from working properly?
Problems with the immune system fall into three categories: hypersensitivity,
immunodeficiency, and autoimmune diseases. Hypersensitivity occurs when the
immune system overreacts to an antigen; hypersensitivity reactions include anaphylactic
reactions, allergies, and contact sensitivity (e.g., reaction to poison ivy). Immuno-
deficiency means that an individual cannot mount an effective immune response.
Immunodeficiency may be genetic (e.g., SCID or “bubble boy” disease) or induced by a
disease (e.g., immunodeficiency from HIV infection) or by immunosuppressive drugs
(e.g., drugs given after organ transplant to prevent rejection). Autoimmune disease
results from the immune system inappropriately mounting an immune response to
20
TRACKING DISEASE OUTBREAKS
Protocol I: ELISA for Tracking Disease Outbreaks
Instructor’s Guide
This interactive procedure provides a context for introducing a real-world, topical application
of ELISA. Students first model the spread of disease in a population by the sharing of
simulated “body fluids”. Each student is given a sample to share, one or two of which are
positive for the “disease agent”. After students share their “body fluids”, they assay their
shared samples using ELISA.
Students’ ELISA results reveal that a large portion of the class now tests positive for the
disease! This leads to a guided, inquiry-based activity about how the disease has spread
through the population. Because students have a personal connection to the results, this
activity tends to captivate students’ imaginations and is particularly relevant in light of the
recently emerged contagious disease SARS.
Many other diseases also work with this protocol, including West Nile virus, HIV, colds,
influenza, and STDs, to name a few. A review of Appendix C will provide useful information
about appropriate diseases and scenarios for implementation in the classroom. A simple
approach may be to leave the actual disease unspecified for a generic lesson.
Implementation Timeline
Lesson 1 Set the stage Lecture and discussion
Lesson 2 Sharing of simulated body fluids ELISA lab
Lesson 3 Analyze ELISA results Tracking exercise
12
, TRACKING DISEASE OUTBREAKS
Instructor’s Laboratory Overview
Step 1: Students share “body fluids” by mixing their sample with those of other students.
Within each pair of students who share, each student takes back half of the combined sample.
Each student repeats the sharing process with a different student either 1 or 2 more times
(depending on class size) and records sharing partners. Note: To ensure dissemination of the
“disease”, the sharing must be performed in two or three separate rounds.
PROTOCOL I
750 µl
Student A Students A+B Students A+B+C
Student B Student C Student D
Step 2: Using a pipet, 50 µl of each student’s sample (unknowns)
along with positive and negative controls are added to the wells of
the microplate strip and incubated for 5 minutes, allowing proteins in
the sample to bind to the wells. The wells are rinsed with wash
buffer (PBST: phosphate buffered saline containing 0.05%
Tween 20) that also blocks the unoccupied protein binding sites in
the wells.
Step 3: Primary antibody (50 µl) is added to each well of the
microplate strip and incubated for 5 minutes at room temperature.
The primary antibody is an antibody that recognizes and binds to
the “disease agent”/antigen. The wells are rinsed with wash buffer
to remove unbound antibody.
Step 4: Horseradish peroxidase (HRP)-labeled secondary antibody
(50 µl) is added to each well and incubated for 5 minutes at room
temperature. The secondary antibody is antibody that recognizes
and binds to the primary antibody. HRP is an enzyme that will
oxidize a color-producing substrate. Wells are rinsed with wash
buffer to remove unbound secondary antibody.
Step 5: The enzyme substrate (50 µl) is added to each well and
students watch color development. If HRP is present (meaning that
the antigen was present in the sample), the solution in the wells will
turn blue within 5 minutes. If the antigen was not present in the
sample, the wells will remain colorless.
13
,PROTOCOL I
TRACKING DISEASE OUTBREAKS
Typical ELISA results.
Using Students’ ELISA Results to Track the “Disease”
The number of positive tests in the class results will depend on how many positive
samples you released at the beginning. You can now track the progress of the disease
through your class.
You may want the students to work out a method to track the disease to its source by
themselves. For a more guided inquiry approach, we recommend using the class results
table on page 32 in the student manual. Make a transparency from the page with the
students' names listed and place it on an overhead projector. Ask each student to come up
and write a plus (+) or a minus (-) in the second column to indicate if their ELISA tested
positive or negative. Then, depending on whether their ELISA tested positive or negative,
they should also write a plus (+) or a minus (-) respectively next to the names of the students
with whom they shared their sample.
For example, if Kiko tested positive and shared with Alexander, Florence, and Mustafa, she
would write a “+” by Alexander, Florence’s, and Mustafa’s names.
The students with all pluses against their names will be revealed as early sources of the
infection.
Question: Why will the class not be able to track the infection to a single student?
