TM

BIO-RAD

Giant Panda Problem Kit

for AP Biology:

A ThINQ!

™

Investigation

Catalog #17002878EDU

for Bio-Rad Explorer kit curricula.

For technical service, call your local Bio-Rad

offi ce or, in the U.S., call 1-800-424-6723

Dear Instructor

Thank you for inspiring the next generation of scientists, citizens, and decision

makers to be curious about the world around them. Our goal is to provide you

with tools to help your students think like real scientists. Bio-Rad’s ThINQ!

on how the result was obtained. Instead of providing students with explanations

or interpretations, the student manual poses a series of questions to focus and

stimulate thinking about all aspects of the investigation. To facilitate the teacher’s

role, explanations and interpretations are included in the instructor’s guide.

The Giant Panda Problem Kit uses two variations of an enzyme-linked

immunosorbent assay (ELISA) to determine the presence of a pregnancy-

associated disorder and to track reproductive hormones in giant pandas.

Investigation #1 in this kit engages students in technique basics and biochemical

interactions as they learn about humoral immune responses. The inquiry

investigation (#2) following Investigation #1 asks students to apply what they have

bridges to other content areas you may

teach. Connections can be made to ecology,

survival and fi tness, climate change,

and the interrelatedness between body

systems, among others. The Giant Panda

Problem Kit covers those processes in

the context of biological systems using

free energy (AP Big Idea 2) and the

interaction of biological systems

(AP Big Idea 4).

your stories, comments, curriculum extensions, and suggestions.

Special thanks to Tim Guilfoyle, AP Biology and Microbiology teacher, for

proposing the name of this kit!

Bio-Rad Explorer

6000 James Watson Drive, Hercules, CA 94547

[email protected]

www.explorer.bio-rad.com

Kit Storage .....................................................................................................................................................................................1

Important Notes .............................................................................................................................................................................1

Road Map to the Instructor’s Guide ................................................................................................................................................4

Kit Timeline .....................................................................................................................................................................................6

AP Biology Standards and Giant Panda Problem Kit Alignment ......................................................................................................9

Background for Instructors ...........................................................................................................................................................10

Investigation #1: ELISA Antibody Test (Optional) ...........................................................................................................................17

Investigation #2: Hormone Detection ELISA ..................................................................................................................................18

Background ..................................................................................................................................................................................21

Assignment: Investigation #1 Wrap-Up .........................................................................................................................................37

ELISA Paper Model (Optional Activity) ...........................................................................................................................................38

Investigation #2: Hormone Detection ELISA (Structured/Guided/Open Inquiry Activity) .................................................................39

Pre-Investigation Questions ..........................................................................................................................................................40

Post-Lab Assessment ..................................................................................................................................................................42

Appendix A: Quick Guide for Investigation #2 ..............................................................................................................................45

Appendix B: Immunological Concepts .........................................................................................................................................48

Appendix C: Glossary of Terms ....................................................................................................................................................55

Appendix D: Quantitative ELISA Laboratory Activity .....................................................................................................................58

Place the reagent bag in the refrigerator (4°C) upon arrival.

Once stock reagents are prepared, store the diluted/

reconstituted solutions at 4°C to ensure stability.

• Go to

to download the

own ELISA tests to detect hormone levels in simulated giant panda urine samples. The kit contains suffi cient materials for eight

student workstations with four students each to test for the presence of antigen in samples for Investigation #2.

disposable plastic transfer pipets (DPTPs) is required. Alternatively, it is possible to reuse the DPTPs in the kit, provided they are

washed and rinsed well. Reusing DPTPs introduces the possibility of cross contamination and should be undertaken with caution.

• Investigation #1: Digital Animation Activity — ELISA Antibody Simulation

ELISA Antibody test

• Investigation #2: Hormone Detection ELISA

Using a Single Assay to Study Two Body Systems

With this kit, students will investigate two distinct body systems using one central scientifi c concept and technique — an ELISA

this assay is used to determine the presence or absence of an antibody (Investigation #1) or antigen (a hormone in Investigation

#2). In both investigations the interaction between antibodies (primary and secondary) and antigen is critical for diagnosis via a

colorimetric reaction.

