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

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. SUMMARY: The new immunogen conjugated (puerarin-cBSA) via Mannich reaction was successfully in rising of antibody against puerarin (PUE). The obtained anti-PUE polyclonal antibody (PAb) was high sensitivity and specificity to PUE. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed and validated using anti-PUE PAb. The established icELISA was applied to determine PUE content in various tuberous root of Pueraria spp. Moreover, icELISA method can be applicable in Pueraria spp. derived products. Abbreviations used: PUE: Puerarin; PAb: Polyclonal antibody; ELISA: Enzyme-linked immunosorbent assay; icELISA: Indirect competitive ELISA; cBSA: Cationized bovine serum albumin.