ELISA: Immunoassay for the Common Man John Butler

ELISA: Immunoassay for the ELISA: Immunoassay for the Common Man Common Man (Copland)(Copland)

• History and Basic Principles• The Curse of Convenience and Simplicity• The Virtues of Solid-phase Immunoassay • Quantitation: Accuracy, Precision and

Dynamic Range• Data Expression

A. History and Basic Principles“Slowest ship in the immunassay convoy”Heterogeneous ELISAs are solid-phase

immunoassays (SPI)SPIs come in many flavors: Two are ubiquitous

B. The Curse of Convenience and SimplicitySPIs are diffusion dependentSPIs can have complicated kineticsImmobilization alters proteinsMultimolecular systems cause steric hindranceSolid phases contribute background

E

E

CAb

Ag

Sec.System

PrimarySystem

ESec.System

E

AgAb

PrimarySystem

Ubiquitous ELISA ConfigurationsUbiquitous ELISA ConfigurationsSandwich ELISA Specific Antibody Immunoassay

[SpAbI]

µg/ml ELISA Units/ml




Y

Microtiter Well(Top view)

Conventional Solid-phase Immunoassaysare very Diffusion-dependent

Immobilized antigen

IgG antibody; D 20,w

= 4 X 10-7 cm2/sec

Time for all IgG toreach the immobilizedantigen

82 hr=

Y

“Shake it Baby”

1 2

1 2 3

Time to Equilibrium

Static diffusion (B1)=>100hr

Vortex at 1200 rpm (B2)=5hr

Vortex agitation (B2) decreases the solution:solid phase ratio (SOL:SLD)

C1. Plungers, vacuum & centri-fugation reduce SOL:SLD

C2. Microparticles have lowSOL:SLD

C3. Membranes have lowSOL:SLD




Solid-phase Immunoassay involves Multiple Equilibria

AgSLD + AbSOL AgSLD ----------AbSOL

(AgSLD-AbSOL )+ (AgSLD-AbSOL ) (AgSLDAbSOL)n

k1 D k1 R

k1 A

k2D k2R

k2A

*

* Dashed bond = AgSLD & AbSOL have moved to the interfacial reaction volume

Drate = Governs the diffusion-dependent mass transfer phase of the reaction

Rrate = Governs reactions within the true interfacial reaction volume

Arate= Governs the aggregation phase of the interaction resulting in hysteresis

SLD= solid-phase reactant; SOL=diffusing reactant

“Shake it Baby Phase”




The Woody Hayes TheoremThe Woody Hayes Theorem

E

AgChimeric

IgG

LabeledmAb

E

Adsorbed Chimeric

IgG

The Specificity of an Antibody should betested against its Antigen in Native Configuration

Example: Monoclonal Antibody (mAb) to Swine IgG

Correct Orientation Denatured by Adsorption

Anti-lysozyme Pig-Camelid Chimera

Direct Adsorption of Antibody

LabeledmAb

0

50

100

150

200

250

Rb 8 Rb 10 Mouse 2-3-6 9-40 10-25 4-4-20 5-14 5-27

Polyclonal Monoclonal

CA

beqv

Anti-globulin Streptavidin Adsorbed

Directly Adsorbed Antibodieshave greatly reduced Capture Capacity (Cabeqv)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

a-a1 a-a4 a-b8 a-b12 a-e4 a-g1 a-g6 d4 b-b2 b-b6 b-c7 b-e1 b-f5 b-g1 b-h10

Anti-fibrinogen Clone

O.D

. at 4

05nm

Direct Adsorption Streptavidin-biotin Linkage

Monophage Monophage Selectively Recognize EpitopesSelectively Recognize Epitopeson Denatured Proteins on Denatured Proteins




ELISA Titration Plots do not Parallel PrimaryAntibody Binding Plots

Primary Antibody Plot

ELISA Plot

Sterichindrance

region

Non-specificbindingregion

Conjugate Size Determines Dynamic Range and LOD

LargestConjugate

LowestLOD

SmallestConjugate

HighestLOD




ELISA Titration Plots do not Parallel PrimaryAntibody Binding Plots

Primary Antibody Plot

ELISA Plot

Sterichindrance

region

Non-specificbinding (NSB)

region

Blocking Agents may reduce NSB

Surface occupancy of an unsaturated monolayer

Blocking Protein-protein NSB on a saturated monolayer

Surface occupancy around the “cluster monolayer”

Antigen Blocking Protein

The Virtues of SolidThe Virtues of Solid--phase Immunoassaysphase Immunoassays

Hydrophobically-adsorbed proteins are very stabile: “This makes ELISAs work!”

