Muthaiya

ABSTRACT

The Illinois Junior Academy of ScienceThis form/paper may not be taken without IJAS authorization.

CATEGORY : Health Science STATE REGION # 6SCHOOL : Adlai E. Stevenson

High School IJAS SCHOOL # 6092

CITY/ZIP : Lincolnshire, 60069 SCHOOL PHONE # (847) 415-4000SPONSOR: 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.

SAFETY SHEET

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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 Plate4. 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. Retrieved from http://www.medicalnewstoday.com/releases/225318.php

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 Sterility, 94(7), 2636–2641. Retrieved from http://www.sciencedirect.com/science/article/pii/S0015028210006096

4. Immunotherapy. (2012, 5 09). Retrieved from http://www.cancer.org/treatment/treatmentsandsideeffects/treatmenttypes/immunotherapy/immunotherapy-toc

5. Johnson, K. A. (2009, 2). The standard of perfection: Thoughts about the laying hen model of ovarian cancer. Retrieved from http://cancerprevention.aacrjournals.org/content/2/2/97.full

6. Johnson, P. A. (2013). The hen as a model of ovarian cancer.Nat Rev Cancer, Retrieved from http://www.nature.com/nrc/journal/v13/n6/full/nrc3535.html

7. Jackson, E. (2006). Ca125 expression in spontaneous ovarian adenocarcinomas from laying hens.Department of Poultry Science, Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16942793?dopt=Abstract

8. Jackson E, Anderson K, Ashwell C, et al. CA125 expression in spontaneous ovarian adenocarcinomas from laying hens. Gynecol Oncol 2007;104:192–8.

9. Kim, A., Ueda, Y., Naka, T., & Enomoto, T. (2012). Therapeutic strategies in epithelial ovarian cancer. Experimental and Clinical Cancer Research, 31. Retrieved from http://www.jeccr.com/content/31/1/14

10. Medline Plus, (2012). Antibody. Retrieved from U.S. National Library of Medicine website: http://www.nlm.nih.gov/medlineplus/ency/article/002223.htm

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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

13. Slotman , B. J. (1988). Ovarian cancer (review). Etiology, diagnosis, prognosis, surgery, radiotherapy, chemotherapy and endocrine therapy. Anticancer Research. Retrieved from http://europepmc.org/abstract/MED/3291746/reload=0;jsessionid=bxXI0bWGzq7oiufhjjE5.36

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 from http://www.ncbi.nlm.nih.gov/pubmed/18364012

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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