Answer: When a single student who is the primary source of infection first shares his or
her sample with a second student, the second student will also have all pluses. This is
representative of the kind of problem that epidemiologists face in the real world. You may
turn this occurrence to your advantage by discussing why epidemiologists investigate many
factors when tracking diseases, such as patients’ locations, histories, and behaviors, in
addition to testing for the infection. You may also have your students perform a more
detailed analysis involving tracking the order in which the samples were shared and deducing
if some of the students can be eliminated from the pool of students suspected as being the
original source.
Epidemiologists rarely have patient samples prior to the outbreak of infection, and rarely
are they able to track an outbreak to a single source. However, you have the advantage of
keeping a record of which students received the infected samples, which for the sake of
this exercise may prove helpful. Alternatively, for a more anonymous approach, you may
sequentially number all the student samples and record the numbers of tubes that are
“infected”. The source can be revealed at the end of the activity to see if it matches your
students’ data analysis.
14
, TRACKING DISEASE OUTBREAKS
Instructor’s Advance Laboratory Preparation
This section is designed to help you prepare for the laboratory efficiently. We recommend that
you read this section of the manual (Protocol I: Tracking Disease Outbreaks) in its entirety
before beginning your preparation. In addition, if you are choosing to perform a scenario-based
activity (for example, HIV testing), we recommend using the information given in Appendix C
to help plan your lesson.
The most important thing for the students to do is to put the correct components in the
PROTOCOL I
assay wells in the correct order, so having the tubes clearly labeled and properly color-coded
is crucial to a successful outcome.
Objectives
Step 1. Prepare buffers
Step 2. Rehydrate the freeze-dried antigen, primary antibody, and secondary
antibody to make 50x stocks
Step 3. Dilute 50x stock solutions
Step 4. Dispense reagents for student workstations
Step 5. Set out student workstations
Time Required 1–3 hours
Preparation Timeframe
We recommend rehydrating and diluting the antigen and primary antibody no more than
3 days before the lesson, and the secondary antibody less than 24 hours before the lesson.
We also suggest using sterile distilled water to prepare the 1x PBS to avoid contaminating
rehydrated reagents. These reagents must be kept on ice or in the refrigerator if prepared
more than 4 hours before the lesson.
Note: If you are planning to use this kit for multiple lab sessions over a 1- or 2-week
period, we strongly suggest using sterile water to prepare the PBS buffer in order to
avoid contaminating reagents. (Water can be sterilized by boiling it in a microwave oven
for 5 minutes in a loosely capped bottle; after you remove the bottle from the microwave
oven, let it cool, then secure the cap.) Dilute only as much concentrated antibody and antigen
as required for each lab session. The rehydrated antibodies are 50x concentrates. Store
the remaining concentrated antigen and antibodies in the refrigerator at 4°C. We do not
recommend storing the concentrated antibody and antigen for more than 2 weeks, even at
4°C. Do not freeze the solutions.
Volume Measurements
This kit contains graduated disposable plastic transfer pipets (DPTPs) to use for preparing
some of the reagents where volumes between 250 microliters (µl) and 5 milliliters (ml) are
required. In addition, adjustable- or fixed-volume micropipets are needed to measure 50 µl
volumes. The illustration shows the marks on the DPTP corresponding to the volumes to
be measured. Volumes over 1 ml will require multiple additions. For each step of the laboratory
preparation, use a fresh DPTP or a fresh pipet tip.
1 ml
750 µl
500 µl
250 µl
100 µl
Measuring liquids that contain detergents that foam (e.g., the wash buffer) requires that you
read the volume at the interface of the liquid and the bubbles.
15
,PROTOCOL I
TRACKING DISEASE OUTBREAKS
PROTOCOL I: Step-by-Step Instructor’s Advance Preparation Guide
These instructions are for the setup of 12 student workstations of 4 students each.
Supplied Reagents Quantity
Antigen, chicken gamma globulin, freeze-dried 1 vial
Primary antibody, rabbit anti-chicken polyclonal antibody, freeze-dried 1 vial
Secondary antibody, goat anti-rabbit antibody conjugated to (HRP),
freeze-dried 1 vial
HRP enzyme substrate (TMB) 1 bottle
10x phosphate buffered saline (PBS) 1 bottle
10% Tween 20 1 bottle
Required Reagent
Distilled water, sterile is recommended, see note on page 15 1L
Step 1. Prepare buffers.
We recommend you use a 100 ml and a 1 liter (L) graduated cylinder for preparing the
buffer solutions. You will also need 1 L of distilled water.
