Jill Yeakel
History
Forensic Analysis
Screening Tests
Confirmation Tests
Immunoassays
Antibodies
Immunoglobulins
polypeptides
2 Heavy chains
2 Light chains
amino acids
Determines specificity
Antibody Regions
Constant Region
Variable Region
Hypervariable (HV)
Complementarity
antigens surface
HV1, HV2, HV3
Framework (FR)
4
Antigens
Hapten
<2000 Daltons
Small molecular weight compound that elicits immune
response ONLY when attached to large carrier
Necessary to trick animals immune system into reacting by
binding the small molecule with a large antigenic molecule
Epitope
Polyclonal
Monoclonal
Polyclonal Antibody
Production
1.
2.
3.
4.
Inject compounds of
interest (antigen) into
animal
Animal system reacts to
form antibodies to antigen
Antibodies are in animals
blood
Scientists collect animal
blood (serum)
Y Y
Y
Y
Y
Y
Y
Y
Y
Polyclonal Antibodies
Monoclonal Antibody
Production
Spleen of Mouse
Monoclonal Antibodies
Binding Definitions
Immunoassay Uses
Immunoassay Choice
Assay sensitivity
Assay specificity
Format and ease of use
Specimen volumes
Analytical equipment available
Cost
Types of Immunoassays
Labeled Antigens
Methodological Principles
Competitive
Noncompetitive
Methodological Principles
Heterogeneous
Homogeneous
Heterogeneous Assays
ELISA
RIA
ELISA Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.
ELISA
H
NH2
H +
NH2
Tetramethylbenzidine
HRP
OH- + OH-
NH + 2H2O
NH
ELISA
Antibody
Drug
H+
H+
H+
H+
H+
H + HRP
OH- + OH-
2H2O
H2SO4
2H+ + SO4-
ELISA
Drugs Present
Enzyme washed
away, nothing to
oxidize substrate
No color
Enzyme free to
oxidize substrate
Drugs
NOT Present
Color
ELISA
Strengths:
Oriented antibody
Optical enzyme conjugates
Small sample size
Long shelf-life
Non-isotopic
Weaknesses:
RIA Procedure
1.
2.
3.
4.
5.
6.
RIA
Drugs Present
Radioactive antigen
in Solution
Radioactivity in
Solution
No radioactive
antigen in solution
Drugs
NOT Present
No Radioactivity in
Solution
RIA
Strengths:
Sensitive
Resists matrix effect
Weaknesses:
Radioactive waste
Expensive
Short shelf-life
Heterogeneous
False positives from adulterants
Homogeneous Assays
EMIT
FRET
FPIA
KIMS
CEDIA
Lateral Flow
EMIT Procedure
1.
2.
3.
4.
5.
6.
7.
EMIT
EMIT
Enzyme free to
oxidize substrate
Drugs Present
Color
Enzyme CANNOT
oxidize substrate
Drugs
NOT Present
No Color
EMIT
Strengths:
Homogeneous
Long shelf-life
Assays are well established
More specialty assays are available
Weaknesses:
Many interferences
False negatives are common
Tolmetin
metabolites
Aspirin metabolites
FRET Procedure
1.
2.
3.
4.
5.
FRET Principle
FRET
Acceptor and donor
DO NOT interact
Drugs Present
Acceptor quenches
fluorescence
Drugs
NOT Present
Fluorescence
Less Fluorescence
FRET
Strengths:
Homogeneous
Sensitive
Fast
Robust
Accurate
Weaknesses:
FPIA Procedure
1.
2.
3.
4.
5.
FPIA
Unbound
Drugs Present
Depolarized
Emission
Bound
Polarized
Light
Polarized
Emission
Drugs
NOT Present
Step 3: Measure fluorescence of solution
FPIA
Strengths:
Weaknesses:
Expensive
Not easily adapted to automation
False positives:
Ibuprofen
Bile
salts
KIMS Procedure
1.
2.
3.
4.
5.
KIMS
Drugs Present
No Aggregation
No change in
absorbance
Aggregation
Change in
absorbance
Drugs
NOT Present
KIMS
Strengths:
Inexpensive
Homogeneous
Long shelf-life
Microparticle drug conjugate stability
Weaknesses:
CEDIA Procedure
1.
2.
3.
4.
5.
6.
7.
8.
CEDIA
CEDIA
-galactosidase
Chlorophenol red--galactopyranoside
Chlorophenol red
+
-galactopyranoside
Step 4: Observe/measure color
CEDIA
Enzyme donor and
acceptor complement
Drugs Present
RED Color
Cleave substrate
Drugs
NOT Present
NO Color
CEDIA
Strengths:
Homogeneous
Long shelf-life
Specialty assays
Weaknesses:
One-Step Procedure
Patient Info
COCAINE
OPIATES
THC
METH
AMP
C
C
YYYYYYY
T
YYYYYYY
YYYYYYY
C
YYYYYYY
YYYYYYY
Lateral Flow
Strengths:
Fast
Simple
Can be performed at point of collection
Only non-negatives are forwarded to lab for testing
Multiple drugs tested at once
Weaknesses:
Matrix limitations
Subjective
Not automated
IMMUNOASSAY
PERFORMANCE AND
TROUBLESHOOTING
Performance Characteristics
Discrimination Point
Thresholds
Precision and Accuracy
Dynamic Range
Specificity and Cross-Reactivity
Correlation
Interferences
Discrimination Point
Setting Thresholds
Dynamic Range
Specificity
Specificity
Cross Reactivity
Correlation
Interferences
Photometric
Enzyme Activity
Antigen-Antibody Binding behavior
Additional Sources of Interference
Photometric Interferences
Enzyme Interferences
Sources of Interference
Endogenous compounds
Interference from structurally similar compounds
Cross-reactivity from structurally unrelated compounds
Adulterants
High dose hook effect
Endogenous Compounds
Structurally Similar
Compounds
Ephedrine, phenylpropanolamine
Significant improved specificity of amphetamine
immunoassays by introducing monoclonal antibodies
High
Structurally Unrelated
Compounds
Adulterants
Table salt
Bleach
Visine - For EMIT, THCC partitions between the aqueous
solvent and hydrophobic interior of benzalkonium chloride
micelles, reducing availability of THCC to bind to antibody
Applications
Sample Types Urine, but can use oral fluid, hair and sweat
Specimen Volumes Small volume required
Format Qualitative and semiquantitative
Federally regulated labs MUST do initial screen using
immunoassay
In Postmortem
Instrument Selection
Important considerations:
Quality Control
Reagent Considerations
Proficiency Testing
Performance
Troubleshooting
Troubleshooting in ELISA
Troubleshooting in EMIT
Plate Drift