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Principles of

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

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

*AEIC,

1998

Clinical Diagnostic Immunoassays


AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC

u In Use >30 Years


u Basis for Critical Human Health Decisions
l
l
l
l
l

Disease diagnosis (AIDS, Hepatitis, PSA)


Therapeutic drug monitoring
Drugs of abuse screening
Over 70 clinical analytes tested by immunoassay
Home pregnancy tests

u Highly Reliable

*AEIC,

1998

Other Immunoassay Markets


AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC

u Agricultural

u Pharmaceutical

u Environmental

u Veterinary

u Food

u Water Quality

u Industrial

*AEIC,

1998

Clinical vs. Environmental Immunoassay


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u The Sample
l

Clinical

Urine, blood, saliva

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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u Key Reagents in All Immunoassays


u Proteins Produced by Immune System of Higher

Animals
l
l
l

Produced by specific white blood cells


In response to recognition of foreign substances
Examples:
Vaccinations
Response to natural infections (mumps, chicken pox)

u Physically Bind to Antigens

*AEIC,

1998

Antibodies
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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

*AEIC,

1998

Antibody Structure
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Binding Sites

Light Chain
Heavy Chain

*AEIC,

1998

Antibody-Antigen Binding
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*AEIC,

1998

10

Polyclonal Antibodies
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u Animals are injected with analytical target


u Many different antibody-producing cells

make Polyclonal antibodies


u Polyclonal antibodies purified directly
from blood

*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

continuously in culture to form Hybridomas


A single hybridoma produces only one antibody
A single hybridoma divides to produce a large population
of clones all making the same Monoclonal antibody
Living hybridomas are frozen indefinitely in liquid
nitrogen
Indefinite supply of uniform consistency reagent

*AEIC,

1998

12

Monoclonal vs. Polyclonal


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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

Selection is based on application, time and money


*AEIC,

1998

13

Antibody Development
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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

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Immunoassay Visualization
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Analyte bound by antibody


Target Analyte

How to detect binding?

*AEIC,

1998

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Immunoassay Conjugates - Detecting Binding


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Immunoassay
Conjugate

Detectable Label

*AEIC,

1998

Radiolabel (RIA)
Enzyme (EIA)
Fluorescence (FIA)
Luminescence
Electrochemical
Visual
Colloidal gold
Colored latex
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Immunoassay Formats
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u Antibodies attached to a solid phase


l
l
l
l

Plastic wells, tubes, capillaries


Membranes
Latex particles
Magnetic Particles

u Solid phase used to separate bound from free

Assay Conjugate (label)


u Choice of format determined by application

*AEIC,

1998

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Competitive Immunoassay
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Detector and Analyte


compete to bind
with antibody

Assay
Conjugate

Target Analyte
Capture Antibody

*AEIC,

1998

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Competitive Immunoassay
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I. No analyte - high detection signal

II. Analyte present - detection signal reduced

*AEIC,

1998

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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

Double Antibody Sandwich Immunoassay


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Signal

Target
Analyte
Recognition
Sites
(Epitopes)

Detector
Antibody

Capture Antibody
*AEIC,

1998

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Immunoassay Performance Characteristics


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u Sensitivity (LOD, LOQ) - ppb to ppt (10-12M)


u Specificity
l
l
l
l

Families of chemicals vs. single compounds


Commercial products
Metabolites, degradation products
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
*AEIC,

1998

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Required Sensitivity of Some Environmental


Immunoassays
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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

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Sensitivity of Small Molecule Immunoassays

Analyte MDL in Assay [M]

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1E-05
1E-06
1E-07
1E-08
1E-09
1E-10
1E-11
1E-12
50

100

150

200

*AEIC,

250

1998

300

350

400

450

500

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Specificity Considerations for Triazine


Herbicides
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Common to Class
Compound Specific

Simazine

Atrazine

*AEIC,

1998

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Immunoassay Development Process


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u Define Performance Characteristics


l
l

Sensitivity and specificity are determined by the


antibody and the assay conjugate
Format determined by application

u Development Process
l
l
l
l

Antibody and assay conjugate design and


development
Test format development and optimization
Validation
Controlled production, QA/QC

u 1 to 2 Years
*AEIC,

1998

28

Designing Antibodies and Assay Conjugates


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Toluene

Toluene Immunogen

CH
C

H3 C

H3 C

NH

CH2 CH2 NH C

NH CH2 CH2 CH2 CH2 CH


C

NH

S
H3 C

CH
C

H3 C

CH2 CH2 NH C

Different Linkage

CH
C

NH

CH2

NH CH2 CH2 CH2 CH2 CH


C

H3 C

NH

HN CH2 CH2 CH2 CH2 CH

N
C
O

NH

NH

*AEIC,

1998

29

Selecting an Assay Conjugate for Sensitivity


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100

75

50

Analyte

25

0
0.01

0.1

10

100

1000

Inhibitor (ppb)
*AEIC,

1998

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Assay Conjugate Effect on Sensitivity


AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC AEIC
100

75
Analyte

50

Derivative 1

25

0
0.01

0.1

10

100

1000

Inhibitor (ppb)
*AEIC,

1998

31

Designing Broad Reactivity to 16


Polyaromatic Hydrocarbons
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Naphthalene

Benzo[a]anthracene

Acenaphthylene

Chrysene

*AEIC,

1998

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The Effect of the Antibody and Assay


Conjugate Pair on Specificity
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Immunogen
100

Assay Conjugate
10

Fluoranthene

0.1

Naphthalene

*AEIC,

1998

33

The Effect of the Antibody and Assay


Conjugate Pair on Specificity
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Assay Conjugate

Immunogen
100

10

Pyrene

0.1

Naphthalene

*AEIC,

1998

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The Effect of the Antibody and Assay


Conjugate Pair on Specificity
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Immunogen

Assay Conjugate

100

10

Pyrene

0.1

Naphthalene

*AEIC,

1998

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Principles of Immunochemistry
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u Immunoassays are quantitative analytical methods


u Antibodies physically bind target analytes
u Strength of binding determines sensitivity
u Specificity
l
l

Broad or specific (screening or quantitative)


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
*AEIC,

1998

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