A recombinant fab antibody against kwakhurin as a tool for sensitive indirect competitive ELISA

Background: Pueraria candollei var. mirifica (P. candollei), known as White Kwao Krua in Thai, has long been used in traditional Thai medicine for the symptoms of menopause due to the potent estrogenic activity exhibited by the isoflavonoids and chromenes it contains. Recently, health hazards caused by P. candollei-derived products have arisen in Japan, and demands for analytical methods to standardize the P. candollei have been increasing. Previously, we have focused on quantifying the unique P. candollei-derived isoflavonoid kwakhurin (Kwa) and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) using a monoclonal antibody (MAb) against Kwa. However, MAb preparation requires the use of costly culture medium and sophisticated techniques. Objective and Method: In this study, we produced a recombinant antigen-binding fragment (Fab) against Kwa, as an alternative to MAb, for use in icELISA for quantitative analysis of Kwa. The VH-CH1 and VL-CL proteins were individually expressed in Escherichia coli BL21 (DE3) strain and were then refolded to form active anti-Kwa Fab. Results and Conclusion: Characterization of anti-Kwa Fab revealed that it possessed high specificity (cross-reactivities with other Kwa-related compounds, <0.03%) and high sensitivity (limit of detection, 8.16 ng/mL). Additionally, validation analyses indicated that icELISA using anti-Kwa Fab is highly precise, accurate, and sufficiently reliable for use in quantitative analysis of Kwa. Consequently, an icELISA incorporating anti-Kwa Fab was developed for the analysis of P. candollei-derived products, to assure consumer safety.