Immunoassays

Immuno refers to an immune

response that causes the body to
generate antibodies and Assay
refers to a test.
What is Immunoassays?​
Type of Bioanalytical test where the detection
of compound or analyte is depends upon the
specific antigen or antibody reaction.​
It has the ability to detect the trace amount
of drugs and other xenobiotics metabolites
in physiological fluid and organs.​
It also has the ability to detect explosive residue.​
It is also used in toxicological analysis.​
Why is it Bioanalytical Test?​
An immunoassay is a type of bioanalytical test
where the detection of a target compound (analyte)
depends on a specific antibody-antigen reaction.
Analyte means the specimen as a subject to analysis.
Antibodies are large proteins produced by the
immune system in response to the presence of
antigens.
Antigens are foreign compounds that can come from
the environment or originate from within the body.
What is Bioanalytical Test  
 Bioanalytical method is used for
“quantitative determination of drugs
and/or metabolites in biological
matrices such as blood, serum,
plasma, or urine.
 Bioanalytical
testing is important in
drug discovery and development. To
quantify drugs and their metabolites
or relate biomarkers in biological
fluids.
Why does antigen and antibody reaction needed?​
 Immunoassays rely on the ability of an antibody to
recognize and bind a specific macromolecule in
what might be a complex mixture of macromolecules. In
some cases, an immunoassay may use an antigen to
detect for the presence of antibodies, which
recognize that antigen, in a solution.
 When various drugs enter the body, the immune system
develops a protective antibody. Immunoassay
techniques measure the presence of antibodies in the
urine associated with particular drugs.
DIFFERENT TYPES OF IMMUNOASSAYS
1. Radioimmunoassay (RIA)

2. Counting Immunoassay (CIA)

3. Enzyme Immunoassays (EIA) or Enzyme-linked immunosorbent

assays (ELISA)

4. Fluoroimmnoassay (FIA)

5. Chemiluminescence immunoassay(CLIA)
  
1. Radioimmunoassay
 The radioimmunoassay is perhaps the oldest
types of immunoassays. Here, a radioisotope is
attached to an antigen of interest and bound with
its complementary antibody. Then a sample with
the antigen to be measured is added. It competes
with the radioactive antigen, kicks it out of the
binding spot and replaces it. After washing away
unbound antigens the radioactivity of the sample
is measured. The amount of radioactive signal is
inversely related to the amount of target
antigen. The health hazards of using radioactive
substances caused a movement toward safer
methods.
2. Counting Immunoassays
 In a counting immunoassay polystyrene beads are
coated with many antibodies complementary to
the target antigen. During incubation the beads bind
to multiple antigens and group together into a large
mass. Some beads remain unbound. The entire
solution is passed through a cell counter and only the
unbound beads are counted. The number of unbound
beads is inversely proportional to the amount of
antigen.
3. Enzyme-linked immunosorbent assay
 In an ELISA the antibody is linked to an
enzyme. After incubation with the antigen the
unbound antibody is washed away. The bound
antibody-enzyme attached to the target antigen is
observed by adding a substrate to the solution.
The enzyme catalyzes a chemical reaction of the
substrate to produce a quantifiable color change. 
A practical example is a magneto-ELISA system
for the detection of CD4+ cells for the diagnosis
of AIDS.
4. Fluoroimmunoassay
 In a fluoroimmunoassay the antibodies are
labeled with fluorescent probes. After
incubation with antigen the antibody-antigen
complexes are isolated and the fluorescent
intensity is measured.
5. Chemiluminescence immunoassay

