Analytical specificity
The analytical specificity or cross-reactivity of an immunoassay provides an indication of how the assay responds to other drugs relative to the drug used as a calibrator or standard. The cross reactivity profile of the immunoassay is important when assessing whether it is suitable for a particular task. For example, determination of cocaine use by the analysis of a urine specimen requires the immunoassay to exhibit good cross-reactivity to the urinary metabolite benzoylecgonine. By contrast, the assay needs to demonstrate good cross-reactivity to cocaine if the samples to be analysed are saliva or hair extracts, since high concentrations of the parent drug relative to metabolites are found in these specimens.
With classic competitive immunoassays (such as RIA and EIA), cross-reactivity is often calculated relative to the amount of drug that displaces 50% of the antibody-bound signal reagent. In the drug-testing field it is common to add a known amount of drug to the assay and record what response is given. Percentage cross-reactivity is then calculated as:
For example, if in an opiate assay (calibrated with morphine standards) to which a solution of 1000 ng/mL of codeine is added as a test sample, the result from the assay read against the morphine calibration curve is 100 ng/mL, the cross-reactivity of codeine in the assay relative to morphine is 10%, i.e.
However, for many drug immunoassays it is not sufficient to test cross-reactants at a single concentration. Several concentrations must be prepared in the sample matrix under test and the effect on the assay examined, effectively making a calibration or standard curve for each potential cross-reactant. The graph in Figure 1 illustrates curves for five compounds, with compound A used as the calibrator. The important feature of many drug immunoassays is that some drugs show parallel cross-reactivity to the calibrator substance (e.g. B and E in Fig. 1), while others exhibit non parallel cross-reactivity. In the example shown, compound D would have been thought to have low cross-reactivity if it had been tested at high concentrations only (1000 and 2000 lg/L). At a concentration of 10 lg/L, it is closer to 100% cross-reactive, while at low levels (1 lg/L) compound D exhibits 400% cross-reactivity relative to compound A.
Figure 1 Different cross-reactivity reactions using one compound (compound A) as a reference. Concentration is lg/L.
Nonparallel cross-reactivity can be seen within a drug group for both parent drug(s) and metabolite(s). Isomeric forms can also exhibit different cross-reactivity profiles and it is common to see different reactivity between (+)-amfetamine and (-)-amfetamine. Drugs tested for cross-reactivity indicate to the analyst which compounds might be appropriate for subsequent confirmatory analysis after a positive immunoassay result. Drugs that show little or no cross-reactivity in vitro can give strong immunoassay positives from people who take the drug in vivo. For example, benzfetamine is converted into methamfetamine and amfetamine, but the parent drug would not show cross-reactivity in many methamfetamine or amfetamine immunoassays.
The important factor with regard to anti bodies from an analyst’s perspective is to know the characteristics of the antibody used in the immunoassay. Knowledge of the type of immunoassay used is important when considering the next stage of the analytical process, for example gas chromatography–mass spectrometry (GC-MS). If an immunoassay is used to demonstrate the use of a drug by measuring its metabolites in urine, the confirmation method should be configured appropriately. This can be achieved by the introduction of hydrolysis steps so that, for example, glucuronide metabolites, which are detected by the immunoassay screen, can then be analysed as the parent drug compound by GC-MS. Other metabolites may exist to which the immunoassay may be directed, as with the buprenorphine metabolite norbuprenorphine, and consideration must be given to the most appropriate substance to target by GC-MS. It is normal practice to set a lower cut-off for confirmatory analysis than that used for the initial screening method. The above assay design features have import ant consequences when using immunoassay for samples other than urine (e.g. saliva, hair and blood). For example, if the drug in the sample is largely present in parent form in saliva or hair, the antibody and immunoassay must be directed to the parent molecule rather than to the metabolites.