As an extension, another means for connecting these two body systems and their function is via the stress hormone cortisol.

Several studies can support students’ understanding of stress on the body (see bibliography for further reading). Cortisol, the

primary “stress” hormone, is known to reduce the function of the reproductive system by preventing ovulation. This is particularly

problematic in giant pandas living in captivity. As giant pandas experience chronic stress due to limited access to physical

space in their enclosures, access to agreeable food and companions, exposure to unfavorable weather and caretakers and the

general public through viewing at zoos, both their immune system and reproductive system functions may suffer. Caretakers are

ELISA assay and testing simulated panda urine samples.

• ELISA Paper Model

• Investigation #2: Hormone Detection ELISA

of antigen in samples for Investigation #2. There will be some extra components left over (e.g. eight microplate strips) that you may

choose to use for open inquiry experiments with your students.

understanding of body systems, in particular antigen and antibody interactions in the context of immune responses and

endocrinology.

How to Integrate the Inquiry Investigation Labs into Your Schedule

Assessment Questions in the student manual

• Sidebars that call out tips and tricks, teachable moments, and AP Bio curriculum

Once the pre-lab and core Digital Animation Activity: Antibody ELISA Simulation or

Investigation #1 are performed by the entire class, you have the choice of taking a structured,

guided, or open inquiry approach for Investigation #2. If you have not conducted a guided

or open inquiry investigation previously with your students, we suggest that you review the

Teacher Model Process on page 19 for tips on how to support students as they navigate

Investigation #2.

The following is a suggested lab schedule, but it is just one of many possible scenarios. In

activity to AP learning objectives.

AP Big Ideas 2 and 4 are easily

covered in this kit (see AP Biology

Curriculum Alignment on page 9 for

detailed alignments to AP LO, EK,

and SP).

These sidebars suggest teachable

moments to help your students

through the inquiry process.

AP Biology Answer Guide included

in the kit for the answers. These

questions can also be used for

formative assessment.

There are several different types of ELISA protocols. The protocols in this kit rely on indirect

antibody capture ELISA. The steps in this assay are:

binding, after which unbound antigen is washed from the wells with buffer containing

detergent. The detergent also serves as a blocking agent, binding to all unused protein

incubated to allow color to develop. Results of the assay are evaluated. Wells that remain

colorless are negative and wells that turn blue are positive.

To create a relevant and meaningful classroom context for this activity, the in-depth

information in Appendices B and C provides background vocabulary and factual and

conceptual lecture points. In addition, useful reading and web sites are included in

Appendix E. The following section briefl y describes the technical and conceptual points

that are directly related to the investigations in this curriculum.

by hydrophobic interaction. The plates provided in this kit have 96 wells, arranged in

any hypothetical antigen, protein or otherwise. In the Giant Panda Problem Kit the antigen represents a hormone, such as GnRH,

estrogen, FSH, or LH, that indicates the beginning of ovulation.

kit has been optimized so that each incubation can be performed for 5 minutes at room temperature. Exceeding this time or

temperature will cause an increase in color intensity and possibly some background color in the negative controls and samples.

would produce false positive results. The blocking agent may be a protein or a detergent (or both). Common blocking agents

include Tween 20 (a nonionic detergent that is used in this kit), nonfat dry milk, gelatin, and bovine serum albumin (BSA).

this kit, the primary antibodies are polyclonal rabbit antibodies raised against chicken gamma globulin and are called rabbit

anti-chicken antibodies. In the ELISA Antibody Test starting on page 33, this primary antibody represents giant panda antibodies

that are bound to primary antibodies occurs by an enzyme-substrate reaction. In this kit, the secondary antibody is linked to

the enzyme horseradish peroxidase (HRP). In the presence of hydrogen peroxide (H

O

), HRP catalyzes the oxidation of the

chromogenic substrate 3,3',5,5'-tetramethylbenzidene (TMB). This oxidation of TMB by HRP forms a blue product.

the microplate strips sit longer, nonspecifi c color may develop. Color that develops after the 5–10 minute incubation should not be

considered in the assay results. After 20–30 minutes, the blue color may begin to fade as TMB precipitates out of solution.

double cation radical

of TMB

(Blue color)

BACKGROUND FOR INSTRUCTORS

Controls can resolve ambiguous results that occur due to human error or contaminated reagents; controls must be included in any

valid ELISA. For the negative control, the antigen or primary antibody is either omitted (as in this kit) or the antigen is replaced by a

false negative.

immunosuppression (e.g., from drugs given after transplants) or from administering the test too soon after infection with HIV.

