Production of Monoclonal Antibodies
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Transcript of 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
- Most common cause of opportunistic disease in humans
- Serious disease in the immunocompromised can result in death.
www.cbr.ncr.ca
Background cont.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
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
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.
Materials & Methods
• Hybridoma cells
• Error Prone PCR
• Vector with amp marker
• Transformation into E. coli cells
• ELISA assay
Hybridoma cells
Monoclonal antibodies
www.fucell.com www.whfreeman.com
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
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
Transformation
• Electroporation– Mix Vector &
Electrocompetent cells.
– Apply voltage ~ 17000v
– Add SOC media
– Incubate for 1hr
– Plate on selective media.
www.agr.okstate.edu
Transformation cont.
www.eppendorfsi.comwww.eppendorfsi.com
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
ELISA
Www.uoguelph.ca
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.
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8
www.mds.qmw.ac.uk
Results cont.ELISA
01020
30405060
708090
1 2 3 4 5 6 7 8 9 10
Concentration of Ag (micro gram/mL)
Bin
din
g (
%) Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
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.
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.
Acknowledgments
• Dr. Zhang
• Natalie Lucindo
• Howard Hughes Medical Institute
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.