Characterization and validation of fragment antigen-binding (Fab) antibody-based immunoassay for deoxymiroestrol, a potent phytoestrogen from Pueraria candollei

Deoxymiroestrol is the most potent phytoestrogen in chromenes group that has been found in Pueraria candollei, Thai name known as Kwao Krua Khao. Several studies reported estrogenic activity of P. candollei in order to using as hormone replacement therapy for postmenopausal women. Previously, specific determination of deoxymiroestrol content by enzyme-linked immunosorbent assay (ELISA) using polyclonal antibody (pAb) have been reported. However, production of pAb has limitation and variability from different batches. Therefore, in this study, we established quantitative method for determination of deoxymiroestrol using fragment antigen-binding (Fab) antibody-based immunoassay. The developed immunoassay has specificity to deoxymiroestrol with a calibration range of 15.6-1000 ng mL-1. Precision including intra-assay and inter-assay are 1.48-7.11 and 0.58-9.31%, respectively. Accuracy of the assay showed in recovery between 99.77-101.61% when spike deoxymiroestrol standard into the samples. The limit of detection (LOD) is 30.80 ng mL-1. Comparation antibody-based immunoassay for determination of deoxymiroestrol using Fab with pAb was represented consistency (R2 = 0.9807) when analysis roots bark of Pueraria candollei from difference areas. Therefore, this development assay can apply to determine deoxymiroestrol content in the plant samples.