The Immunoassay

Chad Chisolm, Jaclyn Holcombe, Matthew Shelnutt

Objectives
Know that Toxicology is the branch of Forensic Science in
which the immunoassay plays the greatest role.
Know that in the year 1959, the RIA, predecessor of modern
immunoassays, was invented.
Know that the RIA was invented by Drs. Yalow and Berson.
Know that antibodies are proteins that are found in blood or
other bodily fluids that are used by the immune system to
identify and neutralize foreign objects, such as bacteria and
viruses.
Know that an antigen is any substance that causes your
immune system to produce antibodies against it. An antigen
may be a foreign substance from the environment such as
chemicals, bacteria, viruses, or pollen; they may also be
produced inside the body.
Objectives Cont’d
Know that a hybridoma is the hybrid of an antibody and a
myleoma cell, which are cultured to produce antibodies.
Know that immunoassays are typically categorized as either
competitive or noncompetitive.
Know that in noncompetitive immunoassays, the amount of
labeled analyte is directly proportional to the amount of
antigen; in the competitive, it is inversely proportional.
Know that heterogeneous immunoassays depend on the
separation of unbound tracers before the bound signal is
measured; homogeneous immunoassays do not require this
separation because the signal is generated when the binding
occurs.
Know that two specific types of immunoassays include the
EMIT, RIA, ELISA, FPIA.
History and Background

• In the year 1959, Drs.

Rosalyn Yalow & Soloman
Berson invented the
radioimmunoassay, which
applied the use of
radioisotopes in the
measurement of
insulin. Dr. Rosalyn Yalow became the first
female to win a Nobel Prize with
her work on the radioimmunoassay.
• The RIA is the
predecessor of modern
immunoassays.
History and Background Cont’d
• 1972 First Hepatitis RIA
• 1972 EMIT Assay
• 1975 Bead-based Assays
• 1978 Coated-tube assays
• 1985 HIV Immunoassay
• 1993 TroponinAssays
• 1997 Automated Cytokine
Assays

(Evans)
Immunoassays at a Glance
• Immunoassays are a group of sensitive analytical tests that utilize
very specific antibody/antigen complexes to produce a signal that
can be measured and related to the concentration of a compound in
solution (Wild). Immunoassays also produce qualitative data in
terms of the presence (+) or absence (-) of a compound in the body.

• They are used in a lot of laboratories, including hospitals labs, and

have been widely used in the special area of Forensic Toxicology to
screen for drugs and other chemicals in the body.
Antibodies
• Antibodies are produced by the B
lymphocytes. They are
glycoproteins belonging to the
“immunoglobulin supergene family”
(Wild) that are produced in
response to a foreign substance in
the body.

• Antibodies have a generally

common structure, but have
regions that vary among them to
accommodate the specific
antigens (Abbott Diag.).
Antigens
• An antigen is a substance with the ability to induce an immunological
response. They typically enter the body from an infection. They are
recognized at their epitopes by B cells or by the T cell receptor on T cells
(Lee).

• Proteins or glycoproteins make the best antigens because they are the
best at stimulating antigen recognition molecules. Some immunoassays
test for antigens, rather than antibodies (Lee).
Production of Antibodies
• The production of antibodies is an important process in the use of
immunoassays because it is the antibody-antigen complexes that form that
the device uses for its results. Antibodies can be called monoclonal or
polyclonal, depending upon the technique used to produce it.

• Monoclonal antibodies involve these basic steps and result in very

specific antibodies that bind only to one antigen epitope, which in turn
reduces the occurrence of false positives in the immunoassay:

− A mouse (or rabbit) is immunized by being injected with an antigen; the antigen
generates an antibody response in the animal.
− The mouse is sacrificed and the antibody forming cells are isolated from the
mouse's spleen.
− Monoclonal antibodies are produced by fusing single antibody-forming cells
with myeloma cells. The resulting cell is called a hybridoma. This hybridization
makes the cells “immortal.”
− The hybridomas contain large amounts of antibodies, and can easily be
cultured into populations of cells that contain identical antibodies. These
antibodies are called "monoclonal antibodies" because they are produced by
the identical offspring of a single, cloned antibody producing cell. (UCLA)
Production of Antibodies Cont’d

A diagram depicting the steps involved in

monoclonal antibody production.
Production of Antibodies Cont’d
• Polyconal antibodies are more likely to produce a false positive
because they are less specific to antigen epitopes and have varying
binding affinities; they may bind of molecules similar to their
antigens. Production includes these steps:

− A mouse (or rabbit) is immunized by being injected with an antigen;

the antigen generates an antibody response in the animal.
− The animal is bled and the antibodies are collected; the blood
contains a heterogenous mixture of antibodies of varying binding
affinities and specificities. (Wild)
Labels in Immunoassays
• Immunoassays require the use of labeled materials in
order to measure the amount of antigen or antibody
present. A label is a molecule that will react as part of
the assay, and in doing so produce a signal that can be
measured in the solution. Examples of a label include a
radioactive compound, or an enzyme that causes a
change of color in a solution or its fluorescence (Wild).
Competitive Immunoassays
• The measurement of the analyte using the labels is broadly
categorized into competitive and noncompetitive methods.