(Antibodies against HIV do not appear until some weeks after HIV infection; the appearance of specifi c antibodies is called

seroconversion.) Because of this, positive HIV ELISA results are always confi rmed by western blot.

In an ELISA test like that in Investigation #2 (in which antigen concentration is the experimental variable), an appropriate negative

control would be wells with antigen omitted. Any color product in those wells would be the result of either 1) nonspecifi c binding of

the primary or secondary antibody, or 2) experimental error. An appropriate positive control would be a sample known to contain

Label one 50 ml bottle or tube for each of the diluted solutions below. Add the contents of the appropriate 50x concentrated

stock to the corresponding 50 ml bottle or tube.

giant pandas contain disease antibodies. Learning this technique will support their design of an ELISA for the presence of a panda

hormone in Investigation #2.

ols (+) 200 µl, 1x primary antibody

Blue tubes, 8 Negative controls (–) 200 µl, 1x PBS

Green tubes, 8 Purifi ed antigen (AG) 800 µl, 1x antigen

Orange tubes, 8 Secondary antibody (SA) 800 µl, 1x secondary antibody

Brown tubes, 8 Enzyme substrate (SUB) 800 µl, HRP enzyme substrate

Yellow tubes, 1 tube per Positive panda urine samples — 1 per student pair (P1) 200 µl, 1x primary antibody

pair of students (16 max) Negative panda urine sample — 1 per student pair (P2) 200 µl, 1x PBS

We recommend that you design the experiment so that 50% of the urine samples will test positive and 50% of the urine

(decided during the Pre-lab activity). Students will test four simulated giant panda urine samples for the presence or absence of

the hormone.

200 µl, 1x antigen

Blue tubes, 8 Negative controls (–) 200 µl, 1x PBS

Green tubes, 8 Primary antibody (PA) 1 ml, 1x primary antibody

Orange tubes, 8 Secondary antibody (SA) 1 ml, 1x secondary antibody

Brown tubes, 8 Enzyme substrate (SUB) 1 ml, HRP enzyme substrate

Identifying the

components of the

ELISA and explaining

Understanding how

an ELISA for hormone

detection can

determine the fertility

of female pandas

Clarifying goals for the investigation

Presence or absence

of blue color

Analyzing

Evidence

Use of reagents

such as panda urine

samples, antibodies to

panda hormone, and

secondary antibodies

BACKGROUND

in female mammals to determine when and how much of each of the hormones listed in the table above are present in the body.

Tracking the levels of hormones and when they are released provides valuable information for developing tests that can predict

5. Given the information in the table above, identify at least three hormones that, if tracked, would be good indicators that

ovulation is about to occur in a female giant panda.

In order to best support panda reproduction by natural mating and artifi cial insemination, highly sensitive and specifi c tests are

to view the antibody

detection ELISA animation. Download and print the blank animation activity questions sheet

from the student manual from

for your students to use

along with the animation preparing for Investigation #2. The printed Answer Guide can be

found in the kit.

If you are using the extended timeline for this kit, Investigation #1 (page 33) will use a

• Students learn that the structure of antibodies allows for interactions with specifi c antigens

• Students learn how to set up and run an ELISA for antibody detection

• Students refl ect on how to design a scientifi c protocol to answer a research question

• Students learn how to refi ne scientifi c models based on evidence

Refer to the AP Biology Curriculum

Alignment tables on page 9 for more

details on how this activity aligns to

AP Curriculum Learning Objectives

(LO), Essential Knowledge (EK), and

Science Practices (SP).

