conc handbook
TRANSCRIPT
-
8/19/2019 Conc Handbook
1/106
Biacore
Concentration
Analysis Handbook
®
-
8/19/2019 Conc Handbook
2/106
-
8/19/2019 Conc Handbook
3/106
Biacore®®®®
Concentration Analysis
Handbook
-
8/19/2019 Conc Handbook
4/106
©
-
8/19/2019 Conc Handbook
5/106
_________________________________________________________________________Contents
Contents
1. Introduction 1
2. Terminology 5
3. Assay formats 15
4. Surface preparation principles 27
-
8/19/2019 Conc Handbook
6/106
Contents _________________________________________________________________________
5. Surface preparation in practice 37
6. Regeneration 49
7. Developing and running concentration assays 55
-
8/19/2019 Conc Handbook
7/106
_________________________________________________________________________Contents
8. Measuring concentration in GxP environments73
A. The SPR detection principle 79
B. Using binding rates to measure concentration 83
-
8/19/2019 Conc Handbook
8/106
Contents _________________________________________________________________________
-
8/19/2019 Conc Handbook
9/106
______________________________________________________________________ Introduction
1.
Introduction
•
•
•
1.1 Principles of Biacore
1.2
Biacore in concentration measurement
-
8/19/2019 Conc Handbook
10/106
Introduction_______________________________________________________________________
•
•
•
1.3
Why use Biacore?
-
8/19/2019 Conc Handbook
11/106
______________________________________________________________________ Introduction
1.4 Assay development overview
-
8/19/2019 Conc Handbook
12/106
Introduction_______________________________________________________________________
-
8/19/2019 Conc Handbook
13/106
______________________________________________________________________ Terminology
2.
Terminology
•
•
analyte
ligand
analyte
ligand
capturingmolecule
Figure 2-1. Ligand, analyte and capturing molecule in relation to the sensor surface.
•
2.1 Biacore terminology
-
8/19/2019 Conc Handbook
14/106
Terminology ______________________________________________________________________
•
•
•
•
Buffer Sample Buffer Regeneration Buffer
Time
Absoluteresponse (RU)
Report points
Relative
response (RU)
Figure 2-2. Schematic illustration of a sensorgram. The bars below the sensorgram curve indicate the solutions that pass over the sensor surface.
•
-
8/19/2019 Conc Handbook
15/106
______________________________________________________________________ Terminology
2.2.1 Specificity, selectivity and cross-reactivity
2.2 Performance criteria
-
8/19/2019 Conc Handbook
16/106
Terminology ______________________________________________________________________
100
50
0
1 100
Response/MW
Concentration
Compound A
Compound B
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
2.2.3 Precision
•
-
8/19/2019 Conc Handbook
17/106
______________________________________________________________________ Terminology
•
•
( ) ( )
=−
−∑
×=
-
8/19/2019 Conc Handbook
18/106
Terminology ______________________________________________________________________
Response
Concentration
CVresponse
CVdose
Figure 2-4. CV response 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.
2.2.4
Limit of detection (LOD) ×
2.2.5 Limits of quantitation (LOQ)
-
8/19/2019 Conc Handbook
19/106
______________________________________________________________________ Terminology
× ×
Response
Concentration
3xSD
Limit ofdetection
Limits ofquantitation
acceptable precisionand accuracy
Figure 2-5. Limits of detection and quantitation.
-
8/19/2019 Conc Handbook
20/106
Terminology ______________________________________________________________________
2.2.6 Linearity
2.2.7 Range
Response/MW
Concentration
0
100%
50%
B50
Response
Concentration
0
100%
50%
IC50
Figure 2-6. B 50 (direct assays, left panel) and IC 50 (inhibition assays, right panel) are the analyte concentration that gives 50% of the maximum response.
-
8/19/2019 Conc Handbook
21/106
______________________________________________________________________ Terminology
2.2.8 Robustness
2.2.9 Sensitivity
Response
Concentration
Sensitivity= slope
Figure 2-7. Sensitivity is the response per unit analyte concentration, which is the slope of the calibration curve. The sensitivity varies over the range of the assay if the calibration curve is not linear.
-
8/19/2019 Conc Handbook
22/106
Terminology ______________________________________________________________________
-
8/19/2019 Conc Handbook
23/106
____________________________________________________________________ Assay formats
3.
Assay formats
•
•
•
-
8/19/2019 Conc Handbook
24/106
Assay formats_____________________________________________________________________
analyte
ligand
competinganalyte
Surface competition assay
analyte
ligand
Direct binding assaywith enhancement
enhancementmolecule
1 2
analyte
ligand
Direct binding assay
analyte
ligand
detecting
molecule
Inhibition assay(solution competition)
Figure 3-1. Schematic illustration of four different approaches to concentration measurement with Biacore.
