Preparation of a highly specific single chain variable fragment antibody targeting miroestrol and its application in quality control of Pueraria candollei by enzyme-linked immunosorbent assay

Benyakan Pongkitwitoon, Panitch Boonsnongcheep, Tharita Kitisripanya, Gorawit Yusakul, Seiichi Sakamoto, Hiroyuki Tanaka, Satoshi Morimoto, Waraporn Putalun

Research output: Contribution to journalArticle


Introduction: Miroestrol is the potent phytoestrogen isolated from White Kwao Krua (Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, a Thai traditional medicinal plant. Nowadays, various health supplementary products featuring White Kwao Krua are available worldwide. A sensitive and rapid analytical method for quantification of miroestrol is necessary for quality control of these products. Objectives: To prepare a single-chain variable fragment (scFv) antibody specific to miroestrol and develop a scFv-based enzyme-linked immunosorbent assay (ELISA) for quantitative analysis of miroestrol in plant materials and health supplementary products. Methods: A gene encoding anti-miroestrol scFv antibody was constructed and expressed in Escherichia coli SHuffle T7 strain. Anti-miroestrol scFv antibody was characterised and applied to ELISA. The developed scFv-based ELISA method was validated for its sensitivity, specificity, accuracy and precision. Results: Anti-miroestrol scFv antibody was highly specific to miroestrol. The scFv-based ELISA was applied to determine miroestrol in the range 0.06–7.81 μg/mL, with the limit of quantification of 0.06 μg/mL miroestrol. The accuracy of the assay was validated by its 95.08–103.99% recovery from the spiked miroestrol recovery experiment and in good correlation with the results from the monoclonal antibody-based ELISA. The relative standard deviation of the intra- and inter-assay were less than 6.0%. Conclusion: The developed scFv-based ELISA was sensitive, specific, accurate, and precise for determination of miroestrol and useful for quality control of P. candollei plant raw materials and supplementary products.

Original languageEnglish
Pages (from-to)600-608
Number of pages9
JournalPhytochemical Analysis
Issue number6
Publication statusPublished - Nov 1 2019


All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Food Science
  • Biochemistry
  • Molecular Medicine
  • Plant Science
  • Drug Discovery
  • Complementary and alternative medicine

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