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Cytotechnology. 2004 Mar;44(3):115-23. doi: 10.1007/s10616-004-1179-3.

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

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1
Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.

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 mug ml(-1) possessing the detection limit of 1.56 mug ml(-1). The established ELISA using MAb 2A1-9E-1 was applied for the survey of isoquinoline alkaloids in various medicinal plants.

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