Here are the materials used in this experiment: - Chicken serum samples from hens with and without ovarian cancer - Purified recombinant Selenium Binding Protein 1 (SBP1) as an antigen - Anti-chicken immunoglobulin G horseradish peroxidase (IGY_HRP) as a secondary antibody - ELISA plates - Bicarbonate buffer, ELISA buffer, TMB (3,3',5,5'-tetramethylbenzidine), and sulfuric acid solutions - Spectrophotometer - Pipettes, tubes, gloves, lab coat for handling materials safely Procedure: 1. Serum samples were collected from hens at various time points as they developed or did

1. Muthaiya
ABSTRACT
The Illinois Junior Academy of Science
This form/paper may not be taken without IJAS authorization.
CATEGORY : Health Science STATE REGION # 6
SCHOOL : Adlai E. Stevenson
High School
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CITY/ZIP : Lincolnshire, 60069 SCHOOL PHONE # (847) 415-4000
SPONSOR: Mrs. Palffy
MARK ONE: EXPERIMENTAL INVESTIGATION
NAME OF SCIENTIST: Monica Muthaiya GRADE : 11th
* If this project is awarded a monetary prize, the check will be written in this scientist's name, and it will be his/her responsibility to
distribute the prize money equally among all participating scientists.
PROJECT TITLE: The Effect of Ovarian Cancer Development on SELENBP1 Optical
Density Concentration.
Purpose: The purpose of this experiment is to determine the effect of ovarian cancer
development on the antibody Selenium Binding Protein 1’s optical density concentration. The
aim was to determine if the OD concentration of the SELENBP1 autoantibody changes as the
hens develop ovarian tumors. By tracking the changes over time, scientists and researchers can
determine whether SELENBP1 is a valid biomarker of ovarian cancer.
Procedure: At the completion of the study (6-14 months) hens were euthanized, according to the
approved Institutional Animal Care and Use Committee (IACUC) protocol. Ovarian tissue and
blood was collected. The presence of a tumor was determined by gross morphology and
histology. Standard immunoassays were conducted to measure autoantibody against SBP1.
Purified recombinant SBP1 as an antigen was used to capture the serum antibody, and anti-
chicken IGY_HRP (horseradish peroxidase) as the secondary antibody to detect bound serum
antibodies. The optical density was read using a Spectrophotometer with wavelength set to a
450nm and 580nm as a reference wavelength.
Conclusion: Based solely off the data extracted in this experiment, there is no significant
difference in percent change OD values between the hens with OVCA and the control group. The
percent change in OD Value remained fairly close to 1. The hypothesis is mainly refuted, as the
titer didn’t significantly increase or decrease. Further experiments will need to be executed to
come to a decisive conclusion. However, various papers written by the Department of
Pharmacology at RUSH University suggest that SELENBP1 autoantibody may be a potential
biomarker for Ovarian Cancer.
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SAFETY SHEET
The Illinois Junior Academy of Science
Directions: The student is asked to read this introduction carefully, fill out the bottom of
this sheet, and sign it. The science teacher and/or advisor must sign in the indicated space.
Safety and the Student: Experimentation or design may involve an element of risk or injury to
the student, test subjects and to others. Recognition of such hazards and provision for adequate
control measures are joint responsibilities of the student and the sponsor. Some of the more
common risks encountered in research are those of electrical shock, infection from pathogenic
organisms, uncontrolled reactions of incompatible chemicals, eye injury from materials or
procedures, and fire in apparatus or work area. Countering these hazards and others with suitable
controls is an integral part of good scientific research.
In the box below, list the principal hazards associated with your project, if any, and what specific
precautions you have used as safeguards. Be sure to read the entire section in the Policy and
Procedure Manual of the Illinois Junior Academy of Science entitled "Safety Guidelines for
Experimentation" before completing this form.