 The principles of a chemiluminescent immunoassay

 are the same as an ELISA or fluoroimmunoassay,
but the reporter is different. Luminescence is the
release of light due to an electron being kicked up to a
higher energy state and emitting a photon as it relaxes
down. This is the same principle as fluorescence.
The difference lies in the mechanism of kicking the
electron up to a higher energy in the first place. In
fluorescence this is achieved with certain frequencies
of light. In chemiluminescence this is achieved by a
chemical reaction. These reactions require an
emitter and a coreactant. A magneto-actuated
chemiluminescence assay was developed to detect the
presence of  Zika virus in patient samples.
Why do we need to detect the amount
of drugs?​
We need to detect the amount of drugs to identify the
possible overdose and avoid too much usage of drugs.
Alcohol, medications, illegal drugs and some herbal
remedies can cause harm if you take too much. This is
called overdose.
Your risk of overdose increases when you take more than
one of these substances at a time, so the amount of drugs
should be detected.
How to detect the amount of drugs?​
By measuring the amount of labeled drug
bound to the antibody, the analyst can calculate
the amount of unlabeled drug in the biological
specimen.
A toxicology screen is a test that determines the
approximate amount and type of legal or illegal drugs
that you've taken. It may be used to screen for drug
abuse, to monitor a substance abuse problem, or to
evaluate drug intoxication or overdose​.
Xenobiotics Metabolite in Physiological
Fluids and Organs
 Xenobioticsare molecules that are introduced into the body from
the environment and subsequently metabolized by the body.
 Xenobiotic metabolism strongly impacts the body’s oxidative
status.
 Xenobioticmetabolism can be defined as chemical
transformation by a biological system usually via DMEs,
which typically converts relatively lipophilic compounds into
more readily excreted hydrophilic metabolites.
Immunoassays ability to detect
explosive residue
The small amount of solution required for each assay
allows for several analyses. Results of
immunoassays confirmed that they were suitable to
detect post-blast residues in soil and target materials
and post transfer residues on hands.
Why do we need to detect the
explosive residue?
Itis of great importance for the explosive and their
post-blast residues analysis to assist forensic
scientist in determining the origin of explosive
material used in the bombing and eventually help
the law enforcement in narrowing down the
investigation.
How immunoassays detect explosive
residue
 Immunoassays are simple and selective analytical tests able to
detect molecules and their immunoreactions can occur in
portable formats for use on-site. This work demonstrates the
application of three immunochemical assays capable of detecting
TNT(Trinitrotuluene) to typical forensic samples from
experimental tests: an indirect competitive ELISA with
chemiluminescent detection (CL-ELISA). 
 ELISA with chemiluminescent detection
(CL-ELISA)
  A commercially available monoclonal antibody was used and 13
specially synthesized conjugates were tested. We optimized the assay by
determining the optimal concentration of monoclonal antibody and
conjugates and the influence of various non-specific factors such as:
tolerance to organic solvents at different concentrations, the washing and
competitive step time, and the cross-reactivity with related compounds.
 It has been applied to real samples of various materials involved in a
controlled explosion of an "improvised explosive device". Three
extraction procedures were tested on these samples, all employing
methanol as the solvent.
 UNDER THE CATEGORY OF HIGH
EXPLOSIVE
 Primary high explosives will explode if they are heated up or if they are
subjected to some kind of shock. In fact, they are more sensitive to shock,
heat, and friction than secondary explosives are. A couple of explosives in
this category are nitrogen sulfide and mercury fulminate.
 Secondary high explosives aren't as sensitive to friction, heat, and shock as
primary high explosives are. These explosives may be added to blasting
caps to boost power. A couple of explosives in this category are dynamite
and TNT.
Immunoassays in Forensic Toxicology
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.
Immunoassays used in Toxicological Analysis

 Forensictoxicologists 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 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.
Immunoassays in Philippines
 Quantitative detection of Pf HRP2 in saliva of malaria patients in
the Philippines
 quantitativeinsight on the availability of biomarkers and the
dynamics of immunoassay in saliva.
 This pilotstudy measured the levels of the Pf HRP2 in patient saliva
to inform the development of salivary diagnostic tests for malaria.
 Matched samples of blood and saliva were collected between
January and May, 2011 from eight patients at Palawan Baptist
Hospital in Roxas, Palawan, Philippines. Parasite density was
determined from thick-film blood smears. Concentrations
of Pf HRP2 in saliva of malaria-positive patients were measured
using a custom chemiluminescent ELISA in microtiter plates.
SUMMARIZATION
THANK YOU, NATAPOS GID!

ANG PAMANGKOT