Proteins adsorb as “active islands”

Solid-phase antibody-antigen reaction mayexperience a two log avidity increase

Flow-through technology allows real-time kinetic measurements

20 mg/ml IgG1 No IgG1

Soak Time (min)

Imm 2/PBS-T86% Bd

91% Bd

PEP/PBS-T

86% Bd

90% Bd

Soak Time (min)

PEP/PBS

Soak Time (min)

98.7% Bd

99.1% Bd

Soak Time (min)

Imm 2/PBS

91% Bd

98.5% Bd

Ng

Rel

ease

d

0

1

2

3

1 10 100 1000

Ng

Rel

ease

d

0

3

6

9

12

1 10 100 1000

Ng

Rel

ease

d

0

3

6

9

12

1 10 100 1000

Ng

Rel

ease

d0

1

2

3

1 10 100 1000

Stability of IgG1 Adsorbed on Polystyrene and Silicone

Imm2= Immulon 2(Dynatech)

PEP= Silicone elastomer(Dow-Corning)

PBS= Phosphate-bufferedsaline

PBS-T= PBS plus 0.05%\Tween 20

%Bd= Measured at 1000 minutes


Hydrophobically-adsorbed proteins are very stabile


Solid-phase antibody-antigen reaction mayexperience a two log avidity increase


Adsorptive Surface are not SmoothAdsorptive Surface are not Smooth

Proteins may be Adsorbed as Active IslandsProteins may be Adsorbed as Active Islands

Proteins may be Adsorbed as Active IslandsProteins may be Adsorbed as Active Islands

Blocking Agents may reduce NSB

Surface occupancy of an unsaturated monolayer

Blocking Protein-protein NSB on a saturated monolayer

Surface occupancy around the “cluster monolayer”

Antigen Blocking Protein




Solid-phase antibody-antigen reaction mayexperience a two log avidity increase:“Another reason why they work!”


The Aggregation Phase of SPIThe Aggregation Phase of SPIResults in HysteresisResults in Hysteresis

(AgSLD--AbSOL )+ (AgSLD--AbSOL ) (AgSLDAbSOL)n

k1 A

k2A

AgSLD + AbSOL AgSLD--AbSOL

k1R

k2R

Ka= k1R 5 X 10 6 m / seck2R 1 X 10 -2 sec = 5 X 108 l/m

Ka= k1A 5 X 10 6 m / sec

k2A 1 X 10 -4 sec= 5 X 1010 l/m

=

=

Translational Diffusion Phase




Solid-phase antibody-antigen reaction mayexperience a two log avidity increase:“Another reason why they work!”


Real-time Reaction Kinetics with Plasmon Surface Resonance

BiaCore from Pharmacea

Quantitation:Accuracy, Precision and Dynamic RangeDynamic range as a function of the

detection system

Capture antibody affinity determines dynamic range

Parallelism determines accuracy in equilibirum-based SPIs

Precision depends on internal standardsand technical skill

Conjugate Size Determines Dynamic Range and LOD

LargestConjugate

SmallestConjugate


detection system




1 10 100 1 10 100 1000 10000

Low

Moderate

High

O.D.O.D.