Buffer Volume Reagent Used for
1x PBS, 100 ml 90 ml Distilled water • Rehydrating antigen,
10 ml 10x PBS Primary and secondary
antibodies to make 50x
reagent stock solutions
• Diluting 50x antigen to make
positive control and “infected”
student samples
• Negative control
• Negative student samples
Wash Buffer, 900 ml 805 ml Distilled water • Dilution of 50x antibody
stocks
90 ml 10x PBS • Plate washing
4.5 ml 10% Tween 20
Step 2. Rehydrate the freeze-dried antigen, primary antibody, and secondary
antibody.
Carefully remove the stoppers from the three freeze-dried reagents and use a fresh DPTP to
add 0.5 ml 1x PBS to each. Close the stoppers and shake to mix. These solutions are 50x
concentrates, or stock solutions. NOTE: You must not use wash buffer in this step.
Freeze-Dried Reagent Protocol for 50x Stock Solution Used for
Antigen Add 0.5 ml of 1x PBS to vial • Positive control
• “Infected” student samples
Primary antibody Add 0.5 ml of 1x PBS to vial • Primary antibody
Secondary antibody Add 0.5 ml of 1x PBS to vial • Secondary antibody
16
, TRACKING DISEASE OUTBREAKS
Step 3. Dilute 50x stock reagents.
Label one 30 ml bottle for each of the diluted solutions below. Use a fresh DPTP to add the
contents of the appropriate 50x concentrated stock to the corresponding 30 ml bottle.
Diluted solution Volume Reagent Used for
Positive control (1x antigen), 7.5 ml 1x PBS Positive control
label one 30 ml bottle 150 µl 50x antigen
stock
PROTOCOL I
NOTE: you must not add any buffer containing Tween 20 to
the antigen, or the experiment will not work.
1x primary antibody, 24.5 ml Wash buffer Primary antibody
label one 30 ml bottle 0.5 ml 50x primary
antibody stock
• Use the DPTP to rinse out the vial with some of the diluted
reagent to ensure that all of the stock solution is used.
• Close the cap and shake to mix.
1x secondary antibody, 24.5 ml Wash buffer Secondary antibody
label one 30 ml bottle 0.5 ml 50x secondary
antibody stock
• Dilute the secondary antibody less than 24 hours before the start
of the lesson. Use the DPTP to rinse out the vial with some of the
diluted reagent to ensure that all of the stock solution is used.
• Close the cap and shake to mix.
Step 4. Dispense reagents for student workstations.
Tubes Description Label Contents (Each Tube)
Violet tubes, 12 Positive control “+” 0.5 ml positive control solution
(1x antigen)
Blue tubes, 12 Negative control “–” 0.5 ml 1x PBS
Green tubes, 12 Primary antibody “PA” 1.5 ml 1x primary antibody solution
Orange tubes, 12 Secondary antibody “SA” 1.5 ml 1x secondary antibody solution
Brown tubes, 12 Enzyme substrate “SUB” 1.5 ml HRP enzyme substrate (TMB)
Note: TMB is light sensitive, so it is important to use the dark tubes to store this
reagent.
Yellow tubes, # “Infected” student Determined 100 µl 50x antigen stock solution
depends on # of sample(s) (6.6x antigen) by 650 µl 1x PBS
students (1–3 instructor
tubes)
The number of students in the class will determine the number of “infected” samples
you mix with the blanks. For a result where about half the students become infected,
we recommend making one infected sample per 16 students. (Note: if your class is
less than 10 students, use a single sample and perform just 2 rounds of sharing.)
Note: You must not add any buffer containing Tween 20 to the antigen, or the
experiment will not work. You may want to keep the infected samples separate
from the blank samples until the lesson to keep track of who receives them.
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TRACKING DISEASE OUTBREAKS
Tubes Description Label Contents (Each Tube)
Yellow tubes, # Blank (uninfected) Determined 750 µl 1x PBS
depends on # of student samples by
students instructor*
Make enough blank yellow tubes for your student number minus the "infected"
samples. Note: You must use 1x PBS and not wash buffer for the blank
students samples, or the experiment will not work.
* (Optional) For your own information, you may wish to number each tube of student sample (infected
and blank) from 1 to 48 and record which numbered tubes contain the infected samples.
Step 5. Set out student workstations.
Student Workstation Checklist
One workstation serves 4 students.
