Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 2004 Mar;44(3):115–123. doi: 10.1007/s10616-004-1179-3

Development of monoclonal antibody against isoquinoline alkaloid coptisine and its application for the screening of medicinal plants

Jun-Sik Kim 1, Hiroyuki Tanaka 1, Chun-Su Yuan 2, Yukihiro Shoyama 1,✉
PMCID: PMC3449483  PMID: 19003234

Abstract

In the development of immunoassay technique, the design of hapten containing a functional group suitable for protein conjugate is the key step for the preparation of antibodies against small molecules. Coptisine (MW 320), a bioactive constituent of Berberis and Coptis species, is small as an immunogen. In addition, coptisine has no reactive group in molecule for conjugating with a protein. To overcome this problem, 9-O-carboxymethyl-berberrubine was designed and conjugated with carrier protein. In order to confirm its immunogenicity, the ratio of hapten in the conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting antibodies against coptisine were produced by fusing splenocytes with mouse myeloma cell line, P3-X63-Ag8-653. Among hybridomas, the clone 2A1 secreting anti-coptisine monoclonal antibody (MAb) 2A1-9E-1 was obtained through the limited dilution method. The MAb-based enzyme-linked immunosorbent assay (ELISA) against coptisine was developed and characterized. The linear range of the assay in this ELISA method was extended from 1.56 to 25 μg ml−1 possessing the detection limit of 1.56 μg ml−1. The established ELISA using MAb 2A1-9E-1 was applied for the survey of isoquinoline alkaloids in various medicinal plants.

Keywords: Coptisine, ELISA, Isoquinoline alkaloid, Monoclonal antibody, Small hapten

Full Text

The Full Text of this article is available as a PDF (156.5 KB).

References

  1. Colombo M.L., Bugatti C., Mossa A., Pescalli N., Piazzoni L., Pezzoni G., Menta E., Spinelli S., Johnson F., Gupta R.C., Dasaradhi L. Cytotoxicity evaluation of natural coptisine and synthesis of coptisine from berberine. Il Famaco. 2001;56:403–409. doi: 10.1016/S0014-827X(01)01121-1. [DOI] [PubMed] [Google Scholar]
  2. Galfre G., Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 1981;73:3–46. doi: 10.1016/0076-6879(81)73054-4. [DOI] [PubMed] [Google Scholar]
  3. Goding J.W. Antibody production by hybridomas. J. Immunol. Meth. 1980;39:285–308. doi: 10.1016/0022-1759(80)90230-6. [DOI] [PubMed] [Google Scholar]
  4. Hammock B.D., Mumma R.O. Potential of immunochemical technology for pesticide analysis. In: Harvey J., Zweig G., editors. Recent Advances in Pesticide Analytical Methodology. Washington: American Chemical Society; 1980. pp. 321–352. [Google Scholar]
  5. Hirano H., Osawa E., Yamaoka Y., Yokoi T. Gastric-mucous membrane protection activity of coptisine derivatives. Biol. Pharm. Bull. 2001;24:1277–1281. doi: 10.1248/bpb.24.1277. [DOI] [PubMed] [Google Scholar]
  6. Ishiyama M., Shoyama Y., Murakami H., Shinohara H. Production of monoclonal antibodies and development of an ELISA for solamargine. Cytotechnology. 1996;18:153–158. doi: 10.1007/BF00767762. [DOI] [PubMed] [Google Scholar]
  7. Iwasa K., Kamigauchi M. 13-hydroxylation of tetrahydroberberine in cell suspension cultures of some corydalis species. Phytochemistry. 1996;41:1511–1515. doi: 10.1016/0031-9422(95)00813-6. [DOI] [Google Scholar]
  8. Marco M.P., Gee S., Hammock B.D. Immunochemical techniques for environmental analysis II. Antibody production and immunoassay development. Trends Anal. Chem. 1995;14:415–425. [Google Scholar]
  9. Sadykov Y.D., Davidyants S.B., Poroshin K.T., Grigina I.N. Synthesis of esters and ethers from the alkaloid berberine. Dokl. Akad. Nauk Tadzh SSR. 1967;10:18–21. [Google Scholar]
  10. Shoyama Y., Sakata R., Isobe R., Murakami H. Direct determination of forskolin–bovine serum albumin conjugate by matrix assisted laser desorption ionization. Org. Mass. Spectr. 1993;28:987–988. doi: 10.1002/oms.1210280910. [DOI] [Google Scholar]
  11. Shoyama Y., Tanaka H., Fukuda N. Monoclonal antibodies against naturally occurring bioactive compounds. Cytotechnology. 1999;31:9–27. doi: 10.1023/A:1008051618059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Tedder J.M., Nechvatal A. and Murray A.W. 1972. Amino-acids and proteins. In: Basic Organic Chemistry. John Wiley & Sons, Chapter 6, pp. 305–342.
  13. Weiler E.W., Zenk M.H. Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. Phytochemistry. 1976;15:1537–1545. doi: 10.1016/S0031-9422(00)88933-5. [DOI] [Google Scholar]
  14. Yoshida A., Takagaki Y. Detection of food additives by enzyme immunoassay. Nippon Rinsho. 1995;53:2316–2321. [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES