Download Immunoassays - OldForensics 2012-2013

Immunoassays
Katarzyna, Reyna, Becca, Ismael
History
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The first immunoassay was described by Berson and Yalow in 1959. Their work
resulted in their receipt of the Nobel Prize in Medicine in 1977. Since this
introduction, immunoassays have evolved considerably.
The first homogenous immunoassay (no separation step required) was the Enzyme
Multiplied Immunoassay Technique (EMIT), which enabled adaptation of this assay
onto automated chemistry platforms.
Today, immunoassays play a prominent role in the analysis of many clinical
laboratory analytes such as proteins, hormones, drugs, and nucleic acids.
Although unskilled technicians can run immunoassays, the development of these
tests requires knowledge in many areas of immunology and protein chemistry.
Syva immunoassays which were commercially produced first were developed to be
used in the methadone maintenance treatment programs. When the US military
explored methods to curb the drug addiction problem in troops from Vietman,
further development in drug testing emerged and expanded.
Major developments in homogenous immunoassays have been due to the
development of illicit drug testing and therapeutic drug testing programs.
Immunoassay Tests
• 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 Blymphocytes (immune cells) in response to stimulation by an
antigen. Immunoassays measure the formation of antibodyantigen complexes and detect them via an indicator reaction.
• Immunoassays may be qualitative (positive or negative) or
quantitative (amount measured).
• There are several different methods used in immunoassay
tests.
Immunoassay Tests Continued
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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
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Particle immunoassays This allows rapid and sensitive detection of antibodies that are markers of
such diseases, as infectious mononucleosis and rheumatoid arthritis.
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Immunonephelometry.. The antigen concentration can be determined within minutes of the
reaction.
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.
• They are also used to detect antigens associated with
organisms that are difficult to culture, such as hepatitis
B virus and Chlamydia trichomatis.
How They Are Collected
• Blood samples are collected by vein puncture
with a needle.
• Urine samples are acceptable for drug assays
Homogenous & Heterogeneous
Methods
• Homogeneous immunoassays do not require
separation of unbound complexes from the
bound complexes, and thus are faster and
easier to perform then heterogeneous
immunoassays.
• Heterogeneous immunoassays require the
separation of unbound complexes, often
utilizing a solid phase reagent such as a
magnetic particle or plastic bead.
Normal Results
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Immunoassays which are qualitative are reported as positive or negative.
Quantitative immunoassays are reported in mass units
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Although immunoassays are both highly sensitive and specific, false positive and
negative results may occur.
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False-negative results may be caused by improper sample storage or treatment,
reagent deterioration, or improper washing technique.
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False-positive results are sometimes seen in persons who have certain antibodies,
especially to mouse immunoglobulins (immune cells) that may be used in the test.
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Immunoassay is an in vitro procedure, and is therefore not associated with
complications
Advantages of Immunoassays
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High Sensitivity – Low detection limit
High Specificity - Detect specific compound
Safe and simple
Fast Tests (between 5 minutes and 1 hour)
Cost effective
Field-portable tests
Tests can yield quantitative or qualitative data
Disadvantages of Immunoassays:
• They have limitations
• Negative results don’t always rule out the
presence of a drug
• May not be sensitive to be certain compounds
• Some chemists are reluctant to use
immunoassays due to its biological basis and
their unfamiliarity with it
Common Immunoassay Types:
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Enzyme-linked immunosorbent assay (ELISA)
Western blot
Immunofluorescent antibody assay (IFA)
Electrochemoluminescence
Magnetic immunoassays
Advantage of Each Type
• ELISA- Widely-used, high-throughput,
inexpensive
• Western Blot- Highly sensitive and specific
• IFA- simple to perform
• Electrochemoluminescence- very high
sensitivity
• Magnetic immunoassays- produce
quantitative results
Disadvantage of Each Type:
• ELISA- Requires temperature control, more
qualitative results, time-and-labor intensive
• Western Blot- Has a complicated lab analysis
• IFA- less sensitive than ELISA, also requires use of
a microscope
• Electrochemoluminescence- More applicable to
commercial labs
• Magnetic immunoassays- requires a magnetic
reader that limits its field application
Case #1
• Immunoassay is used in examining hair for
drug investigations.
- Hair analysis was performed in order to
determine and establish inter and intraday
variabilities.
Case #1
• Being examined:
- 2 suicide cases
- 1 allege date rape
- Benzodiazepines user
• Results: All hair specimens screened positive for
benzodiazepines using micro-plate enzyme
immunoassay.
• More Results: 2 cases (including date rape) were
negative for FN and 7-AFN (aminoflunitrazepam).
2 post mortem samples were positive for FN
and its metabolite.
Case #2
• Post mortem false positive reactivity
• Tool Utilized: Enzyme-multiplied urine
phencyclidine (PCP) immunoassay
• Why? A single-agent fatal tramadol overdose
(Tramadol level of 14.0 mg/L while
therapeutic range is 0.1 to 0.3 mg/L)
• Results: The urine yielded positive for PCP at
88 mAU/min.
Case #2
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BUT…
The family was like, “Nawwww”.
False positive
Conclusion: Immunoassay of tramadol and its
metabolites in aggregate is responsible for the
PCP immunoassay interference and falsepositive result.
Works Cited
1) Wild, David E. Immunoassay Handbook, third edition. Oxford: Elsevier Ltd, 2005. online
2) "What Is the History of Immunoassays?" Apoptosis, Cell Death, Caspases, Necrosis, Assay
Development, Blocking Buffer, ELISA Buffer. N.p., n.d. Web. 02 Apr. 2013.
3) "Surge" Immunoassay Tests. N.p., n.d. Web. 02 Apr. 2013.
4) http://www.seedtechnology.net/genetic_wrk/2006%20IAWG%20%20Basics%20of%20Immunoassay%20Presentation.pdf
5) http://www.bvgh.org/Biopharmaceutical-Solutions/Global-HealthPrimer/Targets/cid/ViewDetails/ItemID/13.aspx
6) http://www.princeton.edu/~ota/disk2/1988/8830/883006.PDF
7) https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CDkQFjAB&
url=http%3A%2F%2Fwww.cap.org%2Fapps%2Fdocs%2Fnewspath%2F1112%2Fbenzodiazepin
es.doc&ei=-5FbUd79DqSCyAHGqYHoAg&usg=AFQjCNHlIsalpYnszYIY2mudvn2k7dPFQ&sig2=wWCzEOSPPk_bKJypEmanqg&bvm=bv.44697112,d.aWM&cad=rja
Review Questions
1) What do immunoassay tests detect?
2) What are some advantages/disadvantages of
immunoassays?
3) What is the difference between homogenous
and heterogeneous immunoassays?
4) What are normal results of immunoassays?
5) What are the common immunoassay types?