principles of immunoassay - aeic · immunoassay performance characteristics u sensitivity (lod,...
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2*AEIC, 1998
Immunoassay
An immunoassay is an analyticalmethod which uses antibodies asreagents to quantitate specificanalytes
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Immunoassays
u $6 Billion Industry Worldwide
u 2.5 Billion Tests Sold Annually
u Highly Quantitative
u Regulatory Approved
u Flexible Test Formats
u Diverse Markets and Applications
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Clinical Diagnostic Immunoassays
u In Use >30 Years
u Basis for Critical Human Health Decisionsl Disease diagnosis (AIDS, Hepatitis, PSA)
l Therapeutic drug monitoring
l Drugs of abuse screening
l Over 70 clinical analytes tested by immunoassay
l Home pregnancy tests
u Highly Reliable
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Other Immunoassay Markets
u Agricultural
u Environmental
u Food
u Industrial
u Pharmaceutical
u Veterinary
u Water Quality
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Clinical vs. Environmental Immunoassay
u The Samplel Clinical
² Urine, blood, saliva
l Environmental, Agriculture²Water
² Soil extracts
² Plant extracts
² Animal products/tissues - blood, urine, milk, meat
² Food
² Industrial processes and effluents
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Antibodies
u Key Reagents in All Immunoassays
u Proteins Produced by Immune System of HigherAnimalsl Produced by specific white blood cells
l In response to recognition of “foreign” substances
l Examples:² Vaccinations
² Response to natural infections (mumps, chicken pox)
u Physically Bind to “Antigens”
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Antibodies
u Tightly Bind Only to Substance Which ElicitedProduction
u Strength of Binding (Affinity) DeterminesSensitivity of Method
u Specificity Allows Detection in Complex Matrix- Minimum Sample Preparation
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Antibody Structure
Binding Sites
Heavy ChainLight Chain
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Polyclonal Antibodies
u Animals are injected with analytical target
u Many different antibody-producing cellsmake “Polyclonal” antibodies
u Polyclonal antibodies purified directlyfrom blood
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Monoclonal Antibodies
u Mice are injected with the analytical target
u Antibody producing cells are taken from the animals
u Antibody-producing cells are fused with cells that growcontinuously in culture to form “Hybridomas”
u A single hybridoma produces only one antibody
u A single hybridoma divides to produce a large populationof ‘clones’ all making the same “Monoclonal” antibody
u Living hybridomas are frozen indefinitely in liquidnitrogen
u Indefinite supply of uniform consistency reagent
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Monoclonal vs. Polyclonal
u Monoclonall Lot-to-lot consistencyl Indefinite supplyl Highly specificl Longer lead timel Higher initial costs
u Polyclonall Lot-to-lot variabilityl More broadly reactivel Often more sensitivel Shorter lead timesl Lower initial costs
Selection is based on application, time and money
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Antibody Development
u Immune System Responds Only to High Molecular Weight“Immunogens”
l M.W. Typically > 10,000
u Agrochemicals and Environmental Pollutants Mostly SmallMolecules
l M.W. Typically < 1,000
u Agrochemicals Require Preparation of Suitable Immunogen
l Couple chemical to carrier protein
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Immunogens
Target Analyte
Carrier Protein
Linker
Derivative
Immunogen
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Immunoassay Visualization
Target Analyte
Analyte bound by antibody
How to detect binding?
