Synthesis of Hapten and Development of Immunoassay Based on Monoclonal Antibody for the Detection of Dufulin in Agricultural Samples

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the quantitative detection of the antiviral agent against tobacco mosaic virus (TMV). Hapten 6-[2-((methyllbenzothiazol-2-yl)-1-(2-ethoxy)-O, O-diethyl-α-aminophosphonate)acetamido)] hexanoic acid (DHS) was prepared from commercial chemicals and incorporated into the spacer arm through a carbon–carbon single bond. The prepared hapten was then coupled to carrier proteins keyhole limpet hemocyanin (KLH) to be used as an immunogen for monoclonal antibody (mAb) production together with ELISA development. This assay was further optimized by the assessment of the dependence of assay parameters on organic solvents, pH, and ionic strength. The IC50 values of the optimized assay for Dufulin and the calculated limit of detection in phosphate-buffered saline (PBS) were 9.6 ± 0.59 and 0.3 ± 0.05 ng/mL, respectively. Using the optimized assays Dufulin residues in soil and tobacco samples were determined with recovery values ranging from 81.5 to 95.3%, intra-assay variation ranging from 2.88 to 6.10%, and interassay variation ranging from 6.11 to 9.42%.