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Production of Monoclonal Antibodies

Production of Monoclonal Antibodies. Jose Baeza California State University, Long Beach Mentor: Dr. Zhang. Introduction. Background Main goals of the experiment Materials and Methods Results Conclusions Acknowledgements. Background. Candida albicans

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Production of Monoclonal Antibodies

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  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.com www.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 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. Color indicates the there is antibodies. They have different affinities. Clone #5 has the greatest affinity. Clone #4 has no affinity for the antigen. Results 1 2 3 4 5 6 7 8 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.

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