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8/10/2019 Immunochemistry Principles
1/35
Principles of
Immunochemistry
8/10/2019 Immunochemistry Principles
2/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
2*AEIC,
1998
Immunoassay
An immunoassay is an analytical
method which uses antibodies asreagents to quantitate specific
analytes
8/10/2019 Immunochemistry Principles
3/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
3*AEIC,
1998
Immunoassays
u $6 Billion Industry Worldwide
u 2.5 Billion Tests Sold Annuallyu Highly Quantitative
u Regulatory Approved
u Flexible Test Formats
u Diverse Markets and Applications
8/10/2019 Immunochemistry Principles
4/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
4*AEIC,
1998
Clinical Diagnostic Immunoassays
u In Use >30 Years
u Basis for Critical Human Health Decisions
l 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
8/10/2019 Immunochemistry Principles
5/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
5*AEIC,
1998
Other Immunoassay Markets
u Agricultural
u Environmentalu Food
u Industrial
u Pharmaceutical
u Veterinaryu Water Quality
8/10/2019 Immunochemistry Principles
6/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
6*AEIC,
1998
Clinical vs. Environmental Immunoassay
u The Sample
l Clinical
Urine, blood, saliva
l Environmental, Agriculture
Water
Soil extracts
Plant extracts
Animal products/tissues - blood, urine, milk, meat Food
Industrial processes and effluents
8/10/2019 Immunochemistry Principles
7/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
7*AEIC,
1998
Antibodies
u Key Reagents in All Immunoassays
u Proteins Produced by Immune System of Higher
Animalsl Produced by specific white blood cells
l In response to recognition of foreign substances
l Examples:
Vaccinations
Response to natural infections (mumps, chicken pox)
u Physically Bind to Antigens
8/10/2019 Immunochemistry Principles
8/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
8*AEIC,
1998
Antibodies
u Tightly Bind Only to Substance Which Elicited
Production
u Strength of Binding (Affinity) Determines
Sensitivity of Method
u Specificity Allows Detection in Complex Matrix
- Minimum Sample Preparation
8/10/2019 Immunochemistry Principles
9/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
9*AEIC,
1998
Antibody Structure
Binding Sites
Heavy Chain
Light Chain
8/10/2019 Immunochemistry Principles
10/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
10*AEIC,
1998
Antibody-Antigen Binding
8/10/2019 Immunochemistry Principles
11/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
11*AEIC,
1998
Polyclonal Antibodies
u Animals are injected with analytical target
u Many different antibody-producing cells
make Polyclonal antibodies
u Polyclonal antibodies purified directly
from blood
8/10/2019 Immunochemistry Principles
12/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
12*AEIC,
1998
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 grow
continuously in culture to form Hybridomas
u A single hybridoma produces only one antibody
u A single hybridoma divides to produce a large population
of clones all making the same Monoclonal antibody
u Living hybridomas are frozen indefinitely in liquid
nitrogen
u Indefinite supply of uniform consistency reagent
8/10/2019 Immunochemistry Principles
13/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
13*AEIC,
1998
Monoclonal vs. Polyclonal
u Monoclonal
l Lot-to-lot consistency
l Indefinite supplyl Highly specific
l Longer lead time
l Higher initial costs
u Polyclonal
l Lot-to-lot variability
l More broadly reactivel Often more sensitive
l Shorter lead times
l Lower initial costs
Selection is based on application, time and money
8/10/2019 Immunochemistry Principles
14/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
14*AEIC,
1998
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
8/10/2019 Immunochemistry Principles
15/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
15*AEIC,1998
Immunogens
Target Analyte
Carrier Protein
Linker
Derivative
Immunogen
8/10/2019 Immunochemistry Principles
16/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
16*AEIC,1998
Immunoassay Visualization
Target Analyte
Analyte bound by antibody
How to detect binding?
