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Immunochemistry
Immunoassay
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
An immunoassay is an analytical
method which uses antibodies as
reagents to quantitate specific
analytes
*AEIC,
1998
Immunoassays
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
*AEIC,
1998
u Highly Reliable
*AEIC,
1998
u Agricultural
u Pharmaceutical
u Environmental
u Veterinary
u Food
u Water Quality
u Industrial
*AEIC,
1998
u The Sample
l
Clinical
Environmental, Agriculture
Water
Soil extracts
Plant extracts
Animal products/tissues - blood, urine, milk, meat
Food
Industrial processes and effluents
*AEIC,
1998
Antibodies
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Animals
l
l
l
*AEIC,
1998
Antibodies
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Production
u Strength of Binding (Affinity) Determines
Sensitivity of Method
u Specificity Allows Detection in Complex Matrix
- Minimum Sample Preparation
*AEIC,
1998
Antibody Structure
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Binding Sites
Light Chain
Heavy Chain
*AEIC,
1998
Antibody-Antigen Binding
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
*AEIC,
1998
10
Polyclonal Antibodies
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
*AEIC,
1998
11
Monoclonal Antibodies
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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
u
u
u
u
*AEIC,
1998
12
u Polyclonal
u Monoclonal
l Lot-to-lot variability
l Lot-to-lot consistency
l More broadly reactive
l Indefinite supply
l Often more sensitive
l Highly specific
l Shorter lead times
l Longer lead time
l Lower initial costs
l Higher initial costs
1998
13
Antibody Development
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
u Immune System Responds Only to High Molecular Weight
Immunogens
l M.W. Typically > 10,000
u Agrochemicals and Environmental Pollutants Mostly Small
Molecules
l M.W. Typically < 1,000
u Agrochemicals Require Preparation of Suitable Immunogen
l Couple chemical to carrier protein
*AEIC,
1998
14
Immunogens
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Carrier Protein
Immunogen
Linker
Derivative
Target Analyte
*AEIC,
1998
15
Immunoassay Visualization
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*AEIC,
1998
16
Immunoassay
Conjugate
Detectable Label
*AEIC,
1998
Radiolabel (RIA)
Enzyme (EIA)
Fluorescence (FIA)
Luminescence
Electrochemical
Visual
Colloidal gold
Colored latex
17
Immunoassay Formats
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*AEIC,
1998
18
Competitive Immunoassay
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Assay
Conjugate
Target Analyte
Capture Antibody
*AEIC,
1998
19
Competitive Immunoassay
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
*AEIC,
1998
20
Competitive Immunoassay
Data Format
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Competitive Immunoassay Data
Percent of Maximum Absorbance
100
90
80
70
60
50
40
30
20
10
0
0.1
10
100
1000
Concentration of Analyte
*AEIC,
1998
21
Signal
Target
Analyte
Recognition
Sites
(Epitopes)
Detector
Antibody
Capture Antibody
*AEIC,
1998
22
1998
23
Compound
Benzene
TCE
PAH
PCB
RDX
TNT
Detection Level
ppb
[M]
78
106
137
202
324
222
227
100
100
1
1
*AEIC,
1.3E-06
9.4E-07
7.3E-08
5.0E-08
3.1E-08
4.5E-09
4.4E-09
1998
8E+05
1E+06
1E+07
2E+07
3E+07
2E+08
2E+08
24
100
150
200
*AEIC,
250
1998
300
350
400
450
500
25
Common to Class
Compound Specific
Simazine
Atrazine
*AEIC,
1998
26
u Development Process
l
l
l
l
u 1 to 2 Years
*AEIC,
1998
28
Toluene
Toluene Immunogen
CH
C
H3 C
H3 C
NH
CH2 CH2 NH C
NH
S
H3 C
CH
C
H3 C
CH2 CH2 NH C
Different Linkage
CH
C
NH
CH2
H3 C
NH
N
C
O
NH
NH
*AEIC,
1998
29
75
50
Analyte
25
0
0.01
0.1
10
100
1000
Inhibitor (ppb)
*AEIC,
1998
30
75
Analyte
50
Derivative 1
25
0
0.01
0.1
10
100
1000
Inhibitor (ppb)
*AEIC,
1998
31
Naphthalene
Benzo[a]anthracene
Acenaphthylene
Chrysene
*AEIC,
1998
32
Immunogen
100
Assay Conjugate
10
Fluoranthene
0.1
Naphthalene
*AEIC,
1998
33
Assay Conjugate
Immunogen
100
10
Pyrene
0.1
Naphthalene
*AEIC,
1998
34
Immunogen
Assay Conjugate
100
10
Pyrene
0.1
Naphthalene
*AEIC,
1998
35
Principles of Immunochemistry
AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
1998
36