LO 2.43 [EK 2.D.4 & SP 7.2]

LO 4.8 [EK 4.A.4 & SP 3.3]

LO 4.9 [EK 4.A.4 & SP 6.4]

LO 4.10 [EK 4.A.4 & SP 1.3]

LO 4.19 [EK 4.B.3 & SP 5.2]

LO 4.22 [EK 4.C.1 & SP 6.2]

In the Pre-Lab you learned about the reproductive hormones in female mammals, like the

giant panda. This information is needed in order to design a test that tracks reproductive

good place to start when thinking about how you will develop a test to track hormones in a

panda during Investigation #2. In this activity you will view a digital animation of an antibody

detection ELISA.

Go to

bio-rad.com/PandaAPResources

to view the antibody detection ELISA animation. Your

can also download and print the question sheet yourself at

bio-rad.com/PandaAPResources

.

As part of your assignment you will design an ELISA protocol to test panda urine samples for

an ovulation hormone during Investigation #2. Be sure to pr

2. Why is it important to add purifi ed antigen to the wells fi rst?

3. The animation did not include any contr

6. The secondary antibody is attached to an enzyme (HRP) that chemically changes the enzyme substrate, turning it from a

colorless solution to a blue solution. If you ran an antibody detection ELISA with positive control wells, negative control

wells, and experimental wells, predict which wells of your experiment should turn blue, which should remain colorless,

and which wells you are not sure about and why.

7. In the space below, draw and annotate what is happening in wells that turned blue to explain what is causing the

During this activity you observed a digital animation of an antibody detection ELISA. Thinking back to the background reading in

this manual, you learned that ELISAs can also track hormones in the body, like reproductive hormones in female giant pandas.

However, when tracking a hormone, the ELISA would be detecting an antigen (the hormone of choice) instead of the presence or

8. How can antibodies be engineered to detect the presence of a molecule that does not cause disease, such as a hormone?

9. Based on the animation and your answer to question 7 draw a well and label the components in order of addition to detect

11. What is your reasoning for choosing this particular hormone?

ELISA ANTIBODY TEST

1. The yellow tubes contain the urine samples that will be tested for the presence of anti-phospholipid antibodies. Label each

yellow tube to identify the sample being tested.

3. Use a pipet to transfer 50 µl of the purifi ed antigen (AG) from the green tube into the fi rst 8 wells. The antigen in this case

5. Wash unbound antigen out of the wells:

strip a few times upside down on the paper towels to get rid of all the liquid and bubbles in the wells.

neighboring wells. The same transfer pipet will be used for all washing steps. Take care not to touch the tip of the pipet

to the wells of the strip.

7. Use a fresh pipet to transfer 50 µl of the positive control (+) from the violet tube into the two (+) wells. The positive control

contains anti-phospholipid antibodies.

Wash

8. Use a fresh pipet to transfer 50 µl of the negative control (–) from the blue tube into the two (–) wells.

9. Use a fresh pipet to transfer 50 µl of each urine sample (P1 and P2) into the appropriately labeled two wells.

13. Wait 5 min for the secondary antibody to bind to the primary antibody.

15. Use a fresh pipet to transfer 50 µl of enzyme substrate (SUB) from the brown tube into the fi rst 8 wells of the microplate strip.

16. Wait 5 min. Observe and record your results.

ELISA ANTIBODY TEST

These post-investigation questions are designed to prompt students’ refl ections about the protocol they completed in Investigation

#1. There are several ways students can answer the question presented in Investigation #1. Students should be given time to

discuss their thinking and share their ideas with the class.

1.1 What is the purpose of this protocol?

1.3 Why is it important to include a positive and a negative control?

1.4 The secondary antibody is attached to an enzyme (HRP) that chemically changes the enzyme substrate, turning it from

a colorless solution to a blue solution. Predict which wells of your experiment should turn blue, which should remain

colorless, and which wells you are not sure about.

During this investigation, you conducted an antibody detection ELISA. Thinking back to the background reading in this

manual, you learned that ELISAs can also track hormones in the body, like reproductive hormones in female giant pandas.

