elisa: immunoassay for the common man - iowa state ... immunoassay for the common man (copland) •...
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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
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
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
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
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”
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
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
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
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
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
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
The Virtues of SolidThe Virtues of Solid--phase Immunoassaysphase Immunoassays
Hydrophobically-adsorbed proteins are very stabile
Proteins adsorb as “active islands”
Solid-phase antibody-antigen reaction mayexperience a two log avidity increase
Flow-through technology allows real-time kinetic measurements
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:“Another reason why they work!”
Flow-through technology allows real-time kinetic measurements
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
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:“Another reason why they work!”
Flow-through technology allows real-time kinetic measurements
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
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
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
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
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
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
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