Immunoassays in Forensic Toxicology
Authors: Moody David E. 
Published: September 2006

Forensic toxicology encompasses the determination of the presence and concentration of drugs, other xenobiotics and their metabolites in physiological fluids and organs and the interpretation of these findings as they may impact on legal issues. These include medical examiner investigations, driving under the influence and other transportation accident investigations, workplace pre‐employment, random and for‐cause drug testing and judicial monitoring of arrestees and parolees. Similar techniques are employed in emergency room clinical toxicology and to monitor the efficacy of substance abuse treatment. The introduction of immunoassays into forensic toxicology in the early 1970s has had a major impact on the speed and efficiency that samples can be screened for the presence of certain drug classes. For the most part, forensic toxicologists use commercial immunoassays directed primarily towards abused drugs. Commercial immunoassays developed for therapeutic monitoring of other drugs, veterinary drugs and pesticides, as well as immunoassays developed in research laboratories for specialized studies, may find a role in the forensic toxicology laboratory for specialized cases.

Most immunoassays used for forensic toxicology are competitive. An antigen structurally similar to the target compound is conjugated to a signalling molecule and competes with target drug in a sample for antibody binding. Immunoassays are also classified as homogeneous and heterogeneous. Homogeneous assays do not separate the original sample from the final detection sample. They must use a signal that changes when antibody is bound. Homogeneous immunoassays include enzyme immunoassay (EIA) (enzyme activity decreases when bound), fluorescent polarization immunoassay (FPIA) (emission in a polar field increases when bound) and kinetic interaction of microparticles in solution (KIMS) immunoassay (lattice formation inhibited when bound). Heterogenous immunoassays include radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) where unbound radiolabeled antigen and enzyme conjugated antigen, respectively, are removed from the sample before measurement. In general, the homogeneous immunoassays are more ameniable to full automation, and thereby quicker throughput. The heterogeneous immunoassays are less susceptible to matrix interference, and thereby more versatile with nonurine matrices.

While most commercial immunoassays have been developed for a urine matrix, they have been applied by forensic toxicologists to other matrices, including blood, hair, saliva, sweat, tissue homogenates, blood stains and most other physiological samples that may be of value in the investigation. The use of nonurine matrices must contend with two factors. With the exception of parenchymal tissues, the concentration of the target compound is often lower and the sensitivity of the immunoassay may be limiting. In addition, the nonurine matrix usually is much more complex in its composition. Sample pretreatments that range from simple deproteinations to multistep extractions to remove matrix components and/or concentrate the sample are often required. The heterogenous RIAs and ELISAs usually require less rigorous, if any, pretreatments.

Interpretation of immunoassay results must take into consideration the limits of detection of the assay, the cross‐reactivity of the antibody(ies) and the potential for interference. Appropriate controls should be included to demonstrate adequate signal separation from blanks (drug‐free sample in the same matrix). The concentration of the low control is often determined by the sensitivity of the assay, or in workplace testing programs by administratively determined cutoffs that draw the line between a negative and presumptive positive. The ability of the antibody to detect compounds other than the target compound (its cross‐reactivity) can be a useful characteristic or, when the drugs that can be confirmed are limited, a nuisance. The amphetamines, barbiturates and benzodiazepines, in particular, all have a number of licit (and for amphetamines illicit) analogs that could be present in a sample. Cross‐reactivities are antibody‐source (i.e. manufacturer) dependent. Further testing to determine the immunoreactive compound often requires rigorous methodologies. In some instances, the potency of the drug and its poor cross‐reactivity make detection by immunoassay difficult (e.g. nitrosobenzodiazepines). Interference in immunoassays may arise from compounds that appear in the matrix during disease states, or those that are intentionally added to a sample in the hope of negating a positive test. These act through many different mechanisms and may decrease or increase the immunoassay test result.

Immunoassays have added an extremely useful tool to the forensic toxicology investigation. They can be used to screen rapidly a large number of samples for the potential presence of a drug group. With rare exceptions (emergencies, limited sample volume), their use without a confirmation assay (e.g. gas or liquid chromatography/mass spectrometry (LC/MS)) is unwarranted, as it leads to a risk of improper test result interpretation.