Development of homogeneous phase-modulation fluoroimmunoassay for 2,4-dichlorophenoxyacetic acid Original Research Article

This paper describes a homogeneous and competitive fluoroimmunoassay technique using two methods of phase-modulation fluorescence spectroscopy in which non-labelled analyte, 2,4-dichlorophenoxyacetic acid (2,4-D), competes with the fluorescent-labelled antigen (2,4-D-NH2F) for non-labelled monoclonal antibody. In the first method the fluorescence lifetime difference between the free and the monoclonal antibody-bound 2,4-D-NH2F is used to determine both fractions by means of a phase-modulation mathematical approach. The second method is based on the simultaneous equation method using phase-resolved fluorescence spectroscopy. The equation system generated is solved by the pseudo-inverse method. An analytical parameter comparison of the different standard curves obtained by both methods has been made. An assay of specificity of the antiserum has been made using fluorescence lifetime measurements because they provide better results. The technique provides good sensitivity, specificity and MDC.