1. Isolation of Human Anti-Pig Antibodies in Vitro
Ruben Naoye1, Ray Chihara, MD2, Leela Paris, PhD2, Zheng-Yu Wang, PhD2, Richard Sidner, PhD2, Sue Downey2, Lisset Martinez2, and Christopher Burlak, PhD2.
1Brebeuf Jesuit Preparatory School, Indianapolis, IN, and 2Department of Surgery, Indiana University School of Medicine, Indianapolis, IN
Discussion
Abstract
Our aim is to prevent rejection of the pig organ by modifying the genes in the pig. The patient will nourish the pig liver rather than reject it and immunosuppressive therapy may be reduced or avoided. The pigs that are used in our Xenoimmunology lab are called “Gal Knockouts” because the pigs have a gene known as alpha galactose 1, 3 galactose syl transferase taken-out (knocked out). We used genetically modified pigs expressing a gene for a human protein to disguise the pig liver to prevent rejection. We harvested pig organs and used their tissue to examine the cells and their activity with human antibodies. First, the organs were harvested then we isolated protein from biopsies by homogenizing the tissue in the presence of protease inhibitors and 2ME in 1.5 ml tubes. Second, we heated the solution to 95 degrees Celcius, and then the samples were then ready for gel electrophoresis. We collected human antibodies that bound to cultured GTKO pig fibroblast cells to use in our Westernblot analysis. Next, they were placed in tubes to be used for primary antibodies after the membrane transfer was complete. The gel was processed for 45 minutes at 200 volts and a fourth of it was cut off and stained in coomassie blue reagent; the other three fourths of the gel was western blotted for an hour at 100 volts. After the proteins were transferred to the PVDF membrane, it was placed in a blocking buffer of 5% BSA. The primary and secondary antibodies were placed on the membrane in one hour increments. After scanning, our results proved that human antibodies eluted from GTKO pig fibroblasts did attach to the kidney and liver samples. The issue with the attachment was that the antibodies attached more than expected, so specific antibody banding (indicating rejection) could not be isolated.
Methods
After the organs were harvested we ground up the samples and mixed them with chemical 2ME in 1.5 ml tubes
Cell culture samples of fibroblast cells were then needed to have antibodies attached
The solution was heated to 95 degrees Fahrenheit to prepare samples for gel electrophoresis
The gel was processed for 45 minutes at 200 volts and a fourth of it was cut off and stained in a coomassie blue liquid
The other three fourths of the gel was western blotted for an hour at 100 volts
Every 20 minutes we diluted the coomassie blue liquid with sterilized milli-q water proteins could be seen easier.
After the proteins were transferred to the scanning membrane, we placed them in a blocking buffer of 5% BSA
The primary and secondary antibodies were placed on the membrane in one hour increments
The membrane was scanned using an odyssey infrared scanner
Further study needs to be conducted to identify pig genes that are rejected in the human host after xenotransplantation. The knowledge gained will help overcome the obstacles preventing xenotransplantation.
Conclusions
In the end, non-specific antibodies bound to the Western blot and made it difficult to analyze real xenoantigns. More thorough washing conditions and longer incubation times on the Western blot may lead to a better solution.
In the end I was able to grasp the critical thinking skills of a scientist.
Results
Introduction
100,000 Americans awaiting solid organ transplant
Donors January - April ,490
Xenotransplantation (transplanting pig organs) into human hosts may be a solution
Transplants January - April ,055
Current waiting list candidates as of (7/21/11) 72,345
Microscopic pig fibroblast cells
Objective
Our aim is to modify the genes in the pig to prevent rejection of the pig organ
by the human host.
250
250
250
250
150
250
250
150
Protein Molecular Weight Marker and Western blots
150
150
100
150
100
100
150
100 75
100 75
100 75 75 75 50 75 50 50 50 37 50 37 50 37 37 37 37 25 25 25 20 20 25 20 25 25 20 20 15 15 20 15
Acknowledgements 10 15 15 10 15 10 10 10 10
Project SEED
Indiana University Department of Surgery Everyone in the lab
Parents: Marsha/Greg Aunt/Uncle: Glenda/George
Unbound pig Ab
Eluted human Ab
Eluted pig Ab
Unbound human Ab
Eluted PBS (Sterile)
New Human
Dark bands represent secondary human antibodies on Western blot gel electrophoresis
References
Burlak C et al. The fate of human platelets perfused through the pig liver: implications for xenotransplantation. Xenotransplantation.2010;17:
Hunter P. Xeno’s paradox. EMBO Reports ;10(6):554-7.
TECTOR