1. Production of Monoclonal Antibodies Jose Baeza California State University, Long Beach Mentor: Dr. Zhang

2. Introduction Background Main goals of the experiment Materials and Methods Results Conclusions Acknowledgements

3. Background Candida albicans - Most common cause of opportunistic disease in humans - Serious disease in the immunocompromised can result in death. www.cbr.ncr.ca

4. Background cont. The role of Candida-specific antibodies in host defense against disseminated candidiasis is not well understood. Previous Studies –Passive transfer of antibodies failed to protect mice. 1 –Specific antibodies may block phagocytosis of C. albicans, may enhance severity of disease. 2

5. Background Cont. Previous studies cont. –Mourad and Friedman showed that specific antibodies may be protective against disseminated candidiasis. 3 –Persall obtained similar evidence for a protective effect of antibodies. 4

6. Goals of experiment We hypothesize that only certain kinds of surface antigens may induce protective antibody response. Thus the goal of the experiment is to produce antibodies that will bind to surface antigens of C. albicans.

7. Materials & Methods Hybridoma cells Error Prone PCR Vector with amp marker Transformation into E. coli cells ELISA assay

8. Hybridoma cells

9. Monoclonal antibodies www.fucell.comwww.whfreeman.com

10. Error prone PCR Introduces random mutations into the variable regions. –Template DNA –Taq DNA polymerase –Cycle rxn 30X –Isolate product & Digest with appropiate enzyme. Www.nottingham.ac.uk

11. Vector DNA from Error Prone PCR. –Product digested with enzyme such as EcoRI. –Cloned into vector with marker. These procedures can be done with Ligation kits from New England biolabs Www.bbrp.llnl.gov

12. Transformation Electroporation –Mix Vector & Electrocompetent cells. –Apply voltage ~ 17000v –Add SOC media –Incubate for 1hr –Plate on selective media. www.agr.okstate.edu

13. Transformation cont. www.eppendorfsi.com

14. Transformed cells Successful electroporation colonies can then be grown in culture. The supernatent of the culture can then be tested for antibodies. Www.ultranet.com

15. ELISA Www.uoguelph.ca

16. Results Color indicates the there is antibodies. –They have different affinities. –Clone #5 has the greatest affinity. –Clone #4 has no affinity for the antigen. 1234567812345678 www.mds.qmw.ac.uk

17. Results cont.

18. Conclusion The results were as expected, from the ELISA assay it is evident that the mutant antibodies did bind the antigen. The antibodies can now be produced in sizable quantities, so that they can be used in future experiments.

19. Future Experiments One possible experiment would be to see if the different antibodies elicit a protective effect against disseminated candidiasis in mice. If that proves successful than some day the antibodies could serve as a treatment for the immunocompromised patients.

20. Acknowledgments Dr. Zhang Natalie Lucindo Howard Hughes Medical Institute

21. References 1 Banerjee, U., L. N. Mohapatra, and R. Kumar. Infect. Immun 1984;43:966-72 2 Walker, S. M., J. Clin. Pathol. 33:370-372 3 Mourad S, Friedman L. Proc. Soc. Exp. Biol. Med. 1968;6:103- 105. 4 Pearsall N, Adams L, Bunni R. J. Immunol 1978;120:1176-1180.