Download Immunoassays

Director of Poison Control
& Medical Forensic Chemistry Center,
Techniques used
in Toxicology
 Immunoassays
 Chromatographic
 Spectroscopic
(TLC-LC–GC-IC )
( MS-A.A-FTIR-UV)
 Immunoassays
chromatographic methods were employed,
which had good specificity and sensitivity
but were too labor intensive.
The advent of immunochemical assays,
along with automation, meant faster, less
labor intensive and more reliable for TDM
and screening tests (not confirmatory).
Immunoassays principle
are depends on the
reaction of an antigen
(analyte) and an antibody
All immunoassays require the use of labeled
material in order to measure the amount of
antigen or antibody.
Labeled Immunoassays
 Antigen or antibody is labeled ( tagged ) with a substance that
can be detected later on and allows for the detection of an
antibody – antigen reaction
 Types of tags
 Radioactive isotopes
 Enzymes
 Fluorescent molecules
 Luminescent labels
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Types of reactions
 Competitive binding
 Noncompetitive binding
Separation Techniques
Heterogeneous assay
Bound and free antibody must be separated
before label is measured
Example: ELISA (Enzyme Linked
Immunosorbant Assay)
Homogeneous assay
Bound and free antibody do not need to be
separated prior to measurement phase.
Example: EMIT (Enzyme Multiplied
Immunoassay Technique )
Competitive Labeled Immunoassays (RIA, FIA, EIA)
A competition between tagged antigens ( reagent ) and
untagged antigens ( patient )for a limited amount of antibody ( reagent )
 Immunoassays
EMIT: The EMIT (Enzyme Multiplied Immunoassay Technique)
Homogeneous (Bound and free antibody do not need to be
separated prior to measurement phase)
The EMIT (is qualitative and semi-quantitative
technology) is based on competition for the target
analyte antibody binding sites. Analyte in the sample
competes with the drug in the enzyme reagent that
is labeled with G6PDH. Active enzyme G6PDH
converts the coenzyme (NAD) in the antibody
reagent to NADH, resulting in a kinetic absorbance
change that is measured photometrically.
Viva & Viva-E & Viva Twin
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Made by Siemens
Bench top
Relatively small
Measures multiple
therapeutic drugs
quantitatively
Also measures major drugs
of abuse DOA)
Requires little maintenance
Flexible (Easy to use)
Applies EMIT method for all
measurements
Sensitivity is not sufficient;
many interference and falls
positive (Disadvantages !!!)
FPIA: Fluorescence Polarization Immunoassay
This method uses a fluorescent molecule as the label instead of an
enzyme, making it more sensitive. In FPIA, the patient sample is
incubated with a known quantity of the fluorescent-labeled drug and
an antibody specific for the drug. As in EMIT, the labeled and
unlabeled drugs compete for the binding sites of the antibody.
Polarized light is emitted in certain angles depending on whether the
fluorescent-labeled drug is bound to antibodies or not. Since this is a
competitive assay, the greater the amount of drug in the sample, the
lower the amount of fluorescence.
AxSYM
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Made by Abbott
Big instrument
Measures multiple
therapeutic drugs
quantitatively
Also measures major drugs of
abuse DOA)
Easy to use
Requires daily maintenance
Great sensitivity
FPIA is the main technique
for most TDM measurements
Relatively slow
Requires continuous manual
loading of RVs when running
many samples. High cost of
Kit (Disadvantages !!!)
Chemiluminescent microparticle
immunoassay (CMIA)
Chemiluminescence: This is a chemical reaction
that emits energy in the form of light. When used
in combination with immunoassay technology, the
light produced by the reaction indicates the
amount of analyte in a sample
label is a molecule that will react as a part of the
assay, so a change in signal can be measured in
the urine after added reagent solution. CMIA is
non-competitive sandwich assay technology to
measure analytes. The amount of signal is directly
proportional to the amount of analyte present in
the sample
CMIA is non-competitive sandwich assay technology
(Pre-Tigger Solution)
(Tigger Solution)
Activiation
ARCHITECT ci4100
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Made by Abbott
Very big instrument
Measures multiple therapeutic drugs
quantitatively
Also measures major drugs of abuse
DOA (Sensitivity problem for some
tests)
Very fast
Take many unlimited number of
samples (good)
Requires external water supply !!!
