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PM · Lens · A New Highly Selective and Specific Anti‑puerarin polyclonal Antibody for Determination of Puerarin Using a Mannich Reaction Hapten Conjugate

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Article · Volume 13, Issue 52S · pp. S845–S851

A New Highly Selective and Specific Anti‑puerarin polyclonal Antibody for Determination of Puerarin Using a Mannich Reaction Hapten Conjugate

Orapin Udomsin2,3, Supaluk Krittanai2,3, Tharita Kitisripanya2,3, Hiroyuki Tanaka4, Waraporn Putalun2,3

  1. 1Department of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University.
  2. 2Research Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology, National Research University, Khon Kaen University, Khon Kaen 40002, Thailand.
  3. 3Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812‑8582, Japan.

Abstract

Background: Puerarin (PUE) is a phytoestrogen found in Pueraria candollei and Pueraria lobata. These plants are substantial for traditional medicine in various Asian countries. PUE is a key marker that can be found only in the Pueraria species.Objective: To establish the method for determination of PUE content which is required for quality control of pharmaceutical products.Materials and Methods: PUE‑cationized bovine serum albumin conjugate was created via Mannich reaction. After the rabbit immunization, the obtain anti‑PUE polyclonal antibody (PAb) was used to develop an enzyme‑linked immunosorbent assay (ELISA).Results: An anti‑PUE PAb possess a great sensitivity and specificity. The cross‑reactivity analysis shows no cross‑reaction of an established antibody against other substances. In addition, we successfully developed an indirect competitive ELISA (icELISA) for the quantitative analysis of PUE. The result of method validation conforms to acceptance criteria and correlates with high‑performance liquid chromatography, the reference method. The icELISA was applied to determine PUE content in Pueraria spp. plant samples and its derived pharmaceutical products.Conclusion: This highly specific immunogen was created from the Mannich reaction. An icELISA can also be applied to other research propose in the further studies.

Keywords: Enzyme‑linked immunosorbent assay; polyclonal antibody; Pueraria spp; puerarin

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