While conducting this experiment, I handled chicken serum, buffer
solutions, wash solutions, and the ELISA plates with gloves at all times to
prevent the transmission of disease and contamination. I also wore a lab
coat at all times to prevent chemical spills on my body. Long pants and
closed toed shoes were also worn to prevent spills, as well. I kept the
station I worked at very clean and cleaned all the measuring equipment in
a sink with deionized water and antiseptic soap. All the plates and serum
dilutions were properly disposed of in a biohazard bag.
SIGNED _________________________________________________
Student Exhibitor(s)
SIGNED _________________________________________________
Sponsor *
*As a sponsor, I assume all responsibilities related to this project.
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The Effect of Ovarian Cancer
Development on SELENBP1 Optical
Density Concentration
By: Monica Muthaiya
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Table of Contents
Page 1: Abstract
Page 2: Safety Sheet
Page 3: Title Page
Page 5: Acknowledgements
Page 6 & 7: Purpose and Hypothesis
Pages 8 – 12: Review of Literature
Page 13 – 16: Materials and Procedure
Page 17 - 19: Results
Page 20: Conclusions
Page 21 - 22: References
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Acknowledgements
I would like to thank my mentors for the duration of this experiment, Dr.
Luborsky, Dr. Yi Yu, Seby Edassery, and the faculty at the Department of
Pharmacology. Dr. L allowed me to conduct research at Rush University in her
laboratory and I would not have gained this experience or have this project to
showcase if it wasn’t for her. Seby helped me around the lab and ensured that I
knew what I would be doing each day and that I would remain safe at the lab. Dr.
Yi Yu gave me great background knowledge of what I would be doing and helped
me startup my project.
I would also like to thank my sponsor, Mrs. Palffy, for taking time out of her busy
schedule to help me with my project. She was always very patient and helpful. I
am very thankful to have such wonderful sponsor, and even though we were all
stressed the month of science fair, Mrs. Palffy managed to help everyone to the
best of her ability. I would also like to thank my parents, who were encouraging of
doing research at a University and offered constructive criticism.
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Purpose:
The purpose of this experiment was to determine the effect of Ovarian Cancer
development on Selenium Binding Protein 1’s optical density concentration. The aim was
to determine if the OD concentration of the SELENBP1 autoantibody changes as the hens
develop ovarian tumors. By tracking the changes over time, scientists and researchers can
determine whether SELENBP1 is a valid biomarker of ovarian cancer. There is currently
no viable biomarker for ovarian cancer, but research from Dr. Judith Luborsky’s Lab at
RUSH University has shown that SELENBP1 is a possible autoantibody biomarker for
ovarian cancer. If this is confirmed, then there can be tests created in order to detect
ovarian cancer at very early stages in patients. This would greatly reduce the deaths
caused by late detection and ultimately lead to a cure for ovarian cancer.
Hypothesis:
If ovarian cancer develops in a hen, then the optical density (OD) of Selenium
Binding Protein 1 will increase during the progression from normal ovary to tumor
occurrence. As an ovarian tumor develops, the human body makes more autoantibody in
order to protect against the growing disease (ovarian cancer). Since this autoantibody can
be traced in one’s body, SELENBP1 may possibly be considered the first biomarker for
ovarian cancer.
Independent Variables: Hens with Ovarian Cancer vs. Hens without Cancer
Dependent Variables: SELENBP1 Optical Density Concentration (measures
autoantibody level in hens; greater the OD, more auto antibodies in the blood).
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Constants:
• The type of chicken (White Leghorn laying hens (2.5 - 3 years old) ).
• Light regimen (14 h light:10 h dark) with food and water provided ad libitum.
• Serum was serially collected at ~2-4 month intervals. Blood was obtained from
each hen’s wing vein and serum stored at -80°C.
• Amount of purified recombinant SBP1 and anti-chicken IGY_HRP (horseradish
peroxidase).
• Spectrophotometer read with wavelength set to a 450nm and 580nm as a
reference wavelength.