– In competitive formats, unlabelled analyte in the test

sample is measured by its ability to compete with labeled
antigen for a limited number of antibody binding sites
(Bell). The unlabeled antigen blocks the ability of the
labeled antigen to bind because that binding site on the
antibody is already occupied. Thus, in a competitive
immunoassay, less label measured in the assay means
more of the unlabeled (test sample) antigen is present.
The amount of antigen in the test sample is inversely
related to the amount of label measured in the
competitive format (Abbott Diag.). As one increases, the
other decreases.
Noncompetitive Immunoassays
– Noncompetitive (sandwhich) immunoassays generally provide the
highest level of assay sensitivity and specificity. This format is referred
to as a “sandwich” assay because the analyte is bound (sandwiched)
between two highly specific antibody reagents.

– The reaction mixture typically includes an excess of labeled antibody,

so that all drug/metabolite is bound. The amount of antibody-antigen
complex is then measured to determine the amount of drug present in
the sample. The measurement of labeled analyte, usually antibody, is
directly proportional to the amount of antigen present in the sample.
Homogeneous VS Heterogeneous Methods

• Immunoassay methods that require separation of bound Ab-Ag* complex

are referred to as heterogeneous immunoassays. Those that do not
require separation are referred to as homogeneous immunoassays.
• Homogeneous methods have been generally applied to the measurement
of small analytes such as abused and therapeutic drugs. Since
homogeneous methods do not require the separation of the bound Ab-Ag*
from the free Ag*, they are generally much easier and faster to perform.
Types of Immunoassays

• Within the categories of competitive, noncompetitive,

homogenous, and heterogeneous, there are specific
types, which include:

• Radioimmunoassays
(RIAs) utilize a
radioactive label
(usually 125I, 3H or
14C), which emits
radiation that can be
measured with a beta
or gamma counter.
Types of Immunoassays Cont’d
• In the Enzyme Multiplied
Immunoassay (EMIT),
the drug in the sample
and the drug labeled
with G6PD compete for
antibody binding sites.

• Binding inhibits enzyme

activity, while free
enzyme remains active
to interact with.

• Enzyme
activity/absorbance is
directly proportional to
drug concentration.
Types of Immunoassays Cont’d
• Enzyme linked
immunosorbant assay
(ELISA): competitive,
heterogeneous EIA

• Reaction components are

absorbed or bound to the
surface of a solid phase,
commonly a well of a
microtiter plate

• Absorbance is measured
using a micro-plate reader

• Sample absorbance is
inversely proportional to
drug concentration
Types of Immunoassays Cont’d
• In the Fluorescent Polarized
Immunoassay, the drug in
the sample competes with
fluorescein-labeled drug for
antibody binding sites.

• Reaction mixture is excited

by planepolarized light.

• As the tracer returns to a

lower energy state, it emits
light; polarization is • The polarization value of the sample is
measured. inversely proportional to analyte
concentration.
Immunoassay Results
• Qualitative
– Single point calibration at a specific cutoff
– Results are either ‘positive’ or ‘negative’; (i.e.
above or below the cutoff)
– Possible false positives; monoclonal antibodies
restrict this slightly.
• Quantitative
– Provides numeric results that are an estimate of
drug/compound concentration based on the
measurement of labeled analyte in the solution,
and taking into consideration the
competitive/noncompetitive nature of the device.
– In terms of use on drugs, this is sometimes
complicated by possible cross-reactivities.
Immunoassay Results Cont’d
A pregnancy test is an
example of a
commercially produced
immunoassay that
produces a positive or
negative qualitative
response.

Typical 4-parameter logistic

graph for a competitive-format
immunoassay.

Dose-response curve for a non-

competitive CL immunoassay.
Standardization of Immunoassays
• The aim of standardization is to ensure that assays of
the same analyte in the same samples, done at
different places or at different times or both, can be
readily compared (Masseyeff).
• International Standards are prepared by the National
Institute for Biological Standards and Control in the
UK (Wild).
• Preparation of standard proteins are purified, mixed
with an inert carrier compound, divided and freeze-
dried, and sent to laboratories (Wild).
Standardization of Immunassay Cont’d

• Known standards include:

– Cortisol
– Alphafetoprotein (AFP)
– Carcino-embryonic antigen (CEA)
– Human gonadotropin (hCG)

Cortisol
(Wild)
Immunoassays and Forensic Science
• 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.

• 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.
Immunoassays and Forensic Science Cont’d
• 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 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 (Abbot Diag.).
References
• Bell, Suzanne (2006). Forensic Chemistry. Upper Saddle River, New Jersey: Pearson
Prentice Hall.
• Saferstein, Richard (2007). Criminalistics. Upper Saddle River, New Jersey: Pearson
Prentice Hall.
• Wild, David (Ed.). (2005). The Immunoassay Handbook. Kidlington, Oxford: Elsevier.
• Evans, Susan (2004, June 15). Retrieved January 19, 2008, from SACB Online Web site:
http://sacb.org.sg/
• Lee, Tim (2007, May 27). Antigens. Retrieved January 19, 2008, from Immunology
Bookcase Web site: http://pim.medicine.dal.ca/atg.htm
• Monoclonal Antibody Production. Retrieved January 19, 2008, from Lecture Notes Web
site: http://www.college.ucla.edu/webproject/micro7/lecturenotes/finished/monoclonal.html
• Masseyeff , RF (1991).Standardization of immunoassays.. Ann Ist Super Sanita. 27, 427-
436.
• Moody, David E. (2006).Immunoassay in Forensic Toxicology. Encyclopedia of Analytical
Chemistry .
• http://www.troopers.state.ny.us/Forensic_Science/Lab_Sections/Toxicology/
• http://www.abbottdiagnostics.com