Requires complex daily maintenance
Complex software (Requires careful
and long training process)
Chemiluminescent microparticle
immunoassay (CMIA) is the main
technique for most TDM
measurements
 Others Immunoassays
RIA: Radioimmunometric assays
Not commonly used any longer due to waste disposal issues
use radioactivity to detect the presence of the analyte.
A gamma counter is then used to measure the amount of
radioactivity in the sample as counts per minute (CPM).
QC and standards must be performed with each run, leading to
extra labor and cost.
ACMIA: Affinity Chrome-Mediated Immunoassay.
ACMIA is a technique to measure drug concentrations in which
free and drug-bound antibody enzyme conjugates are separated
using magnetic (chrome) particles.
PETINIA: Particle Enhanced Turbidimetric Inhibition
Immunoassay
This method uses the creation of light scattering particles to
measure drug levels. The latex particle-bound drug binds to the
drug-specific antibody, forming insoluble light-scattering
aggregates. This causes an increase in the turbidity of the
reaction mixture.
CEDIA: Cloned enzyme donor immunoassay
(CEDIA) methodology is a novel approach which uses the DNA
technology to produce homogenous enzyme immunoassays for
drugs
The Biochip
The Biochip is the foundation of Biochip Array Technology.
•
A single 9x9mm biochip acts as the reaction vessel, replacing multiple
cuvettes.
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Randox biochips are pre-fabricated with an array of discrete test regions
(DTR’s)
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Randox biochips currently hold up to 25 tests - 23 tests and 2 internal
controls.
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One biochip is used per patient sample.
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Biochip manufacturing processes are accredited to UKAS and ISO13485.
Evidence Biochip Carrier
DRUG DETCTION PERIODS
Sampling
Type of Samples received:
CRUDES, BIOLOGICALS and SEIZED TOOLS
Samples comes from hospitals forensic medicine
administration and from different government
sectors such as:
-Ministry of Interior ( General Directorate of Narcotics Control
and Police offices )
-Customs (Sea & Land ports)
-Other Government sectors
CRUDE SAMPLES
Received from different sectors
Samples of seized crude materials such:
- Khat
- Cannabis
- Captagon tablets
- Alcohols
- Any seized materials and tools etc..
Sampling Specifications:
 Sealed and secure
Privacy (no personal data)
Enough (Fixed amount for each drug)
e.g 5 grams khat
Comes with a letter from requesting sector
Received only from AUTHORIZED person
BIOLOGICAL SAMPLES
Include body fluids (i.e. blood, urine, gastric
lavage, etc.) and tissue samples (Biopsies
and Autopsies)
- Hospitals samples:
TDM , (ET) , Employees screening and police
suspected cases
- Forensic medicine administration samples
BIOLOGICAL SAMPLES (FORENSIC)
Purpose: Employee screening , police suspected cases,
forensic medicine (FM).
Urine sample is the main sample for Narcotics analysis
Blood sample is the main sample for alcohol analysis
For FM, all body fluids (urine, blood, Vitreous humor) and
any other body parts or excretions can be received.
NOTE: Crude samples can be received in some occasions
to help in the detection of the death cause !!
BIOLOGICAL SAMPLES (FORENSIC) cont.
Sampling specifications for employee screening:
2 urine samples
Request form
Labeled correctly
Biohazard bag and cold container
Authorized person
Sampling specifications for police cases:
2 urine samples for narcotics and 2 blood samples for alcohol
All papers completed (request from police, request from hospital,
sample collection witness form)
Labeled correctly
Finger prints of the suspect on the samples and request
No delay
Biohazard bag and cold container
Authorized person
BIOLOGICAL SAMPLES (FORENSIC) cont.
• Sampling specifications for FM:
• Any human or crude samples can be received
• Request form completed by forensic doctor (dead
person information, description of the body during
sampling, any related information)
• Labeled correctly
• No delay
• Biohazard container for tissue transportation and
cold container
• Authorized person
Five “Rights”
Right
Right
Right
Right
Right
Sample
drug
dose
route
time
(in case of Emergency Toxicology & TDM)
Conclusion
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Immunoassay excluded negative samples.
Immunoassay evolves continuously.
Result obtained within short-time (minute).
Easy to use.
No require for extraction procedure.
No hazard chemicals used.