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Review of Literature
Cancer is defined as a malignant growth or tumor caused by abnormal and
uncontrolled cell division that may profuse to other parts of the body through the
lymphatic system or the blood stream. Ovarian cancer is a type of cancer that forms in
tissues of the ovary, in which the ova are formed (Tan & Zhang, 2008). Ovarian cancer
occurs when ovarian cells develop in an unrestrained & unnatural manner (Kim, 2012).
Ovarian cancer is most often seen in women between the ages of 40 to 60 and rarely in
adolescents. Ovarian cancer has the highest mortality of all cancers concerning the
female reproductive system. This is largely due to the lack of early symptoms and
effective OVCA screening tests. Thus, ovarian cancer often is diagnosed at very
advanced stages, after the cancer has metastasized and spread beyond the ovary (Ovarian
Spores, 2012)
The survival rate for ovarian cancer, in humans, is very low. Most women are
diagnosed at late stages after tumor metastasis and less than 20% survive 5 years. Early
detection methods are clearly needed. A substance that could be measured in blood
would be efficient, but most tumor proteins have not proven useful. However, patients
have a characteristic immune response to cancer, that can be measured in blood as
autoantibodies. Information on the behavior of specific antibodies and their ability to
predict ovarian cancer is needed for antibodies to be used clinically. Studies over time
are difficult in humans (Slotman, 1988). Many new treatment options for some women
with aggressive types of ovarian cancer have proven ineffective. The symptoms of
onset/early OVCA include, abdominal pain, bloating, and feeling full. These symptoms
are very nonspecific to solely OVCA, and that is what researchers are having troubles
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with. Using these nonspecific symptoms to detect OVCA would be inadequate, as these
symptoms can be due to various other diseases and conditions, as well (OVCA:
Prevention, genetics, causes, n.d.).
There are no screening tests for OVCA, but the test OVA1 allows for women who
have ovarian cancer to be given a risk label. This test measures the levels of four basic
proteins in the blood. The levels of these proteins, when looked at combined, place the
women into low risk and high risk categories. The women labelled high risk patients are
more likely to have a cancer, and should consider treatment options that are available.
New chemotherapy (chemo) drugs and drug combinations are being tested. The drugs
trabectedin (Yondelis®) and belotecan have shown promise in some studies. Targeted
therapy is a newer type of cancer treatment that uses drugs or other substances to identify
and attack cancer cells while doing little damage to normal cells. Each type of targeted
therapy works differently, but they all attack the cancer cells' inner workings − the
programming that makes them different from normal, healthy cells (Diagnosis, 2011).
These are some treatment options available, but the detection of OVCA is a prevalent
problem today. Researchers have been looking into antibodies and autoantibodies as
biomarkers for OVCA in humans, and some have proven to be effective.
An antibody is a protein produced by the body's immune system when it detects
harmful proteins/substances, called antigens. Antigens include mainly microorganisms
(bacteria, fungi, parasites, and viruses) and chemicals (Medline Plus, 2012). Antibodies
are large Y-shaped proteins which help identify and remove foreign antigens. Each type
of antibody is unique and defends the body against a specific type of antigen. This is
because the two tips of its “Y” are specific to each antigen, allowing different antibodies
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to bind to different foreign antigens (What are antibodies?, n.d.). Antibodies can create
large clumps of antigens by creating a chain. This allows for macrophages, white blood
cells within tissues, and killer T cells, a type of lymphocyte (itself a type of white blood
cell) that play a central role in cell-mediated immunity, to later come by and easily kill
these bacteria. Immunotherapy is defined as the treatment of disease by inducing,
enhancing, or suppressing an immune response (Immunotherapy, 2012). There are
currently many antibody-based immunotherapies for ovarian cancer. As mortality from
advanced OVCA in humans remains abnormally high, new therapies are required to be
innovative, using an array of approaches to treat advanced stages. Immunotherapy
presents an alternative and rational unique approach for ovarian cancer, based on
supporting a protective role of the immune system against these cancers and “on the
clinical success of immunotherapy in other malignancies (Tse, 2013).” The future of
OVCA immunotherapy is unclear, but seems to hold a promising new outlook on the use
of antibodies treat OVCA.
