Development and characterisation of highly specific monoclonal antibody‐based immunoassays for the detection and quantification of genistein‐7‐O‐[α‐rhamnopyranosyl‐(1→6)]‐β‐glucopyranoside in Derris scandens (Roxb.) Benth.

Abstract

AbstractIntroductionThe stem of the plant species Derris scandens (Roxb.) Benth. (DS) contains genistein‐7‐O‐[α‐rhamnopyranosyl‐(1→6)]‐β‐glucopyranoside (GTG), which is a unique marker. Previous analyses of GTG using antibody‐based immunoassays were compromised because of their high cross‐reactivity with structurally related compounds of DS, thereby limiting their applicability in DS quality control.ObjectiveConjugation of GTG with carrier proteins was achieved using the Mannich reaction to produce a highly specific monoclonal antibody (mAb) targeting GTG (anti‐GTG mAb).MethodsThe anti‐GTG mAb was generated using hybridoma technology and characterised using an indirect competitive enzyme‐linked immunosorbent assay (icELISA). Both lateral‐flow immunoassay (LFIA) and icELISA were developed to detect and quantify GTG in DS raw materials and associated products.ResultsicELISA using the anti‐GTG mAb showed 100% specificity for GTG, with only 1.77% cross‐reactivity with genistin and less than 0.01% cross‐reactivity with other compounds. icELISA demonstrated a linear range for GTG determination between 62.5 and 2000 ng/mL. The limits of detection (LOD) and quantification were 49.68 and 62.50 ng/mL for GTG, respectively. The precision of the analysis ranged from 1.28% to 4.20% for repeatability and from 1.03% to 7.05% for reproducibility. The accuracy of the analysis ranged from 101.97% to 104.01% for GTG recovery. GTG levels determined via icELISA were consistent with those confirmed via high‐performance liquid chromatography (HPLC) (R2 = 0.9903). Moreover, the LOD of LFIA for GTG was 500 ng/mL.ConclusionImmunoassays utilising specific anti‐GTG mAbs were successfully developed, including LFIA for rapid GTG detection and icELISA for GTG quantification.