1. Monoclonal Antibody Production Against Synthetic Peptides Representing PrP c and Recombinant Prion Proteins (rPrP) By Kaitlin McDaniel, Dept. of Molecular Biology and Microbiology -with- Dr. E. Lee Belden, Dept. of Veterinary Sciences and Matt Hille, Dept. of Veterinary Sciences

2. Introduction Chronic wasting disease (CWD) is a slow, fatal neurodegenerative disorder that affects deer, elk, and moose Chronic wasting disease (CWD) is a slow, fatal neurodegenerative disorder that affects deer, elk, and moose CWD is thought to be caused by misfolded infectious isoforms of certain cellular proteins called prions CWD is thought to be caused by misfolded infectious isoforms of certain cellular proteins called prions Photo: Delaware Game and Fish Department

3. Prion Protein Mule Deer prion protein variant Mule Deer prion protein variant Amino acid position 225 – phenylalanine (F)/serine (S) Amino acid position 225 – phenylalanine (F)/serine (S) Homozygous deer (225SS) are 30 times more likely to be CWD positive than heterozygous deer (225SF). Homozygous deer (225SS) are 30 times more likely to be CWD positive than heterozygous deer (225SF). 225SS deer also have a shorter incubation period 225SS deer also have a shorter incubation period Diagrams courtesy of: http://wps.prenhall.com/wps/media/objects/476/488316/Instructor_Resources/Chapter_19 /FG19_06-10T02.JPG

4. Importance of Study The purpose of this study is to answer a question – Can monoclonal antibodies be produced against proteins encoded by the two alleles? The purpose of this study is to answer a question – Can monoclonal antibodies be produced against proteins encoded by the two alleles? Currently not possible to immunologically distinguish between the two proteins Currently not possible to immunologically distinguish between the two proteins If we can distinguish between the two proteins, we will be able to study the difference in the susceptibility of mule deer to CWD If we can distinguish between the two proteins, we will be able to study the difference in the susceptibility of mule deer to CWD

5. Previous Work We began using synthetic peptides that represented the prion protein as the immunogen We began using synthetic peptides that represented the prion protein as the immunogen Hybridomas -Two mice were vaccinated with synthetic peptides Peptide S: Q-M-C-I-T-Q-Y-Q-R-E- S -Q-A-Y-Y-Q-R-G-A-S Peptide F: Q-M-C-I-T-Q-Y-Q-R-E- F -Q-A-Y-Y-Q-R-G-A-S Previous Work

6. Methods Harvested splenocytes were fused with SP2 myeloma cells to produce hybridomas Harvested splenocytes were fused with SP2 myeloma cells to produce hybridomas Diagram from: http://www.bio.davidson.edu/Courses/Molbio/MolStudents/01kewestbrook/ hybridoma.gif

7. Methods Indirect ELISA used to screen for antibodies Indirect ELISA used to screen for antibodies Diagram from: http://www.abcam.com/index.html?pageconfig=resource&rid=12065 Synthetic Peptide Produced Monoclonal Antibody Conjugated Secondary Antibody Horseradish Peroxidase

8. Methods Western Immunoblots Western Immunoblots Samples included recombinant proteins, PrP 225F (rPrP 225F) and PrP 225S (rPrP 225S) Samples included recombinant proteins, PrP 225F (rPrP 225F) and PrP 225S (rPrP 225S) Diagram from: http://www.genscript.com/images/L00204-1.http://www.genscript.com/images/L00204-1. Protein samples are separated by gel electrophoresis Proteins are transferred to a PVDF membrane Monoclonal antibodies Conjugated secondary antibody Development of blot

9. Previous Results ELISA ELISA Wells from primary plates identified and expanded Limiting dilutions performed ELISA screen on limiting dilutions Monoclones identified and expanded Monoclones screened for cross reactivity Clones with mono- specificity were determined

10. Western Immunoblots Western Immunoblots The antibodies do recognize both recombinant alleles The antibodies do recognize both recombinant alleles Previous Results “A” rPrP 225F “A” rPrP 225S “B” rPrP 225F “C” rPrP 225F “D” rPrP 225F “E” rPrP 225F “F” rPrP 225F “B” rPrP 225S “C” rPrP 225S “D” rPrP 225S “E” rPrP 225S “F” rPrP 225S Monoclonal Antibodies rPrP 225F rPrP 225S Negative Control 60 kDa 20 kDa 30 kDa 40 kDa 50 kDa 20 kDa 30 kDa 40 kDa 50 kDa

11. Conclusion ELISA – Antibodies recognize but do not distinguish between the two synthetic peptides ELISA – Antibodies recognize but do not distinguish between the two synthetic peptides Western Immunoblots – Antibodies recognize but do not distinguish between the two recombinant proteins Western Immunoblots – Antibodies recognize but do not distinguish between the two recombinant proteins Why did we get these results? Why did we get these results?

12. Hypothesis Hypothesis 1: We hypothesized that monoclonal antibodies can be produced against rPrP. Hypothesis 1: We hypothesized that monoclonal antibodies can be produced against rPrP. Hypothesis 2: We also hypothesized that the monoclonal antibodies will be capable of distinguishing between the two alleles. Hypothesis 2: We also hypothesized that the monoclonal antibodies will be capable of distinguishing between the two alleles.

13. Current Results Screening of primary plates Screening of primary plates 14 F clones 14 F clones 12 S clones 12 S clones Limiting Dilutions Limiting Dilutions

14. Further Work Cross reactive ELISA Cross reactive ELISA Use Western blots to confirm ELISA results Use Western blots to confirm ELISA results Can it be used as a marker for PrP in diagnosis? Cost efficient for Wyoming State Vet Lab? Can it be used as a marker for PrP in diagnosis? Cost efficient for Wyoming State Vet Lab?

15. Acknowledgements Dr. E. Lee Belden – Project Mentor, Department of Veterinary Sciences, University of Wyoming Dr. E. Lee Belden – Project Mentor, Department of Veterinary Sciences, University of Wyoming Matt Hille – Graduate Student Mentor, Department of Veterinary Sciences, University of Wyoming Matt Hille – Graduate Student Mentor, Department of Veterinary Sciences, University of Wyoming Dr. Jean Jewell – Senior Research Scientist, Department of Veterinary Sciences, University of Wyoming Dr. Jean Jewell – Senior Research Scientist, Department of Veterinary Sciences, University of Wyoming Zackie Salmon – McNair Scholars Program Zackie Salmon – McNair Scholars Program Susan Stoddard – McNair Scholars Program Susan Stoddard – McNair Scholars Program University of Wyoming NSF EPSCoR Program University of Wyoming NSF EPSCoR Program Family and Friends Family and Friends

16. Questions?