*Note: All values shown as calculated for Intercept and not whole oral fluids. Multiply the values by 3 to obtain whole oral fluid values. TCH, tetrahydrocannabinol.

*Note: All values shown as calculated for Intercept and not whole oral fluids. Multiply the values by 3 to obtain whole oral fluid values. TCH, tetrahydrocannabinol.

4.1. Analytical Sensitivity/Limit of Detection

The limit of detection (LOD) is defined from the signal-to-noise ratio at the zero-drug concentration as the mean zero absorbance (A0) minus three times the noise level (LOD = A0 - 3SD). The LOD was determined by obtaining the average absorbance value for 80 readings of blank oral-fluid diluent and calculating the standard deviation (SD) and three times the standard deviation (3SD) of the absorbance. The absorbance value minus 3SD was then extrapolated from the curve and represents the sensitivity of the assay. The LOD range of calibrations and cutoff for each Intercept assay are listed in Table 1. The assay cutoffs are separately determined through clinical testing. It is important to note the separation of the cutoffs used from the LOD. It would not be appropriate for a routine screening technique to use the LOD also as its cutoff, because other performance characteristics such as precision would most likely not be acceptable at the LOD.

4.2. Precision

The precision of the OraSure Technologies Inc. (OTI ) Intercept MicroPlate EIAs was assessed by testing oral-fluid diluent containing various concentrations of the target drug. The intra-assay precision was determined by analyzing each level 16 times per run for four runs. The inter-assay precision was determined by analyzing two samples at each level twice per day for 5-20 d, depending on the assay. The oral-fluid diluent used for these tests is carried in a phosphate buffer, which adjusts collected oral-fluid specimens to neutral pH. All tests were performed at room temperature. It should be noted that absolute absorbance values for microplates will be affected by room temperature; this should be considered when performing inter-assay or inter-day precision analysis. The results of this testing are shown in Table 2.

4.3. Cross-Reactivity

The cross-reactivity was determined for each assay for analogous and ubiquitous compounds. Analogous compounds that were cross-reactive in each of the Intercept assays are shown in Fig. 5. Cross-reactivity was determined by spiking various concentrations of each tested compound into the Intercept diluent fluid. A sample that showed a response was compared with each assay standard curve in order to calculate the percent cross-reactivity. For example, a test compound that showed equal immunoassay response to the cutoff concentration in a particular assay would be judged as showing 100% cross-reactivity. Some compounds shown have calculated cross reactivities that are very low. These values are included to show that extraordinarily large concentrations of such drugs would be required to elicit a response in the immunoassay. Thus, those performing a secondary confirmation by GC-MS-MS would not target confirming such compounds in presumptively positive clinical specimens.

4.4. Interferents

The effect of interfering substances or adulterants was examined in the Intercept Micro-Plate EIAs. Testing interferents by spiking them into buffer or some other artificial matrix is not relevant. Therefore, samples from volunteers were used after they consumed a potential interferent. In this experiment, five subjects consumed 1 oz of each adulterant, and oral-fluid samples were collected from each volunteer using the Intercept oral-fluid collection device after a 5-min and 10-min period following consumption. Samples were processed and pooled for each interferent and collection time. Aliquots from each sample pool were spiked with various concentrations of target drug and tested in the assay. The signals obtained for samples containing only the adulterants were used to assess any effects that may lead to false-positive results. The signals of samples containing drug in the presence of each adulterant were used to assess the overall effects of the adulterant. The substance was considered not to interfere if, after the 10-min waiting period, the samples containing 0 or the cutoff level of drug produced absorbance readings greater than the cutoff and if the samples containing 1.5 and 2.0 times the cutoff produced absorbance readings less than the cutoff. Data generated for an Intercept secobarbital assay are

Table 2

Precision of Intercept® Micro-Plate Enzyme Immunoassays

Concentrations Intra-assay Inter-assay ng/mL %CV (n = 64) %CV (n = 4/d, 20 d)


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