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.

1

2

3

4

5

6

7

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.