MATERIALS AND METHODS (cont.)

Assays were performed using the 96-well plate

format of the P450RGS screening assay1 with

2,3,7,8-TCDD as a standard (Figure 2) (Appen-

dix II); results are expressed as TCDD TEQs. Re-

sults for standard curves and sediment extracts for

the 96-well plate format and the standard 6-well

plate format (McFarland et al. 1999) were com-

pared. The limit of detection (LOD) was deter-

mined with the same methods used in standard

chemical analysis, i.e., the LOD is calculated as

three times the average replicate standard deviation

above background level. The LOD will vary from

day to day depending on numerous random vari- Figure 2. The P450RGS assay: 96-well plate

ables; the LODs reported in Table 1 are averages

from three separate assays.

RESULTS AND DISCUSSION: The P450RGS assay has been shown to have good reproduci-

bility between laboratories and within a laboratory (McFarland et al. 1999). In the first version of

the assay used at the U.S. Army Engineer Research and Development Center, Environmental

Laboratory (EL), Vicksburg, MS, the cell exposures were conducted in 6-well plates, after which

the cells were lysed and transferred to microcentrifuge tubes. After centrifugation the lysate

supernatants were transferred to a 96-well plate to be read. The protocol for the P450RGS assay

given in Appendix II incorporates advances developed at EL in which all operations are carried out

on the same 96-well plate (Figure 3). These changes reduce the time required for the assay by about