Conc Handbook

Biacore
Concentration
Analysis
Handbook
Biacore
Concentration Analysis
Handbook

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_________________________________________________________________________ Contents
Contents
1.
Introduction

2.

Terminology

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Surface preparation in practice
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Developing and running concentration assays 55

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______________________________________________________________________ Introduction
1. Introduction
1.1 Principles of Biacore
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1.2 Biacore in concentration measurement

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Introduction_______________________________________________________________________
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1.3 Why use Biacore?

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______________________________________________________________________ Introduction
1.4 Assay development overview

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Introduction_______________________________________________________________________

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______________________________________________________________________ Terminology
2. Terminology
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2.1 Biacore terminology

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analyte
analyte
ligand
ligand
capturing
molecule
Figure 2-1. Ligand, analyte and capturing molecule in relation to the

sensor surface.
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Terminology ______________________________________________________________________
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Absolute
response (RU)
Report points
Relative
response (RU)
Buffer
Sample
Buffer
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Buffer
Time
Figure 2-2. Schematic illustration of a sensorgram. The bars below the

sensorgram curve indicate the solutions that pass over the sensor
surface.
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______________________________________________________________________ Terminology
2.2 Performance criteria

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2.2.1 Specificity, selectivity and cross-reactivity
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Terminology ______________________________________________________________________
Response/MW
Compound A
100
Compound B
50
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1
100
Concentration
Figure 2-3. Cross-reactivity in a direct assay is determined from calibration

curves of molecular weight-corrected response against concentration. In this
illustration, compound B shows 1% cross-reactivity with compound A.
2.2.2 Accuracy
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Terminology ______________________________________________________________________
Response
CVresponse
CVdose
Concentration
Figure 2-4. CVresponse is an indication of the variability in the response for a

given analyte concentration. CV dose is an indication of the variability in
calculated concentration derived from a given set of measurements.
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Response
acceptable precision
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Limit of
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Figure 2-5. Limits of detection and quantitation.

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Terminology ______________________________________________________________________
2.2.6 Linearity
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Response/MW
Response
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100%
50%
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B50
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IC50
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Figure 2-6. B50 (direct assays, left panel) and IC50 (inhibition assays, right
panel) are the analyte concentration that gives 50% of the maximum
response.

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______________________________________________________________________ Terminology
2.2.8 Robustness
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Response
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Figure 2-7. Sensitivity is the response per unit analyte concentration, which
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the assay if the calibration curve is not linear.

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Terminology ______________________________________________________________________
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____________________________________________________________________ Assay formats
3. Assay formats

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enhancement
molecule
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Direct binding assay
2
Direct binding assay
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analyte
analyte
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ligand
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Surface competition assay
Figure 3-1. Schematic illustration of four different approaches to

concentration measurement with Biacore.
3.1 Direct binding assays

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2
Response
Time
Concentration
Figure 3-2. A single step direct assay gives an increasing response with
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to approach equilibrium will give a higher sensitivity at low analyte
concentrations. The inset shows the sensorgrams from which the calibration
curves are derived.
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Figure 3-3. Sensorgrams (inset) and calibration curves for a direct binding
assay with enhancement. : primary response (analyte), : secondary
response (enhancement reagent).
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3.2 Indirect assays

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Response
Concentration
log[concentration]
Figure 3-4. Inhibition and surface competition assays give a calibration

curve where the response is inversely related to the analyte concentration.
Calibration curves are often shown on a log[concentration] scale (right
panel) to expand the low concentration region.

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Increasing affinity
Increasing concentration
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Figure 3-5. Increasing the affinity of the detecting molecule moves the
operating range to lower analyte concentration and also narrows the range.
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3.2.2 Surface competition
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3.3 Binding rate assays

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Time
Figure 3-6. Binding rate and binding level measurements.
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3.4 Choice of assay technique

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3.5 Choice of ligand and other reagents

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________________________________________________________ Surface preparation principles
4. Surface preparation principles

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Figure 4-1. Schematic illustration of the structure of the sensor chip surface.

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Surface preparation principles ________________________________________________________
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3

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Figure 4-2. Amine coupling of ligands to the sensor surface.
=" %!$$

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Figure 4-3. PDEA Thiol coupling reagent, 2-(2-pyridinyldithio)ethaneamine

hydrochloride.

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Figure 4-4. Ligand thiol and surface thiol coupling of ligands to the sensor
surface.
/!% "%!$! "

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Figure 4-5. Aldehyde coupling of ligands to the sensor surface.
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4.2.4 Streptavidin-biotin capture
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4.3.1 Buffer conditions and pre-concentration
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ligand
isolectric point pI
ligand
isolectric point pI
pH < 3.5
ligand
isolectric point pI
pH > pI
3.5 < pH < pI
surface
pKa 3.5
surface
pKa 3.5
surface
pKa 3.5
Figure 4-6. Ligand is concentrated on the surface through electrostatic

attraction when the pH lies between the isoelectric point of the ligand and
the pKa of the surface. If the pH is too low or too high, ligand will not be
concentrated on the surface.