Autoantibodies are antibodies formed in response to and reacting against an
antigenic constituent of one's own tissues or proteins. Usually, autoantibodies are
consistently eliminated by the immune system’s self-regulatory process. This process
sometimes fails, and antibodies will begin to react to self constituents. Autoantibodies
damage body tissues by bringing phagocytosis (ingestion) or lysis (bursting) of healthy
cells, often targeting blood cells (Autoantibody, n.d.).
The optical density of a material relates to the sluggish tendency of the atoms of a
material to maintain the absorbed energy of an electromagnetic wave in the form of
vibrating electrons before reemitting it as a new electromagnetic disturbance. Optical
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density, measured in a spectrophotometer, can be used as a measure of the concentration
of protein in a sample. As visible light passes through a well, the light is scattered.
Greater scatter indicates that more bacteria or other material is present. The amount of
light scattered can be measured in a spectrophotometer. Typically, when working with a
particular type of serum, a researcher determines the optical density at a particular
wavelength, which correlates with the different phases of growth.
As previously mentioned, a spectrometer is an instrument used to measure
properties of light over a specific portion of the electromagnetic spectrum, typically used
in spectroscopic analysis to identify materials or optical density values of certain protein
samples.
Hens are the only known spontaneous animal model of ovarian cancer that
develop the cancer at a high rate. Using hens allows researchers to identify processes
involved in spontaneous tumor formation and progression, and to identify possible
biomarkers that predict the tumors. Hens ovulate regularly and this is one of the
appealing aspects (to researchers) in using a hen model for OVCA. Around 50% of hens
will spontaneously develop ovarian cancer due to ovulating regularly. This makes them
ideal models, since there is a balance of test subjects (cancerous and normal hens) and the
ovulation rate is highly correlated with the risk of human OVCA. Likewise in women,
OVCA in hens is highly age-related. OVCA in hens is also grossly and histologically
similar to that in humans. In both subjects, the cancer metastasizes to similar tissues,
demonstrating a rapid growth commonly seen in OVCA tumors (Johnson, 2013). Cancer
prevention scientists have used animal models to study the role of genes, molecular
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pathways and networks, and environmental factors that relate to carcinogenesis. These
studies are also designed to qualify the animal model as an eventual source of preclinical
evidence that will justify the introduction of promising cancer prevention agents into
clinical trials (Johnson, 2009). Despite the similarities of ovarian carcinogenesis between
chickens and humans, there is some doubt as to whether hens will serve as the best model
for OVCA in humans. (Jackson, 2007) The two have very different biological complexes
and researches are unsure as to whether chickens and humans are sufficiently biologically
similar to allow for the identification of OVCA to be determined (Jackson, 2006).
Dr. Luborsky’s laboratory showed for the first time that SELENBP1 is an auto
antigen in ovarian failure associated with an autoimmune disease in young women.
(Edassery, 2010) They also showed that SELENBP1 is expressed in both normal ovaries
and ovarian tumors in the hen. (Stammer, 2008) Similar to humans, hens with ovarian
tumors make autoantibodies to SELENBP1 making it possible to examine the antibody
response as hens develop tumors. The antibody titer is a measurement of how much
antibody an organism has produced, expressed as the greatest dilution that still gives a
positive result.