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Immunoassay Conjugates - Detecting Binding
Detectable Label
• Radiolabel (RIA)
• Enzyme (EIA)
• Fluorescence (FIA)
• Luminescence
• Electrochemical
• Visual
• Colloidal gold
• Colored latex
ImmunoassayConjugate
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Immunoassay Formats
u Antibodies attached to a solid phasel Plastic wells, tubes, capillaries
l Membranes
l Latex particles
l Magnetic Particles
u Solid phase used to separate bound from freeAssay Conjugate (label)
u Choice of format determined by application
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Competitive Immunoassay
Target Analyte
AssayConjugate
Capture Antibody
Detector and Analyte
compete to bind
with antibody
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Competitive Immunoassay
I. No analyte - high detection signal
II. Analyte present - detection signal reduced
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Competitive ImmunoassayData Format
Competitive Immunoassay Data
0
10
20
30
40
50
60
70
80
90
100
0.1 1 10 100 1000
Concentration of Analyte
Per
cen
t o
f M
axim
um
Ab
sorb
ance
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Double Antibody Sandwich Immunoassay
TargetAnalyte
Capture AntibodyCapture Antibody
DetectorDetectorAntibodyAntibody
Signal
RecognitionSites
(Epitopes)
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23*AEIC, 1998
Immunoassay Performance Characteristics
u Sensitivity (LOD, LOQ) - ppb to ppt (10-12M)
u Specificityl Families of chemicals vs. single compounds
l Commercial products
l Metabolites, degradation products
l Process by-products, intermediates
u Precision - Quantitative or qualitative
u Accuracy - Recovery and false negative/positive rates
u Matrix Effects/Interfering Substances
u Linear Range
u Stability, Reliability, Robustness
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Required Sensitivity of Some EnvironmentalImmunoassays
Compound Detection Level ppb [M]
Benzene 78 100 1.3E-06 8E+05106 100 9.4E-07 1E+06
TCE 137 7.3E-08 1E+07PAH 202 5.0E-08 2E+07PCB 324 3.1E-08 3E+07RDX 222 1 4.5E-09 2E+08TNT 227 1 4.4E-09 2E+08
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Sensitivity of Small Molecule Immunoassays
1E-12
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
1E-05A
naly
te M
DL
in A
ssay
[M
]
50 100 150 200 250 300 350 400 450 500
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26*AEIC, 1998
Specificity Considerations for TriazineHerbicides
Atrazine Simazine
Common to Class
Compound Specific
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Immunoassay Development Process
u Define Performance Characteristicsl Sensitivity and specificity are determined by the
antibody and the assay conjugate
l Format determined by application
u Development Processl Antibody and assay conjugate design and
development
l Test format development and optimization
l Validation
l Controlled production, QA/QC
u 1 to 2 Years
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29*AEIC, 1998
Designing Antibodies and Assay Conjugates
H3C H3C
S
CH2 CH2 NH C NH CH2 CH2 CH2 CH2 CH
NH
C O
CHR
C O
NH
H3C
CH2 CH2 CH2 CH2 CH
NH
C O
CHR
C O
NH
HN
SN
O
O
CH2 C
O
S
H3C CH2 CH2 NH C NH CH2 CH2 CH2 CH2 CH
NH
C O
CHR
C O
NH
H3C
Toluene
Different Linkage
Toluene Immunogen
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Selecting an Assay Conjugate for Sensitivity
0
25
50
75
100
0.01 0.1 1 10 100 1000
Inhibitor (ppb)
Analyte
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Assay Conjugate Effect on Sensitivity
0
25
50
75
100
0.01 0.1 1 10 100 1000
Inhibitor (ppb)
Derivative 1
Analyte
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Designing Broad Reactivity to 16Polyaromatic Hydrocarbons
Chrysene
Benzo[a]anthracene
Acenaphthylene
Naphthalene
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33*AEIC, 1998
The Effect of the Antibody and AssayConjugate Pair on Specificity
0.1
1
10
100
Nap
htha
lene
Fluo
rant
hene
Assay Conjugate
Immunogen
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34*AEIC, 1998
The Effect of the Antibody and AssayConjugate Pair on Specificity
0.1
1
10
100
Nap
htha
lene
Pyre
ne
Immunogen Assay Conjugate
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35*AEIC, 1998
The Effect of the Antibody and AssayConjugate Pair on Specificity
0.1
1
10
100
Nap
htha
lene
Pyre
ne
Assay ConjugateImmunogen
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36*AEIC, 1998
Principles of Immunochemistry
u Immunoassays are quantitative analytical methods
u Antibodies physically bind target analytes
u Strength of binding determines sensitivity
u Specificityl Broad or specific (screening or quantitative)
l Allows detection in complex matrix²Minimum sample preparation
² Field-portable tests
u Sensitivity and specificity determined by antibody andassay conjugate pair
u Flexible format provides for diverse applications