8/10/2019 Immunochemistry Principles
17/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
17*AEIC,1998
Immunoassay Conjugates - Detecting Binding
Detectable Label
Radiolabel (RIA) Enzyme (EIA)
Fluorescence (FIA)
Luminescence
Electrochemical
Visual Colloidal gold
Colored latex
Immunoassay
Conjugate
8/10/2019 Immunochemistry Principles
18/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
18*AEIC,1998
Immunoassay Formats
u Antibodies attached to a solid phase
l Plastic wells, tubes, capillaries
l
Membranesl Latex particles
l Magnetic Particles
u Solid phase used to separate bound from free
Assay Conjugate (label)
u Choice of format determined by application
8/10/2019 Immunochemistry Principles
19/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
19*AEIC,1998
Competitive Immunoassay
Target Analyte
Assay
Conjugate
Capture Antibody
Detector and Analyte
compete to bind
with antibody
8/10/2019 Immunochemistry Principles
20/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
20*AEIC,1998
Competitive Immunoassay
I. No analyte - high detection signal
II. Analyte present - detection signal reduced
8/10/2019 Immunochemistry Principles
21/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
21*AEIC,1998
Competitive Immunoassay
Data Format
Competitive Immunoassay Data
0
10
20
30
40
50
60
70
80
90
100
0.1 1 10 100 1000
Concentration of Analyte
Perc
entofMaximumA
bso
rbance
8/10/2019 Immunochemistry Principles
22/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
22*AEIC,1998
Double Antibody Sandwich Immunoassay
Target
Analyte
Capture AntibodyCapture Antibody
DetectorDetector
AntibodyAntibody
Signal
Recognition
Sites(Epitopes)
8/10/2019 Immunochemistry Principles
23/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
23*AEIC,1998
Immunoassay Performance Characteristics
u Sensitivity (LOD, LOQ) - ppb to ppt (10-12M)
u Specificity
l Families of chemicals vs. single compounds
l Commercial productsl 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
8/10/2019 Immunochemistry Principles
24/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
24*AEIC,1998
Required Sensitivity of Some Environmental
Immunoassays
Compound Detection Levelppb [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+07
RDX 222 1 4.5E-09 2E+08TNT 227 1 4.4E-09 2E+08
8/10/2019 Immunochemistry Principles
25/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
25*AEIC,1998
Sensitivity of Small Molecule Immunoassays
1E-12
1E-11
1E-10
1E-09
1E-08
1E-07
1E-06
1E-05
A
nalyteMDLinA
ssay[M]
50 100 150 200 250 300 350 400 450 500
8/10/2019 Immunochemistry Principles
26/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
26*AEIC,1998
Specificity Considerations for Triazine
Herbicides
Atrazine Simazine
Common to Class
Compound Specific
8/10/2019 Immunochemistry Principles
27/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
28*AEIC,1998
Immunoassay Development Process
u Define Performance Characteristics
l Sensitivity and specificity are determined by the
antibody and the assay conjugate
l Format determined by applicationu Development Process
l 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
8/10/2019 Immunochemistry Principles
28/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
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
8/10/2019 Immunochemistry Principles
29/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
30*AEIC,1998
Selecting an Assay Conjugate for Sensitivity
0
25
50
75
100
0.01 0.1 1 10 100 1000
Inhibitor (ppb)
Analyte
8/10/2019 Immunochemistry Principles
30/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
31*AEIC,1998
Assay Conjugate Effect on Sensitivity
0
25
50
75
100
0.01 0.1 1 10 100 1000
Inhibitor (ppb)
Derivative 1
Analyte
8/10/2019 Immunochemistry Principles
31/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
32*AEIC,1998
Designing Broad Reactivity to 16
Polyaromatic Hydrocarbons
Chrysene
Benzo[a]anthracene
Acenaphthylene
Naphthalene
8/10/2019 Immunochemistry Principles
32/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
33*AEIC,1998
The Effect of the Antibody and Assay
Conjugate Pair on Specificity
0.1
1
10
100
Naphthalene
Fluoranthene
Assay Conjugate
Immunogen
8/10/2019 Immunochemistry Principles
33/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
34*AEIC,1998
The Effect of the Antibody and Assay
Conjugate Pair on Specificity
0.1
1
10
100
Naphthalene
Pyrene
Immunogen Assay Conjugate
8/10/2019 Immunochemistry Principles
34/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
35*AEIC,1998
The Effect of the Antibody and Assay
Conjugate Pair on Specificity
0.1
1
10
100
Naphthalene
Pyrene
Assay ConjugateImmunogen
8/10/2019 Immunochemistry Principles
35/35
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
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 matrix
Minimum sample preparation
Field-portable tests
u Sensitivity and specificity determined by antibody and
assay conjugate pair
u Flexible format provides for diverse applications