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Materials:
- 1L bicarbonate buffer
- 1L ELISA buffer
- 100mL TMB
- 30 mL sulfuric acid
- 1L Baxter Water (shown at bottom)
- anti chicken IGY_HRP (10 ul)
- cancerous chicken sera (480 ul from 3 separate hens)
- normal chicken sera (480 ul from 3 separate hens)
- VersaMax ELISA Microplate Reader (upper right picture)
- (-80 degrees Celsius) freezer
- Pipets (10 ul, 50 ul, 100 ul) (upper left picture)
- Pipet tips
- Incubation box to be kept at room temperature (include pic)
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IMMUNOASSAY PROCEDURE:
1. Setting up Antigen Solution on ELISA Plate (Known as Capturing the
Antigen):
• Shown in the table below are the arrangements of reagents on the ELISA Plate.
The rows are labeled with letters and columns with numbers to help specify and
make clear which solution goes in which well.
1. Make Solution 1 (200ng): Create 2 n/ul Solution: Take 48 ul of SBP (250
ng/ul) in 6ml of bicarbonate buffer to create 200ng/100ul per well
2. Make Solution 2 (100ng): Create 1 n/ul Solution: Take 2 ml of the above
solution in 1 ml of bicarbonate buffer to create 100ng/100ul per well
3. Leave columns 1-4 blank (0ng solution) on the ELISA Plate
4. Add 100 ul of the according solutions in each well as designated by the
table below.
5. Incubate overnight in 4 degrees Celsius
2. Washing & Blocking:
1. After incubation, quickly turn the plate upside down in the sink to let all
unbound protein out
2. Place 200 ul of Baxter water in each well then turn the plate upside down
in the sink again
3. Flip the plate upside down on some paper towels to let the leftover water
run out
4. Place 200 ul of 5% BSA in each well, then incubate the plate in room
temperature for two hours
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3. Create Dilutions of Seras:
• Make the dilutions during the blocking
• Shown below are the instructions to make a set of dilutions for one serum
(extracted from hen ovaries) in a 2 ml tube
• Make the dilutions during the blocking
• Repeat this step with the 4 different sera to get a total of 16 dilutions in 16
2ml tubes
• S1 = DOD 23
• S2 = DOD 23 3rd
Scan
• S3 = DOD 17
• S4 = DOD 17 3rd
Scan
• Make sure to label your dilution bottles with either s1,s2,s3 or s4 and the
dilution (200,1000,2000, or 5000)
4. Washing & Loading Dilutions of Primary in Wells:
• Take the plate and quickly turn it upside down in the sink and wash each well 300
ul of the ELISA wash buffer
• Repeat the washing 2 more times
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• Load 100 ul/well of the dilutions shown above
• Incubate in room temperature for 90 minutes
5. Washing & Adding Secondary in Wells:
• Take the plate and quickly turn it upside down in the sink and wash each well 300
ul of the ELISA wash buffer
• Repeat the washing 2 more times
• Add 100 ul of the secondary (anti chicken IGY_HRP that is diluted 1:5000 in
ELISA WASH buffer) to each well
• Incubate plate at room temperature for 60 minutes
6. Washing & Reading Concentrations:
• Take plate and quickly turn upside down in sink
• Wash the plate 2x with 300 ul of the ELISA buffer in each well
• Wash the plate 4x with 300 ul of water in each well
• Add 100 ul of TMB in each well
• Incubate plate for 15 minutes in the dark (drawer)
• Take plate out and add 50 ul of Sulfuric Acid (.2n) to each well
• Read plate at 450/580 wavelength
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Results
• The x-axis represents the individual chicken number. The Department of Defense
funded this research, so all chickens were numbered with DOD then a number.
• The y-axis represents the antigen dilution.
• The figure above shows the final data collected from this experiment. It shows the
average percent change in SELENBP1 OD (autoantibody) value from the first to
last scan in hens with ovarian cancer and normal hens within each dilution.
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• A consistent change of at least 3% is considered to be a significant increase in OD
concentration.
As the antigen dilution increases, the autoantibody OD signal decreases, as
expected. By testing different antigen dilutions, it can reduce errors in experimental
design. Since the signal fades out with higher dilutions, it is ensured that there are not
too many errors with the plate and sera solutions.
