Immunoassay test and forensic toxicology

1. Immunoassay tests and
forensic toxicology
By
Yasmin Aziz Badi
Higher diploma

2. Immunoassays
Immunoassays are chemical tests used to detect or quantify a specific substance,
the analyte , in a blood or body fluid sample, using an immunological reaction.
Immunoassays are highly sensitive and specific.
Their high specificity results from the use of antibodies and purified antigens as
reagents. An antibody is a protein (immunoglobulin) produced by B-lymphocytes
(immune cells) in response to stimulation by an antigen.
immunoassays measure the formation of antibody-antigen complexes and detect
them via an indicator reaction.
High sensitivity is achieved by using an indicator system (e.g., enzyme label) that
results in amplification of the measured product.

4. Immunoassays may be qualitative (positive or negative) or quantitative (amount
measured). An example of a qualitative assay is an immunoassay test for
pregnancy.
Pregnancy tests detect the presence of human chorionic gonadotropin (hCG) in
urine or serum. Highly purified antibodies can detect pregnancy within two days
of fertilization. Quantitative immunoassays are performed by measuring the
signal produced by the indicator reaction. This same test for pregnancy can be
made into a quantitative assay of hCG by measuring the concentration of product
formed.

5. Purpose
The purpose of an immunoassay is to measure (or, in a qualitative assay, to
detect) an analyte . Immunoassay is the method of choice for measuring
analytes normally present at very low concentrations that cannot be
determined accurately by other less expensive tests. Common uses include
measurement of drugs, hormones, specific proteins, tumor markers, and
markers of cardiac injury.
Qualitative immunoassays are often used to detect antigens on infectious
agents and antibodies that the body produces to fight them.

6. For example, immunoassays are used to detect antigens on Hemophilus,
Cryptococcus , and Streptococcus organisms in the cerebrospinal fluid (CSF) of
meningitis patients. They are also used to detect antigens associated with
organisms that are difficult to culture, such as hepatitis B virus and Chlamydia
trichomatis . Immunoassays for antibodies produced in viral hepatitis, HIV, and
Lyme disease are commonly used to identify patients with these diseases.

7. Description
There are several different methods used in immunoassay tests.
Immunoprecipitation.
The simplest immunoassay method measures the quantity of precipitate, which
forms after the reagent antibody (precipitin) has incubated with the sample and
reacted with its respective antigen to form an insoluble aggregate.
Immunoprecipitation reactions may be qualitative or quantitative.
Particle immunoassays. By linking several antibodies to the particle, the particle
is able to bind many antigen molecules simultaneously. This greatly accelerates
the speed of the visible reaction. This allows rapid and sensitive detection of
antibodies that are markers of such diseases, as infectious mononucleosis and
rheumatoid arthritis.
Immunonephelometry. The immediate union of antibody and antigen forms
immune complexes that are too small to precipitate. However, these complexes
will scatter incident light and can be measured using an instrument called a
nephelometer. The antigen concentration can be determined within minutes of the
reaction.

8. 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.

9. Immunoassays used for forensic
toxicology
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

10. Heterogenous immunoassays include radioimmunoassay (RIA) and enzyme‐linked
immune sorbent 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 non urine matrices.
Immunoassays used for
forensic toxicology

11. Immunoassays used for forensic
toxicology
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 non urine 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 non urine 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 hetero genous RIAs and ELISAs usually require less rigorous, if
any, pretreatments.

12. Interpretation of immunoassay results
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

13. Interpretation of immunoassay results
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.

14. In the end
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.

15. Reference
1- Nadine Theofel, Sonja Roscher, Stefan Scholtis and Michael Tsokos, Wenn Leichen auf
Gerichtsmediziner treffen, Chemie in unserer Zeit, 53, 1, (40-49), (2019).Wiley Online
Library
2- Kronstrand R., Roman M., Dahlgren M., Thelander G., Wikström M., Druid H.. A
cluster of deaths involving 5-(2-aminopropyl)indole (5-IT), Journal of Analytical
Toxicology, 2013, vol. 37 (pg. 542-546)
3-Seetohul L.N., Pounder D.J.. Four fatalities involving 5-IT, Journal of Analytical
Toxicology, 2013, vol. 37 (pg. 447-451)