However, when tracking a hormone the ELISA would be detecting an antigen (the hormone of choice) instead of the

presence or absence of antibodies to a particular disease.

1.6 What changes might you make to this protocol to track the presence of a hormone in

a urine sample instead of the presence of an antibody?

It may be useful to hold a whole

an investigation question, and

careful collection of data, among

others. This list could be posted

on the classroom wall to serve as

a reminder in future investigations

when students generate their own

protocols.

1.7 During the Pre-Lab you identifi ed at least three hormones that could be tracked to determine the onset of ovulation

in a giant panda. Given the information in the graph below, which of the three reproductive hormones would provide the

greatest accuracy for onset of ovulation?

Please refer to the printed copy of the Giant Panda Problem Kit for AP Biology Answer Guide included in the kit for

Please refer to the printed copy of the Giant Panda Problem Kit for AP Biology Answer Guide included in the kit for

1.10 Using your response to question 1.6, what controls would you need to include to test for the presence or absence of your

hormone in samples from giant pandas?

The data you generated during this investigation provide insight about how an ELISA can be used to determine if a panda has

ELISA ANTIBODY TEST

3. What do these data tell you about the condition of the two lemurs that tested positive for antibodies to T. gondii?

Protocol

strip a few times upside down on the paper towels to remove all liquid and bubbles from the wells.

e. Discard the wet paper towels.

8. Wait 5 min for the primary antibody to bind.

10. Use a fresh pipet to transfer 50 µl of secondary antibody (SA) from the orange tube into all 12 wells of the microplate strip.

11. Wait 5 min for the secondary antibody to bind to the primary antibody.

Wash

The secondary antibody is attached to an enzyme (HRP) that chemically changes TMB (the enzyme substrate), turning it

from a colorless solution to a blue solution. Predict which wells of your experiment should turn blue and which should remain

colorless and which wells you are not sure about and state why.

13. Use a fresh pipet to transfer 50 µl of enzyme substrate (SUB) from the brown tube into all 12 wells of the microplate strip.

Antigens can be microorganisms (e.g., viruses and bacteria), microbial products (e.g., toxins produced by some bacteria, or

protein components of the microbes), foreign proteins, DNA and RNA molecules, drugs, and other chemicals. Antibodies are

proteins also called immunoglobulins (Ig), that are produced by B cells and can remain attached to B cells or become freely

in serum and is responsible for the primary immune response. IgA is found in external secretions such as tears, saliva, milk, and

mucosal secretions of the respiratory, genital, and intestinal tracts and is a fi rst line of defense against invading microorganisms.

IgA is also the only antibody passed from mother to infant. IgD may be involved in regulating the immune response, and IgE is a

primary component in allergic reactions.

Epitopes are the specifi c parts of antigens that are recognized by antibodies. Each antibody recognizes a single epitope, thus

multiple antibodies may recognize and bind to different epitopes on a single antigen. For example, an HIV virus particle (virion)

has many potential epitopes on its surface that may be recognized by many different antibodies. One particular antibody may

recognize the amino terminus of p24, an HIV capsid protein, while another may recognize the carboxy terminus of p24. Immune

cells are the soldiers of the acquired immune response. Macrophages serve two primary functions: 1) removing foreign cells and

molecules from the blood, and 2) processing antigens and presenting them on their cell surfaces. Macrophages present antigenic

epitopes on their cell surfaces to be recognized by T cells. The T cells draw more immune cells to the site of infection, causing

infl ammation. Both B cells and T cells are lymphocytes (white blood cells), and each recognizes a single specifi c epitope. T cells

mature in the thymus, and B cells mature in the bone marrow. B cells produce antibodies; antibodies make up to 15% of your

We depend on our immune system to protect us from disease, but when the immune system fails to function correctly, it

can cause severe health problems. These problems fall in to three basic categories: hypersensitivity, immunodefi ciency, and

dermatitis after exposure to chemicals or environmental agents ranging from metallic nickel to cosmetics).