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4.4 Strategy for surface preparation

4.4.1 Amount of immobilized ligand
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_______________________________________________________ Surface preparation in practice
5. Surface preparation in practice

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Surface preparation in practice _______________________________________________________
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5.2.6 Results of immobilization
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Response
Amount
bound
NHS/EDC
Ligand
Amount
immobilized
Ethanolamine
Time
Figure 5-1. Sensorgram from a typical amine coupling, illustrating the

distinction between the amount of ligand bound and the amount
immobilized.
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5.3 Testing the surface

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Response
3
2
1
4
Maximum
binding
capacity
Time
Figure 5-2. Repeated injections of analyte without regeneration can be used
to estimate the maximum analyte binding capacity of the surface.

"
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5.4 Troubleshooting surface preparation

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5.4.1 Low immobilization levels
9$!"$ 8 &

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Response
NHS/EDC
Ligand
Ethanolamine
Time
Figure 5-3. Inadequate binding of ligand to the surface is seen as a poor

increase in response after the ligand injection (the illustration shows a
sensorgram for amine coupling).
-2

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NHS/EDC
Ligand
Ethanolamine
Time
Figure 5-4. Inadequate immobilization of ligand is seen as a large drop in

response as a result of the final washing step (the illustration shows a
sensorgram for amine coupling).
5.4.2 Low analyte responses
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_____________________________________________________________________ Regeneration
6. Regeneration
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6.1 Regeneration scouting strategy

6.1.1 Choice of regeneration solution
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Regeneration _____________________________________________________________________
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_____________________________________________________________________ Regeneration
Response
bad
good
Time
Response
good
bad
Cycle 1
Cycle 2
Time
Figure 6-1. Efficient regeneration removes all bound analyte. Inefficient

regeneration leaves analyte on the surface (top panel). A second injection of
analyte reveals whether the ligand is still fully active (bottom panel).
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Regeneration _____________________________________________________________________
6.2 Interpreting regeneration scouting

6.2.1 Report point placing
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_____________________________________________________________________ Regeneration
Baseline response
(absolute)
Starting values
(cycle 1)
Sample response
(relative)
Cycle number
Figure 6-2. Scouting for regeneration conditions. The report points from the
first cycle give the starting values: thereafter the points are grouped
according to the regeneration conditions tested. See text for interpretation.
= baseline response; x = sample response.
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Regeneration _____________________________________________________________________
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"
___________________________________________ Developing and running concentration assays
7. Developing and running

concentration assays
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Developing and running concentration assays ____________________________________________
7.2 General practical considerations

7.2.1 Preparing samples
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Report point placing
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Response
Response
Analyte binding
Observed sensorgram
Bulk refractive
index
Time
Time
Figure 7-1. The observed response results from a combination of analyte

binding to the sensor surface and differences in bulk refractive index
between the sample and running buffer.
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Response
Analyte response
+ bulk
Analyte
response
Time
Figure 7-2. Place report points for measuring response after the sample
injection so that the measured response is not affected by the bulk refractive
index of the sample.
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Response
Increasing
affinity
Concentration
Figure 7-3. Using a ligand with a higher affinity moves the calibration curve
towards lower analyte concentrations.
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Response
Increasing
amount of ligand
Concentration
Figure 7-4. Higher amounts of immobilized ligand increase the response but
have little effect on the shape of the calibration curve.
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Response
2
Response
Time
Concentration
Figure 7-5. Longer contact times allow measurement at lower analyte
concentrations. The inset shows the sensorgrams from which the calibration
curves are derived.

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Response
Increasing affinity
Increasing concentration
log[concentration]
Figure 7-6. Increasing the affinity of the detecting molecule moves the
operating range to lower analyte concentrations and also narrows the range.
Increasing the concentration of detecting molecule moves the operating
range to higher concentrations.
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Response
Decreasing concentration
log[concentration]
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7.4 Unwanted binding of analyte or detecting molecule

.& $ $
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7.4.1 Unwanted binding in direct binding assays
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7.4.2 Unwanted binding in inhibition and surface

competition assays
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___________________________________________ Measuring concentration in GxP environments
8. Measuring concentration in

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__________________________________________________________The SPR detection principle
A. The SPR detection principle

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The SPR detection principle __________________________________________________________
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____________________________________________Using binding rates to measure concentration
B. Using binding rates to measure

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Mass transport
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late in the injection.
B.2 Affinity-independent assays

B.2.1 Advantages of affinity-independent assays
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____________________________________________________________________________Index
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Index ____________________________________________________________________________

www.biacore.com
BR-1005-12

Conc Handbook

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Conc Handbook

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Biacore

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'%  ( ")   

Surface preparation principles

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Surface preparation in practice

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Developing and running concentration assays 55

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Measuring concentration in GxP environments 73

The SPR detection principle

6*  

Using binding rates to measure concentration 83

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"  % !"$$ !

1.2 Biacore in concentration measurement

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1.3 Why use Biacore?

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1.4 Assay development overview

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2.1 Biacore terminology

Figure 2-1. Ligand, analyte and capturing molecule in relation to the

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 ! $ " %$ !

Figure 2-2. Schematic illustration of a sensorgram. The bars below the

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2.2 Performance criteria

2.2.1 Specificity, selectivity and cross-reactivity

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Figure 2-3. Cross-reactivity in a direct assay is determined from calibration

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&!E% $ % 

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   

Figure 2-4. CVresponse is an indication of the variability in the response for a

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!% !!  %!!$ $ 

2.2.4 Limit of detection (LOD)

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2.2.5 Limits of quantitation (LOQ)

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Figure 2-5. Limits of detection and quantitation.

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