Item (Label) Contents Number ✔)
(✔
Yellow tubes Student test samples (0.75 ml) 4 (1 per student) ❑
Violet tube (+) Positive control (0.5 ml) 1 ❑
Blue tube (–) Negative control (0.5 ml) 1 ❑
Green tube (PA) Primary antibody (1.5 ml) 1 ❑
Orange tube (SA) Secondary antibody (1.5 ml) 1 ❑
Brown tube (SUB) Enzyme substrate (1.5 ml) 1 ❑
12-well microplate strips 2 ❑
50 µl fixed-volume micropipet 1 ❑
or 20–200 µl adjustable micropipet
Yellow tips 10–20 ❑
Disposable plastic transfer pipets 5 ❑
70–80 ml wash buffer in beaker Phosphate buffered saline 1 ❑
with 0.05% Tween 20
Large stack of paper towels 2 ❑
Black marking pen 1 ❑
Note on sharing protocol: Make sure that the students share “body fluids” with students
from other parts of the room, not just with their near neighbors. The best way to do this is to
have orderly sharing: Tell the students to share with one other person, then return to their
lab station. After all students are finished with the first sharing and are back in place, then
tell them to share with a different person. The degree of sharing will depend on your class
size. We recommend one "infected" sample per 16 students. If your class size is less than
10, use a single "infected" sample and perform just two rounds of sharing.
Stopping points: Although this procedure is designed to fit into a single lesson period, you
may stop the laboratory activity after sharing the “body fluids” and place all the reagents in
the refrigerator at 4°C overnight. Alternatively, if you wish to stop during the ELISA you may
add wash buffer to the microplate wells at any stage after the addition of antigen and prior
to the addition of enzyme substrate. Place the microplate strips and all the reagents in the
refrigerator at 4°C overnight.
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, TRACKING DISEASE OUTBREAKS
Setting Up The Activity to Test for a Specific Disease (e.g., HIV)
Appendix C provides information on a variety of diseases that can be diagnosed using
ELISA. In addition, for each disease, we provide a table describing what the reagents for
the activity represent in a real-world diagnostic ELISA. Below is an example of a diagnostic
test to detect HIV viral proteins in a patient's blood sample.
Detecting p24 HIV-1 Capsid Protein.
PROTOCOL I
Tube Actual Tube
Tube Description Color Contents Simulated Tube Contents
Student samples Yellow 1x antigen or Sample derived from patient's blood
(unknowns) 1x PBS
Primary antibody Green 1x primary Anti-p24 capsid protein antibody from
antibody mouse
Secondary Orange 1x secondary Anti-mouse immunoglobulin antibody
antibody antibody conjugated to HRP
Positive control Violet 1x antigen Heat-inactivated viral antigen (p24 protein)
Negative control Blue 1x PBS HIV negative human serum
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Instructor’s Answer Key and Discussion Points
Pre-Lab Focus Questions
1. How does the immune system protect us from disease?
The immune system includes physical barriers, such as the skin and mucous membranes
that prevent pathogens from entering the body, and cellular responses, such as
circulating macrophages that respond to foreign invaders. Our acquired immune system
mounts a specific antibody response when the body is exposed to a foreign invader,
and our immune cells attack the invader.
2. How do doctors use the immune response to protect you from disease?
Doctors use the immune response when we are vaccinated against diseases. Our
immune system remembers the pathogens to which we have been exposed, and the
next time we are exposed to the pathogens our immune system attacks them more
quickly and efficiently. Doctors take advantage of this priming effect by exposing us to
inactivated pathogens (killed or weakened organisms that cannot make us sick) so that if
we are later exposed to the live pathogen, our body will mount a strong and immediate
antibody response, reducing or eliminating the chance that it will make us sick.
3. What are some ways that diseases spread?
Diseases Can Spread Through: Examples:
Exchange of bodily fluids • HIV, SARS, Epstein-Barr virus (cause of
mononucleosis), STDs
Ingestion of contaminated • E. coli O157:H7, prions that cause
food or water Creutzfeldt-Jakob and mad cow diseases,
protozoa that cause giardiasis, nematodes
that cause trichinosis
Inhalation • Viruses that cause the flu, bacteria that
cause tuberculosis
Vector transfer • Mosquito-borne diseases (malaria, West Nile
virus, dengue fever, yellow fever), tick-borne
diseases (Lyme disease, Rocky Mountain
spotted fever)
4. What is an example of a disease that attacks the human immune system?
Diseases that attack the immune system include autoimmune diseases (e.g.,
rheumatoid arthritis, lupus, asthma, eczema, SCID) and AIDS. An extensive list can be
found in Appendix A.
5. What problems can prevent the immune system from working properly?
Problems with the immune system fall into three categories: hypersensitivity,
immunodeficiency, and autoimmune diseases. Hypersensitivity occurs when the
immune system overreacts to an antigen; hypersensitivity reactions include anaphylactic
reactions, allergies, and contact sensitivity (e.g., reaction to poison ivy). Immuno-
deficiency means that an individual cannot mount an effective immune response.
Immunodeficiency may be genetic (e.g., SCID or “bubble boy” disease) or induced by a
disease (e.g., immunodeficiency from HIV infection) or by immunosuppressive drugs
(e.g., drugs given after organ transplant to prevent rejection). Autoimmune disease
results from the immune system inappropriately mounting an immune response to
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