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Production of monoclonal antibodies and enzyme immunoassay for typical adenylate cyclase activator, Forskolin

Abstract

The ratio of hapten to bovine serum albumin in an antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against forskolin was produced by fusing splenocytes immunized with a forskolin-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.57%. A very small cross-reaction appeared with 1-deoxy, 9-deoxy and 1,9-dideoxy forskolin derivatives. The full measuring range of the assay extends from 6 ng to 200 ng of forskolin. The competitive ELISA assay used for this analysis was found to be more sensitive than TLC (10 μg), GLC (30 ng) and HPLC (1 μg) methods.

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References

  • Bhat SV, Bajwa BS, Dornauer H, de Sousa NJ and Fehlhabar HW (1977) Structures and sterochemistry of new labdane diterpenoids fromColeus forskohlii Briq. Tetrahedron Letters 1669–1672.

  • Eto N, yamada K, Koga A, Shirahata S and Murakami H (1991) Establishment and characterization of monoclonal antibodies against Chuzan virus K-47. Cytotechnology 6: 13–21.

    Google Scholar 

  • Galfre G and Milstein C (1981) Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73: 3–46.

    Google Scholar 

  • Hashimoto T, Horie M, Tori M, Taira Z and Asakawa Y (1992) Absolute configuration of forsklin-related compounds isolated from liverwortPtychanthus striatus. The 112nd Annual Meeting of Pharmaceutical Society of Japan, Abstract p. 220.

  • Hatano K, Shoyama Y and Nishioka I (1989) Clonal propagation ofAtractylodes japonica andA. ovata by tip tissue culture and the atractylon content of clonally propagated plants. Planta Medica, 56: 131–132.

    Google Scholar 

  • Ho LT and Ho RJ (1987) Production and assay of antibodies to an activator of adenylate cyclase, forskolin. J. Cyclic Nucletide Protein Phosphorylation Res. 11: 421–432.

    Google Scholar 

  • Inamdar PK, Kanitkar PV, Reden J and de Souza NJJ (1984) Quantitative determination of forskolin by TLC and HPLC. Planta Medica 51: 30–34.

    Google Scholar 

  • Inamdar PK, Dornauer H and de Souza NJ (1980) GLC method for assay of forskolin, a novel positive inotropic and blood pressurelowering agent, J. Pharmaceutical sciences. 69: 1449–1451.

    Google Scholar 

  • Krombholz M, Mersinger R, Kreis W and Reinhard E (1992) Production of forskolin by axenicColeus forskohlii roots cultivated in shake flasks and 20-1 glass jar bioreactors. Planta Medica 58: 328–333.

    Google Scholar 

  • Langone JJ (1982) Use of protein A to remove immunoglobulins from serum in hybridoma culture media. J. Immunol. Meth. 51: 33–45.

    Google Scholar 

  • Matsumoto M, Shoyama Y, Nishioka I, Iwai H and Wakimoto S (1989) Identification of viruses infected inRehmannia glutinosa Libosch. varpurpurea Makino and effect of virus infection on root yield and irridoid glycoside contents. Plant Cell Reports 7: 636–638.

    Google Scholar 

  • Mersinger R, Dornauer H and Reinhard E (1988) Formation of forskolin by suspension cultures ofColeus forskohlii. Planta Medica 54: 200–204.

    Google Scholar 

  • Metzger H and Lindner E (1981) The positive inotropic-acting forskolin, a potent adenylatecyclase activator. Drug Research 31: 1248–1250.

    Google Scholar 

  • Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15: 473–497.

    Google Scholar 

  • Pfeuffer E, Dreher RM, Metzger H and Pfeuffer T (1985) Catalytic unit of adenylate cyclase: purification and identification by affinity crosslinking. Proc. Natl. Acad. Sci. USA, 82: 3086–3090.

    Google Scholar 

  • Sen J, Sharma AK, Sahu NP and Mahato SB (1992) Production of forskolin inin vitro cultures ofColeus forskohlii. Planta Medica 58: 324–327.

    Google Scholar 

  • Reed RG, Puntma FW and Peters Jr T (1980) Sequence of residues 400–403 of bovine serum albumin. Biochem. J. 191: 867–868.

    Google Scholar 

  • Shah V, Bhat SV, Bajwa BS, Domauer H and de Souza NJ (1980) The occurrence of forskolin in the Labiatae. Planta Medica 39: 183–185.

    Google Scholar 

  • Shoyama Y, Nishioka I and Hatano K (1991) Biotechnology in Agriculture and Forestry Vol. 15: Springer-Verlag, Berlin, pp 58–72.

    Google Scholar 

  • Shoyama Y, Nishioka I and Hatano K (1992) Biotechnology in Agriculture and Forestry Vol. 19; Springer-Verlag, Berlin, pp 464–480.

    Google Scholar 

  • Weiler EW (1990) Chemistry of Plant Protection; Springer-Verlag, Berlin, pp 145–220.

    Google Scholar 

  • Vishwakarma RA, Tyagi BR, Ahmed B and Husain A (1988) Variation in forskolin content in the roots ofColeus forskohlii. Planta Medica 57: 471–472.

    Google Scholar 

  • Weiler EW and Zenk MH (1976) Radioimmunoassay for the determination of digoxin and related compounds inDigitalis lanata. Phytochemistry 15: 1537–1545.

    Google Scholar 

  • Yamada Y, Shoyama Y and Nishioka I (1991) Clonal micropropagation ofGentiana scabra Bunge var.buergeri Maxim. and examination of the homogeneity concerning gentiopicroside content, Chem. Pharm. Bull. 39: 204–206.

    Google Scholar 

  • Yamaguchi I, Nakagawa R, Kurogochi S, Murofushi N, Takahashi N and Weiler EW (1987) Radioimmunoassay of gibberellins A5 and A20. Plant Cell Physiol. 28: 815–824.

    Google Scholar 

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Sakata, R., Shoyama, Y. & Murakami, H. Production of monoclonal antibodies and enzyme immunoassay for typical adenylate cyclase activator, Forskolin. Cytotechnology 16, 101–108 (1994). https://doi.org/10.1007/BF00754612

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  • DOI: https://doi.org/10.1007/BF00754612

Key words

  • Adenylate cyclase activator
  • Coleus forskohlii
  • enzyme immunoassay
  • forskolin
  • monoclonal antibody
  • mass spectrometry