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Quantitation ofAcidothermus cellulolyticus E1 endoglucanase andThermomonospora fusca E3 exoglucanase using enzyme-linked immunosorbent assay (ELISA)

  • Rafael A. Nieves
  • Yat-Chen Chou
  • Michael E. Himmel
  • Steven R. Thomas
Session 2 Past, Present, and Emerging Concepts in Applied Biological Research

Abstract

Two distinct quantitative indirect ELISAs were developed to determine the concentration of recombinant cellulase enzymes in culture filtrates. A monoclonal antibody (E1P7) was used as the primary antibody in developing an ELISA specific forAcidothermus cellulolyticus E1 endoglucanase. Likewise, a polyclonal rabbit serum (Ab684) was used to develop an ELISA specific forThermomonospora fusca E3 exoglucanase. Dose-response curves indicated a dynamic range for both assays between 0.01 and 0.08 μg/mL (1–8 ng/assay) when purified enzymes were used as standards. These assays have been used to estimate concentrations of secreted recombinant E1 and/or E3 in culture supernatants ofStreptomyces lividans strain TK24 in which the corresponding genes have been cloned and expressed.

Index Entries

Cellulases Acidothermus cellulolyticus Thermomonospora fusca antibodies ELISA 

References

  1. 1.
    Coughlan, M. P. (1985),Biotechnol. Genet. Eng. Rev. 31, 39–109.Google Scholar
  2. 2.
    Puls, J. and Wood, T. W. (1991),Biotechnology 36, 15–19.Google Scholar
  3. 3.
    Woodward, J. (1991),Bio/Technol. 36, 67–75.Google Scholar
  4. 4.
    Rapp, P. and Beerman, A. (1991), inBiosynthesis and Biodegradation of Cellulose, Haigler, C. H. and Weimer, P. J., eds., Marcel Dekker, New York, pp. 535–597.Google Scholar
  5. 5.
    Coughlan, M. P. (1985),Biochem. Sci. Trans. 13, 405–416.Google Scholar
  6. 6.
    Mandels, M. (1985),Biochem. Sci. Trans. 13, 414–416.Google Scholar
  7. 7.
    Grohmann, K., Wyman, C. E., and Himmel, M. E. (1992), inEmerging Technologies for Materials and Chemicals from Biomass, ACS Symposium Series 476, American Chemical Society, Washington, DC, pp. 354–392.Google Scholar
  8. 8.
    Wilson, D. B. (1988),Methods Enzymol. 160, 314–323.CrossRefGoogle Scholar
  9. 9.
    Tucker, M. P., Mohagheghi, A., Grohmann, K., and Himmel, M. E. (1989),Bio/Technology 7, 817–820.CrossRefGoogle Scholar
  10. 10.
    Wood, T. M. and Bhat, K. M. (1988),Methods Enzymol. 160, 87–135.CrossRefGoogle Scholar
  11. 11.
    Perlmann, P. and Kaltenbach, J. C. (1957),Expt. Cell Res. 12, 185–188.CrossRefGoogle Scholar
  12. 12.
    Oudin, J. (1958),J. Immunol. 81, 376–388.Google Scholar
  13. 13.
    Hitzig, W. H., Schiedegger, J. J., Butler, R., Gugler, E., and Hassig, A. (1959),Helv. Med. Acta. 26: 142–151.Google Scholar
  14. 14.
    Butler, J. E., ed. (1991)Immunochemistry of Solid Phase Immunoassay, Chemical Rubber Company, Boca Raton, FL.Google Scholar
  15. 15.
    Berson, S. A. and Yallow, R. S. (1959),J. Clin. Invest. 38, 1996–2016.CrossRefGoogle Scholar
  16. 16.
    Ekins, R. P. (1960),Clin. Chim. Acta 5, 453–459.CrossRefGoogle Scholar
  17. 17.
    Kohler, G. and Milstein, C. (1975),Nature 256, 495–497.CrossRefGoogle Scholar
  18. 18.
    Riske, F., Labudova, I., Miller, L., Macmillan, J. D., and Eveleigh, D. E. (1987), inBiomass Conversion Technology, Principles and Practice, Moo-Young, M., ed., Pergamon, New York, pp. 167–176.Google Scholar
  19. 19.
    Mischak, H., Hofer, F., Messner, R., Weissinger, E., Hayn, M., Tomme, P., Esterbauer, H., Kuchler, E., Claeyssens, M., and Kubicek, C. P. (1989),Biochim. Biophys. Acta 200, 643–649.Google Scholar
  20. 20.
    Nieves, R. A., Himmel, M. E., Todd, R. J., and Ellis, R. P. (1990),Appl. Environ. Microbiol. 56, 1103–1108.Google Scholar
  21. 21.
    Aho, S., Olkkonen, V., Jalava, T., Paloheimo, M., Bühler, R., Nikv-Paavola, M.-L., Bamford, D. H., and Korhola, M. (1991),Eur. J. Biochem. 200, 643–649.CrossRefGoogle Scholar
  22. 22.
    Oh, T. K. and Kim, S. H. (1986),Biotechnol. Lett. 8, 403–406.CrossRefGoogle Scholar
  23. 23.
    Kolbe, J. and Kubicek, C. P. (1990),Appl. Microbiol. Biotechnol. 34, 26–30.CrossRefGoogle Scholar
  24. 24.
    Riske, F. J., Eveleigh, D. E., and Macmillan, J. D. (1990),Appl. Environ. Micro. 56, 3261–3265.Google Scholar
  25. 25.
    Buhler, R. (1991),Appl. Environ. Microbiol. 57, 3317–3321.Google Scholar
  26. 26.
    Chater, K. F., Hopwood, D. A., Kieser, T., and Thompson, C. J. (1982),Curr. Top. Microbiol. Immunol. 96, 69–95.Google Scholar
  27. 27.
    Tsai, J. F.-Y. and Chen, C. W. (1987),Mol. Gen. Genet. 208, 211–218.CrossRefGoogle Scholar
  28. 28.
    Katz, E., Thompson, C. J., and Hopwood, D. A. (1983),J. Gen. Microbiol. 129, 2703–2714.Google Scholar
  29. 29.
    Denis, F. and Brzezinski, R. (1992),Gene 111, 115–118.CrossRefGoogle Scholar
  30. 30.
    Baker, J. O., Adney, W. S., Nieves, R. A., Thomas, S. R., Wilson, D. B., and Himmel, M. E. (1994),Appl. Biochem. Biotechnol. 45/46, 245–256.CrossRefGoogle Scholar
  31. 31.
    Irwin, D. C., Spezio, M., Walker, L. P., and Wilson, D. B. (1993),Biotechnol. Bioeng. 42, 1002–1113.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • Rafael A. Nieves
    • 1
  • Yat-Chen Chou
    • 1
  • Michael E. Himmel
    • 1
  • Steven R. Thomas
    • 1
  1. 1.Alternative Fuels Division, National Renewable Energy LaboratoryApplied Biological Sciences BranchGolden

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