3.1.1 Single step direct measurement
3.1
Direct binding assays
-
8/19/2019 Conc Handbook
25/106
____________________________________________________________________ Assay formats
Response
Concentration
Time
1
2
1
2Response
Figure 3-2. A single step direct assay gives an increasing response with increasing concentration. The range and sensitivity are determined in part by the time at which the response is measured: an interaction that is allowed to approach equilibrium will give a higher sensitivity at low analyte concentrations. The inset shows the sensorgrams from which the calibration curves are derived.
3.1.2 Sandwich methods
-
8/19/2019 Conc Handbook
26/106
Assay formats_____________________________________________________________________
Response
Concentration
Time
1
2
1
2Response
Figure 3-3. Sensorgrams (inset) and calibration curves for a direct binding assay with enhancement. : primary response (analyte), : secondary response (enhancement reagent).
-
8/19/2019 Conc Handbook
27/106
____________________________________________________________________ Assay formats
3.2.1 Inhibition assays
Response
Concentration
Response
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.
3.2 Indirect assays
-
8/19/2019 Conc Handbook
28/106
Assay formats_____________________________________________________________________
+=
Response
log[concentration]
Increasing affinity
Increasing concentrationof detecting molecule
Figure 3-5. Increasing the affinity of the detecting molecule moves the operating range to lower analyte concentration and also narrows the range.
-
8/19/2019 Conc Handbook
29/106
____________________________________________________________________ Assay formats
3.2.2 Surface competition
-
8/19/2019 Conc Handbook
30/106
Assay formats_____________________________________________________________________
Response
Time
Initial bindingrate Binding level
Figure 3-6. Binding rate and binding level measurements.
3.3 Binding rate assays
-
8/19/2019 Conc Handbook
31/106
____________________________________________________________________ Assay formats
3.4 Choice of assay technique
-
8/19/2019 Conc Handbook
32/106
Assay formats_____________________________________________________________________
3.5.1 Practical approaches to reagent selection
3.5.2 Direct binding assays
•
•
3.5 Choice of ligand and other reagents
-
8/19/2019 Conc Handbook
33/106
____________________________________________________________________ Assay formats
•
•
3.5.3 Inhibition assays
-
8/19/2019 Conc Handbook
34/106
Assay formats_____________________________________________________________________
3.5.4 Surface competition assays
-
8/19/2019 Conc Handbook
35/106
________________________________________________________ Surface preparation principles
4.
Surface preparation principles
S e n s o r C h i p
C M 5
C e r t i f i e d G r a d e
Dextranmatrix
Glass
Gold layer
Figure 4-1. Schematic illustration of the structure of the sensor chip surface.
4.1 Sensor surface properties
-
8/19/2019 Conc Handbook
36/106
Surface preparation principles ________________________________________________________
•
•
•
•
•
4.2.1 Amine coupling
4.2 Ligand immobilization methods
-
8/19/2019 Conc Handbook
37/106
________________________________________________________ Surface preparation principles
Figure 4-2. Amine coupling of ligands to the sensor surface.
4.2.2 Thiol coupling
N SSCH2CH2NH2 HCl
Figure 4-3. PDEA Thiol coupling reagent, 2-(2-pyridinyldithio)ethaneamine
hydrochloride.
-
8/19/2019 Conc Handbook
38/106
Surface preparation principles ________________________________________________________
Figure 4-4. Ligand thiol and surface thiol coupling of ligands to the sensor surface.
4.2.3 Aldehyde coupling
Figure 4-5. Aldehyde coupling of ligands to the sensor surface.
-
8/19/2019 Conc Handbook
39/106
________________________________________________________ Surface preparation principles
4.2.4 Streptavidin-biotin capture
4.2.5
General capture methods
-
8/19/2019 Conc Handbook
40/106
Surface preparation principles ________________________________________________________
4.2.6 Immobilizing small molecules
•
•
•
•
-
8/19/2019 Conc Handbook
41/106
________________________________________________________ Surface preparation principles
4.3.1 Buffer conditions and pre-concentration
3.5 < pH < pI pH > pI
ligandisolectric point pI
pH < 3.5
ligandisolectric point pI
ligandisolectric point pI
surface
pK 3.5a
surface
pK 3.5a
surface
pK 3.5a
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 pK a of the surface. If the pH is too low or too high, ligand will not be concentrated on the surface.