Nanograms per ml Nanograms per ml

Monoclonal Polyclonal or monoclonal cocktail

Capture Antibody AffinityDetermines the Dynamic Range of Sandwich ELISAs


detection system




Parallelism and the Danger Parallelism and the Danger of Single Point Determinationsof Single Point Determinations

Midpoint (MT) System Endpoint (ET) System

Parallelism between Standard and Test SamplesParallelism between Standard and Test SamplesDetermines Assay AccuracyDetermines Assay Accuracy

The ELISA “Hook”

Plot “B” aftercompetitor depletion

A= Reference Standard C= Test sample with high NSBB= Titration before competitor depletion B’= Titration after competitor depletion

ELISANALYSIS: ELISANALYSIS: One Approach to the Collection of Reliable DataOne Approach to the Collection of Reliable Data

ELISANALYSIS Printout ResultsELISANALYSIS Printout Results


detection system




A. Principle1. Super-saturating amounts of soluble ligand,

e.g. antibody, drive reaction by Mass Law principles2. Activity determined by rate of reaction, not by

endpoint color development

B. Advantage1. Shortens substrate incubation time & total time2. Measures a wider dynamic range3. Simplifies checking for linearity

C. Disadvantage1. Higher reagent and sample concentrations needed2. Low concentrations may not be detected3. Specialized equipment is required

Rate-based ELISAs

Data ExpressionStandards for Antigen Quantity

Absolute StandardsReference Standards

Standards for Antibody QuantityAbsolute StandardsELISA Units of Antibody Activity

Specific Activity Measurements

Use of Specific Activity MeasurementsUse of Specific Activity MeasurementsDefinition: ELISA Activity per ug of IgG (IgA, IgM, etc)Value of Measurement:

Affinity maturationSecondary responsesResponses in body fluids versus serum

Disadvantage: Can’t compare one antigen to another

S COL F L U Response

024681012

0 1 2 3 4 5

Weeks Post-Infection/Immunization

IgG

ant

i-FLU

Res

pons

e

PGF F L U Response

0100200300400500600700

0 1 2 3 4 5

Weeks Pos t-Infection/Immunization

IgG

ant

i-FLU

Res

pons

e

Colonized Only Piglets PRRSV-infected Piglets

Specific Anti- fluorescein activityAnti-fluorescein ELISA Titer

ELISA & Vaccine Development

1. More and more investigators recognize the importance of parallelism; a positive development.

2. Movement toward the use of absolute versus relativestandards seems to be slow. Reason?

3. Most assays appear to be “stopped” equilibrium-type assays, not kinetic assays.

4. Recombinant antigens have been slow to dominatethe field. Explanation?

5. What programs for “networking” or “workshopping” have been developed among investigators?

ReferencesReferencesButler, J. E. 2004. Solid supports in enzyme-linkedimmunoassay and other solid-phase immunoassays. In:Molecular Diagnosis of Infectious Diseases (J. Decker and U.Reischl, Eds.), Humana Press , pp. 333-372, Totowa,, NJ.

Butler, J. E. 1996. Solid-phases in Immunoassays. In: E. P.Diamandis and T. K. Christopoulos (Eds.), Textbook ofImmunological Assays. Academic Press, San Diego, pp. 205-225.

Butler, J. E. 1993. Enzyme-linked immunosorbent assays.In G. C. Howard [Ed.], Methods in Non-radioactiveDetection, Section 9, pp. 90-109. Appleton & Lange,Norwalk, CT.

Butler, J. E. 1994. Enzyme-linked Immunosorbent Assay.In C. J. Van Oss and M. H. V. Van Regenmortel [Eds.],Immunochemistry. Marcel Dekker, Inc., New York, Chapter29. pp. 759-803.

Butler, J. E., and R. G. Hamilton. 1991. Quantitation ofspecific antibodies: Methods of expression, standards, solid-phase considerations and specific applications. In J. E.Butler [Ed.], Immunochemistry of Solid-phase Immunoassay.Chapter 9. C.R.C. Press. pp. 173-198.

Adsorption of Proteins on Polystyrene

* High pH encourages charge-dependent aggregation and protein unfolding and the proportion bound remains constant until saturation (A and B)

* Adsorption avidity increases with molecular size (B)

* Saturation occurs at calculated monolayer formation (B)

pH 9.6

pH 7.5

pH 4.5

IgM

a-LA

SIgA

Adsorption at pH 9.6

* Numbers on bars indicate the Ng bound/well* Delectability of secondarily-adsorbed IgG decreases as more human IgG is immobilized* Interpretation: Epitopes are lost by direct adsorption when secondarily-adsorbed IgG is

immobilized at high concentrations

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ELISA: Immunoassay for the Common Man John Butler