Immunodefi ciency means that an individual is unable to mount an effective immune response, resulting in increased vulnerability to

opportunistic infections. There are two types of immunodefi ciency: 1) Primary immunodefi ciency has a genetic basis. Severe

combined immunodefi ciency (SCID, “bubble boy” disease) is an example of primary immunodefi ciency. Treatments for primary

immunodefi ciency may include gene therapy. 2) Secondary immunodefi ciency has an external cause and is more common than

primary immunodefi ciency. Secondary immunodefi ciency may be caused by an infection, as in the case of HIV/AIDS, by drug

treatments, such as immunosuppressive drugs given after organ transplant, or by other health factors, such as poor nutrition,

stress, or aging.

Autoimmune disease results from the immune system making a mistake and mounting an immune response against one’s own

body. Some examples of autoimmune disease include systemic lupus erythematosus (lupus, SLE), rheumatoid arthritis, multiple

Laboratory tests cover a wide variety of methods, some of which have been in use for decades and others, like the tests for RNA

and DNA from disease agents, which are very new. Depending on the test and putative diagnosis, laboratory tests may look for

signs of disease in most body fl uids, including blood, urine, stool samples, cerebrospinal fl uid, and saliva. In the US, the Food and

Drug Administration regulates laboratory tests. The fi rst tests for detecting and identifying microorganisms from clinical samples

used antisera directed against specifi c microbes. The antibodies were labeled with a fl uorescent tag, and the microorganisms

could be detected with microscopy when the antibodies bound to them. Other early diagnostic tests include: 1) culture methods,

in which microorganisms from clinical samples are grown on different culture media and their growth and appearance observed

(frequently takes weeks to get results); 2) identifi cation of microbe-specifi c antibodies in serum by immunoassays such as ELISA;

and 3) agar diffusion assays, in which antisera and antigens are placed in holes in agar plates. Both diffuse into the agar, and

where antibodies encounter antigens for which they are specifi c, they bind. Upon antibody-antigen binding, a visible precipitation

1. Live attenuated vaccines are weakened (attenuated) microbes, that are nonpathogenic. Using current technology, deletion or

inactivation of microbial genes weakens the pathogens so they can be used in vaccines; previously, less pathogenic strains

were selected from natural populations. Examples of live vaccines include those against polio (Sabin type), measles, mumps,

and smallpox.

2. Killed or inactivated vaccines are made of microbes killed by heat or chemicals. Killed vaccines are much safer than live

vaccines, particularly for individuals with compromised immune systems, but they do not usually provoke as strong an

immune response as do live vaccines. Examples of killed vaccines include those against rabies, cholera, polio (Salk type),

and infl uenza.

3. Subunit vaccines are made from pieces of microbes. They consist of one or more antigens from either the disease agent or a

microbial product, and they may be derived from the organisms or engineered using molecular biology. Examples of subunit

For example, in 2012 Raxibacumab - a human monoclonal antibody against an antigen involved in anthrax infection - became

available for use in humans.

6. Post-exposure vaccines (immunotherapy) are used to treat a disease. Some immunotherapies have been used for years (e.g.,

administering immune serum globulin after exposure to hepatitis and administering equine antivenin for snakebite), but there

Tapping Nature’s Toolkit: Manufacturing Antibodies

Polyclonal antibodies are generated by immunizing an animal (usually a rabbit, goat, or sheep) and obtaining serum. For example,

purifi ed HIV gp120 protein can be injected into a goat, which will then generate antibodies directed against the many epitopes of

gp120, and the antiserum can be used directly or the antibodies can be purifi ed from it. The antibodies are called polyclonal

because the antibodies are from many (poly) B cell clones (clonal) in the goat’s blood. Polyclonal antiserum has the advantage of

being simple and inexpensive to produce, but the disadvantage is that no two batches, even made in the same animal, will be

exactly the same.

For many antibody applications such as diagnostic tests, polyclonal antibodies are too variable. In these cases, one antibody type

from a single B cell clone is preferable. B cell clones producing single antibodies can be isolated from the spleens of immunized

mice, but these cells die after a few weeks in the laboratory, limiting production of the large amounts of antibody generally needed

for research and commercial applications. However, B cells can be made to live (and produce antibodies) indefi nitely if they are

fused with tumor-like immortal cells. The fusion generates hybrid cells (a hybridoma cell line), which can be cultured indefi nitely;

the monoclonal antibodies generated by the hybrid cells can be collected and purifi ed from the growth medium with almost no

batch-to-batch variability.