In the normal chicken samples (sample of chickens without ovarian cancer), the
regularity between the dilutions remained as expected, solely with DOD63 & DOD33
being exceptions. DOD63 had a 9 month difference from the first scan to the final scan.
The dates were not recorded when collecting the data, so the data from this hen is not
very reliable. In the OVCA samples, the samples were very consistent, significantly
fading out on all but sample DOD62, suggesting that errors may have occurred and the
data from this hen may not be reliable either. DOD62 showed abnormalities within
dilutions, suggesting that errors may have occurred: while creating the dilution, in the
ELISA wells being tested, when extracting a sample from the hen, or within the hen
itself. DOD62 was diagnosed with stage 3 mucinous OVCA, a cancer where the
mucinous tumors are filled with a mucous-like substance. This type of OVCA is
unpredictable and may have led to the increase in signal, as dilution increases. From the
data above, OVCA samples contained very minimal sources of error within dilution
variations.
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The data suggests that there is no significant difference in percent change in OD
value from hens with OVCA and the control group. The normal hens have great
fluctuation and higher signals than the OVCA group. Therefore, normal hens have a
lower OD concentration in comparison to the OVCA. The titer of SELENBP1
autoantibody remains constant as the hens develop tumors.
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Conclusions
The aim was to determine if the titer of SELENBP1 autoantibody changes as the
hens develop tumors. Based solely off the data extracted in this experiment, there is no
significant difference in percent change OD values between the hens with OVCA and the
control group. However, various papers written by the Department of Pharmacology at
RUSH University suggest that SELENBP1 autoantibody may be a potential biomarker
for Ovarian Cancer.
The original hypothesis stated that “If ovarian cancer develops in a hen, then the
titer will increase during the progression from normal ovary to tumor occurrence.” The
percent change in OD Value remained fairly close to 1. The hypothesis is mainly refuted,
as the titer didn’t significantly increase or decrease. Further experiments will need to be
executed to come to a decisive conclusion.
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References
1. Autoantibody. (n.d.). Retrieved from
http://www.britannica.com/EBchecked/topic/44700/autoantibody
2. Diagnosis, Q. (2011, 5 14). Ova1 blood test detects ovarian cancer more accurately
than medically accepted ca 125 method for evaluating women with ovarian mass.
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3. Edassery, S. L., Shatavi, S. V., Kunkel, J. P., Hauer, C., Brucker, C., Penumatsa, K.,
Yu, Y., Dias, J. A., & Luborsky, J. L. (2010). Autoantigens in ovarian autoimmunity
associated with unexplained infertility and premature ovarian failure. Fertility and
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/immunotherapy-toc
5. Johnson, K. A. (2009, 2). The standard of perfection: Thoughts about the laying hen
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11. Ovarian cancer: Prevention, genetics, causes. (n.d.). Retrieved from
http://www.cancer.gov/cancertopics/prevention-genetics-causes/ovarian
12. Ovarian spores. (2012). Retrieved from http://trp.cancer.gov/spores/ovarian.htm
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E5.36
14. Stammer, K., Edassery, S. L., Barua, A., Bitterman, P., Bahr, J. M., Hales, D. B., &
Luborsky, J. L. (2008). Selenium-Binding Protein 1 expression in ovaries and ovarian
tumors in the laying hen, a spontaneous model of human ovarian cancer. Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/18272210
15. Tan , E. M., & Zhang, J. (2008). Autoantibodies to tumor-associated antigens:
reporters from the immune system. Immunological Reviews, 222(1), 328-340. Retrieved
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16. Tse, B. W. C. (2013). Antibody-based immunotherapy for ovarian cancer: where are
we at?. Oxford University Press, Retrieved from
http://annonc.oxfordjournals.org/content/early/2013/11/26/annonc.mdt405.abstract
17. What are antibodies?. (n.d.). Retrieved from http://pdl.com/technology-
products/what-are-antibodies/
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