1. Production of Polyclonal Antibody against Mycoplasma ovipneumoniae in Rabbits Saroj Poudel Advisor: Dr. Jeffery J. Adamovicz Contributors: Dr. Gerard P. Andrews and his lab Department of Molecular Biology, Department of Veterinary Science, Honors Senior Project, UW.

2. Introduction O Big Horn Sheep O Polyclonal antibodies in rabbits O Diagnostic assay

3. Overview Protein extraction  Swabs or pure culture Immunization ELISA Affinity chromatography

4. Attachment to epithelial cells Close association of attachment structure

5. Protein Extraction Specific protein from the pathogen or Membrane protein fractionation SDS Page BCA Lowry absorbance test

6. 15 kd 25 kd 35 kd 50 kd 75 kd 100 kd 150 kd 225 kd Membrane Protein Prep

8. Immunization Adaptation Pre-bleed & Immunize Booster

9. Predicted chart for bleeding and immune response

10. ELISA Indirect Elisa  Primary antibody – rabbit antibody  Secondary antibody – Goat anti-rabbit antibody  TMB substrate

11. Affinity Chromatography Protein A chromatography Sepharose 4B chromatography

12. Conclusion Animal protocol Lab protocol Protein Membrane fractionation Bled Rabbits Immunized

13. Acknowledgements Dr. Jeffery J. Adamovicz Dr. Gerard P. Andrews and his lab members. Jake Berg

14. Supplements M. Pneumonia on Blood Agar Plate