immunochemistry principles

8/10/2019 Immunochemistry Principles

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Principles of

Immunochemistry


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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


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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


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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


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5*AEIC,

1998

Other Immunoassay Markets

u Agricultural

u Environmentalu Food

u Industrial

u Pharmaceutical

u Veterinaryu Water Quality


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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


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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


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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


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9*AEIC,

1998

Antibody Structure

Binding Sites

Heavy Chain

Light Chain


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10*AEIC,

1998

Antibody-Antigen Binding


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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


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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


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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


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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


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15*AEIC,1998

Immunogens

Target Analyte

Carrier Protein

Linker

Derivative

Immunogen


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16*AEIC,1998

Immunoassay Visualization

Target Analyte

Analyte bound by antibody

How to detect binding?


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17*AEIC,1998

Immunoassay Conjugates - Detecting Binding

Detectable Label

Radiolabel (RIA) Enzyme (EIA)

Fluorescence (FIA)

Luminescence

Electrochemical

Visual Colloidal gold

Colored latex

Immunoassay

Conjugate


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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


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19*AEIC,1998

Competitive Immunoassay

Target Analyte

Assay

Conjugate

Capture Antibody

Detector and Analyte

compete to bind

with antibody


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20*AEIC,1998


I. No analyte - high detection signal

II. Analyte present - detection signal reduced


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21*AEIC,1998


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


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22*AEIC,1998

Double Antibody Sandwich Immunoassay

Target

Analyte

Capture AntibodyCapture Antibody

DetectorDetector

AntibodyAntibody

Signal

Recognition

Sites(Epitopes)


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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


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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


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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


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26*AEIC,1998

Specificity Considerations for Triazine

Herbicides

Atrazine Simazine

Common to Class

Compound Specific


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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


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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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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


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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


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32*AEIC,1998

Designing Broad Reactivity to 16

Polyaromatic Hydrocarbons

Chrysene

Benzo[a]anthracene

Acenaphthylene

Naphthalene


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33*AEIC,1998

The Effect of the Antibody and Assay

Conjugate Pair on Specificity

0.1

1

10

100

Naphthalene

Fluoranthene

Assay Conjugate

Immunogen


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34*AEIC,1998



0.1

1

10

100

Naphthalene

Pyrene

Immunogen Assay Conjugate


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35*AEIC,1998



0.1

1

10

100

Naphthalene

Pyrene

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 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

immunochemistry principles

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