Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

Abstract

To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on microtiter plates via rabbit anti-mouse IgG was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 pgIl to 10 ig/1. The center point of the 2,4-D test was found at a concentration of 5 ig/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90%. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.