The document summarizes a study examining whether the titer of autoantibodies against SELENBP1 changes as hens develop ovarian cancer tumors. The study found no significant difference in the percent change of optical density (OD) values between hens that developed ovarian cancer tumors and normal hens. While the hypothesis was that antibody titer would decrease as tumors developed, the titer remained fairly constant. However, other studies from Rush University suggest SELENBP1 autoantibodies may still have potential as an ovarian cancer biomarker. Further experiments are needed to draw definitive conclusions.

1. Autoantibody Biomarkers in Ovarian Cancer
Monica Muthaiya
Department of Pharmacology, Rush University
Faculty Advisor: Dr. Judith Luborsky
Background
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 (5). Ovarian cancer occurs when ovarian cells develop in an
unrestrained & unnatural manner (2). Ovarian cancer is most often seen
in women between the ages of 40 to 60 and rarely in adolescents.
The survival rate for ovarian cancer is 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 auto antibodies.
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 (3).
Hens are the only known spontaneous 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.
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. (1) They also showed that SELENBP1 is expressed in
both normal ovaries and ovarian tumors in the hen. (4) Similar to
humans, hens with ovarian tumors make auto antibodies 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. ELISA is a common procedure in determining antibody
titers.
Purpose & Hypothesis
Purpose: The aim was to determine if the titer of SELENBP1
autoantibody changes as the hens develop tumors.
Hypothesis: If ovarian cancer develops in a hen, then the titer
will decrease during the progression from normal ovary to tumor
occurrence.
Variables
Independent Variables: Hen with Ovarian Cancer or No Cancer
Dependent Variables: SELENBP1 Antibody Level and Titer
Procedure
1. Animals: White Leghorn laying hens (2.5 - 3 years old) were
maintained at the Poultry Research Farm of the University of
Illinois at Urbana-Champaign, under a controlled light regimen
(14 h light:10 h dark) with food and water provided ad libitum.
2. Hen Selection: Based on ultrasound morphology , hens with
normal ovaries were selected.
3. Blood Collection: Serum was serially collected at ~2-4 month
intervals. Blood was obtained from each hen’s wing vein and
serum stored at -80°C.
4. Experiment Completion: 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 as
described previously (ref) .
5. Immunoassay to detect autoantibodies: Conduct a standard
immunoassay to measure autoantibody against SBP1. Use
purified recombinant SBP1 as an antigen to capture the serum
antibody, and anti chicken IGY_HRP (horseradish peroxidase)
as the secondary antibody. to detect bound serum antibodies.
Read the optical density using a Spectrophotometer with
wavelength set to a 450nm and 580nm as a reference
wavelength.
6. Safety procedures: While conducting this experiment, all
safety protocols were followed. All materials were handled with
gloves while wearing a lab coat.
Data
* As the dilution increases, autoantibody signal decreases, like
expected. By testing different dilutions, you can reduce error in your
experimental design. When collecting data for different dilutions, I
observed the fading of the signal.
* In the Normal samples, 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.
* 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 titer in
comparison to the OVCA. The titer of SELENBP1 autoantibody
remains constant as the hens develop tumors.
Conclusion
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
decrease 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.
References
1. 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
2. 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
3. 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
4. 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
5. 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
The Effect of
Ovarian Tumor
Development
on Autoantibody
Titer
The Effect of
Ovarian Tumor
Development
on Autoantibody
Titer
Figure 1. Expression of SELENBP1 in normal hens
ovaries. Each row contains an H&E stained section
(A & D) and a corresponding example of the
SELENBP1 expression (B, C, E & F) at two
magnifications (20X and 120X). SELENBP1 is
predominantly found in surface epithelium cells
(arrows) of normal ovaries.
Acknowledgements:
I would like to extend my thanks to Dr. Judith Luborsky, Seby Edassery, Yi Yu and the faculty at the Department of Pharmacology: they gave me great advice on future plans for research, medicine, and experiments.
Figure 2: The figure on the left shows the
final data collected from this experiment. It
shows the percent change in OD value from
the first to last scan between OVCA and
normal hens within each dilution.
a | A shows an example of a high-grade
carcinoma, morphologically consistent with
the serous subtype, with a small follicle
towards the bottom left-hand corner.
b | B is an example of a low-grade
endometrioid carcinoma showing simple
glandular structures in a back- to-back
arrangement with little stroma intermixed
between the glands (scale bar = 100 μm).