The ability of antibodies to act like magic bullets and home in on their targets makes them ideal candidates for medical therapies.

For example, an antibody that recognizes a tumor antigen can be attached to a chemotherapy drug or radioactive molecule

obviates the sacrifi ce of laboratory animals. Two of the methods used to engineer antibodies are described below.

Recombinant DNA technology allows the antigen recognition site from a known mouse monoclonal antibody to be camoufl aged

within a human antibody by combining part of the mouse gene with the human antibody gene. Bacteria transformed with this

DNA are capable of producing humanized monoclonal antibodies indefi nitely, with the added bonus that culturing bacteria requires

much less time and expense then the culture of a mouse hybridoma cell line.

are being created by inserting shuffl ed antibody genes from billions of human B cells into the genomes of bacteriophage lambda

(bacteriophages, or phages, are viruses that infect bacteria; lambda phage is a specifi c species of phage), so that the lambda

phages display the binding sites from human antibodies on their surfaces. This phage library is screened to fi nd a phage that binds

to a specifi c antigen. The phage can then be used directly as an antibody would be used. Alternatively, the DNA from the selected

phage can be cloned into a human antibody gene and transformed into bacteria. Large amounts of the antibody can then be

produced for therapeutic use. Phage display is a robust methodology used in immunotherapy.

Antibodies are used in diagnosis and research as labeling tools. As labels they have to be made visible, so antibodies are

antibody specifi c for

the antigen and looking for signal.

Secondary antibodies are produced by injecting the antibodies of one animal into a different species of animal. For example, if the

is the specifi c binding of an antibody to its antigen, and there are many ways that binding can be utilized. Here are some of those

uses: Immunostaining localizes antigens in organelles, cells, tissues, or whole organisms, and can also be used to distinguish

one cell type from another. For example, pathologists can identify cancer cells using immunostaining. Cancer cells frequently

look identical to normal cells under the microscope, but when they are immunostained, variations in the amount and kinds of cell

surface proteins (antigens) are revealed. Studying this information helps diagnose cancer, and it can help in our understanding of

how cancer cells cause harm.

Immunostaining tissues or organisms can tell us in what cell types a protein is normally found, which can help us understand

the protein’s function. For instance, immunostaining of plant seedlings at different stages of maturation allows us to follow how a

protein’s abundance and localization change as the plant grows. Antibodies for immunostaining are labeled with either fl uorescent

molecules or enzymes that produce colored signals upon addition of a substrate.

Antigen

Antibody

HRP

Enzyme

Substrate (TMB)

Researchers working with giant panda conservationists were interested in determining which of two female pandas were nearing

their ovulation point.

Urine samples were collected from each panda and the samples were frozen and shipped to the lab for analysis. Once at the lab,

the samples were thawed and an antigen detection ELISA protocol was followed in order to determine the amount of ovulation

indicating hormone in each panda urine sample. The researchers knew that a minimum of 120 ng of ovulation indicating hormone

per ml of urine would be needed to indicate an ovulation event in the next 1–2 days.

The r

produce a color change indicating the presence of ovulation indicating hormone. The researchers used a microplate reader set at

655 nm to determine the absorbance (Abs) of each of the wells.

Results

On the next page, generate a standard curve using the Standard results from the fi rst table above. Use the example on the

previous page as a guide. Then using the results from the Experiment (second table above) determine the concentration of

ovulation indicating hormone in ng/ml for the positive control, negative control, and the two panda urine samples by drawing

vertical lines from their absorbance values on the x-axis to the standard curve and then reading horizontally from the points where

the vertical lines intersect with the standard curve to the concentration values on the y-axis.

Appendix E

Appendix F

The optional paper model activity for Investigation #2 asks students to model their understanding of the ELISA. You can make

ELISA PAPER MODEL

TM

BIO-RAD

Bio-Rad

Laboratories, Inc.

(01)03610521157823