Application of CALUX and TCDD immunoassay

The CALUX bioassay and the immunoassay provide a two tiered screening system that can be utilized as relatively rapid and inexpensive assays for the detection and relative quantitation of TCDD and TCDD-like chemicals in a wide range of samples and sample extracts (Figure 33.4). The CALUX assay

Figure 33.4 Overview of the two-tiered screening system for detection and relative quantitation of TCDD and TCDD-like chemicals in sample extracts.

can be used alone to eliminate samples that do not contain HAHs that bind to the Ah receptor. False negatives at this step could result from the presence of AhR antagonists in a sample; however, the presence of these inhibitory agents can readily be determined by measuring the ability of the sample to reduce the induction response of a known amount of TCDD. With a positive CALUX, the sample can be analyzed directly by chromatographic procedures or taken to a second tier where it is further evaluated by immunoassay. As shown in Figure 33.4, antibodies to TCDD, PCBs or other HAHs can be used to discriminate positives further in the CALUX assay. This second tier will put the sample in one of four categories: containing TCDD, containing PCBs that react with Ah receptor, containing TCDD and PCBs or containing an Ah receptor agonist that is neither TCDD or PCB. In samples where only TCDD or PCBs are known to be the primary contaminant either the CALUX or immunoassay can be used alone. Additionally, the CALUX and immunoas-says can be used to prescreen large numbers of samples in order to identify those that should be subsequently analyzed by the more costly and time consuming GC/MS procedures. With proper controls, obtaining false negatives with the CALUX and ELISA assays is difficult. Thus, these techniques can be used to screen out large numbers of negative samples. Often the most time-consuming part of GC/MS and LC/MS analysis is cleaning an instrument. The CALUX and immunoassays provide a way to rank samples so that samples with suspected low levels of HAHs can be run on the instrument

Figure 33.5 Overview of the CALUX bioassay-driven fractionation scheme for the purification and identification of novel AhR agonists.

when the sensitivity is high and samples suspected of high concentrations of HAHs can be either diluted or run subsequently.

Not only have these assays have been used to detect TCDD and related chemicals in variety of biological, environmental and food samples, but the CALUX bioassay provides an avenue to identify and characterize novel chemicals and classes of chemicals which can bind to and activate the AhR signal transduction pathway. Thus, a positive CALUX that is shown by subsequent immunoassay or immunoprecipitation assay (Figure 33.4) to be due to neither TCDD nor PCB must contain a novel AhR agonist. The identify of the activating chemical(s) can be obtained by using the CALUX bioassay to monitor a classical purification scheme based on differential extraction and column chromatography as presented in Figure 33.5.

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