Immunoanalysis methods for the detection of dioxins and related chemicals

Abstract

With the development of biotechnology, approaches based on antibodies, such as enzyme-linked immunosorbent assay (ELISA), active aryl hydrocarbon immunoassay (Ah-I) and other multi-analyte immunoassays, have been utilized as alternatives to the conventional techniques based on gas chromatography and mass spectroscopy for the analysis of dioxin and dioxin-like compounds in environmental and biological samples. These screening methods have been verified as rapid, simple and cost-effective. This paper provides an overview on the development and application of antibody-based approaches, such as ELISA, Ah-I, and multi-analyte immunoassays, covering the sample extraction and cleanup, antigen design, antibody preparation and immunoanalysis. However, in order to meet the requirements for on-site fast detection and relative quantification of dioxins in the environment, further optimization is needed to make these immuno-analytical methods more sensitive and easy to use.

Figure 1.

Schematic diagram for experimental procedure of indirect and direct competitive ELISA for detecting dioxins. (a) For an indirect competitive ELISA assay, a microplate is coated with appropriate antigens (1). Samples and anti-dioxin antibodies (Abs) are added in to the microplate. Antigens compete with dioxins in the test sample for binding to the Ab (2). After incubation, soluble molecules are washed away. Secondary antibody labeled with enzyme is added and reacts with the immobilized anti-dioxin Abs (3). Substrates are added to detect the immobilized Ab (4). (b) For a competitive ELISA assay, a microplate is coated with anti-dioxin Ab (5). After incubation, soluble Abs are removed by washing. Samples and enzyme-labeled standard congener are added which compete for binding to the anti-dioxin Abs. After incubation, soluble molecules are washed away (6). Substrates are added to detect immobilized standard congener (7).

Figure 2.

Schematic diagram for experimental procedures of Ah-I for detecting dioxins. Microplates are coated with streptavidin (1). Cell lysate containing AhR and ARNT, testing samples containing dioxins, and biotin-labeled DNA fragments containing DRE consensus sequences are added into the microplate. Biotin-labeled DRE binds to streptavidin. AhR activated by dioxins combines with ARNT to form a complex, which is immobilized by binding to DRE (2). Anti-ARNT antibody (3) or anti-AhR antibody (5) is added to recognize the complex. Secondary antibodies and substrates are added for detection (3, 4, 5, 6).