4.3 Conditions for ligand immobilization
-
8/19/2019 Conc Handbook
42/106
Surface preparation principles ________________________________________________________
4.4.1 Amount of immobilized ligand
4.4
Strategy for surface preparation
-
8/19/2019 Conc Handbook
43/106
________________________________________________________ Surface preparation principles
4.4.2 Choice of immobilization method
-
8/19/2019 Conc Handbook
44/106
Surface preparation principles ________________________________________________________
-
8/19/2019 Conc Handbook
45/106
_______________________________________________________ Surface preparation in practice
5.
Surface preparation in practice
•
•
•
5.1
Conditions for immobilization
-
8/19/2019 Conc Handbook
46/106
Surface preparation in practice _______________________________________________________
•
•
•
-
8/19/2019 Conc Handbook
47/106
_______________________________________________________ Surface preparation in practice
5.2.1 Preparing solutions
5.2
Immobilization procedure
-
8/19/2019 Conc Handbook
48/106
Surface preparation in practice _______________________________________________________
5.2.2 Amine coupling
5.2.3 Surface thiol coupling
-
8/19/2019 Conc Handbook
49/106
_______________________________________________________ Surface preparation in practice
-
8/19/2019 Conc Handbook
50/106
Surface preparation in practice _______________________________________________________
5.2.4 Ligand thiol coupling
-
8/19/2019 Conc Handbook
51/106
_______________________________________________________ Surface preparation in practice
5.2.5 Aldehyde coupling
-
8/19/2019 Conc Handbook
52/106
-
8/19/2019 Conc Handbook
53/106
_______________________________________________________ Surface preparation in practice
1
2
3
Response
Time
4
Maximumbinding
capacity
Figure 5-2. Repeated injections of analyte without regeneration can be used to estimate the maximum analyte binding capacity of the surface.
5.3 Testing the surface
-
8/19/2019 Conc Handbook
54/106
Surface preparation in practice _______________________________________________________
5.4.1 Low immobilization levels
•
•
•
•
Response
Time
NHS/EDC Ligand Ethanolamine
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).
5.4 Troubleshooting surface preparation
-
8/19/2019 Conc Handbook
55/106
_______________________________________________________ Surface preparation in practice
•
•
•
•
Response
Time
NHS/EDC Ligand Ethanolamine
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
-
8/19/2019 Conc Handbook
56/106
Surface preparation in practice _______________________________________________________
-
8/19/2019 Conc Handbook
57/106
-
8/19/2019 Conc Handbook
58/106
-
8/19/2019 Conc Handbook
59/106
_____________________________________________________________________ Regeneration
good
bad
Response
Time
Cycle 1 Cycle 2
good
bad
Response
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).
-
8/19/2019 Conc Handbook
60/106
Regeneration _____________________________________________________________________
6.2.1 Report point placing
6.2.2 Trend plots
•
•
•
6.2 Interpreting regeneration scouting
-
8/19/2019 Conc Handbook
61/106
_____________________________________________________________________ Regeneration
Sample response(relative)
Baseline response(absolute)
Cycle number
A B C D
Starting values(cycle 1)
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.
-
8/19/2019 Conc Handbook
62/106
Regeneration _____________________________________________________________________
-
8/19/2019 Conc Handbook
63/106
___________________________________________ Developing and running concentration assays
7.
Developing and running
concentration assays
•
•
•
•
•
•
•
7.1
Assay requirement specification
-
8/19/2019 Conc Handbook
64/106
Developing and running concentration assays ____________________________________________
7.2.1 Preparing samples
•
•
•
7.2 General practical considerations
-
8/19/2019 Conc Handbook
65/106
___________________________________________ Developing and running concentration assays
7.2.2 Report point placing
Response
Response
Time
Time
Analyte binding
Bulk refractive
index
Observed sensorgram
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.
-
8/19/2019 Conc Handbook
66/106
Developing and running concentration assays ____________________________________________
Response
Time
Analyte response+ bulk
Analyteresponse
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.
7.3
Adjusting the operating range
-
8/19/2019 Conc Handbook
67/106
___________________________________________ Developing and running concentration assays
7.3.1 Direct binding assays
•
Response
Concentration
Increasingaffinity
Figure 7-3. Using a ligand with a higher affinity moves the calibration curve towards lower analyte concentrations.
•
-
8/19/2019 Conc Handbook
68/106
Developing and running concentration assays ____________________________________________
Response
Concentration
Increasingamount of ligand
Figure 7-4. Higher amounts of immobilized ligand increase the response but
have little effect on the shape of the calibration curve.
•
Response
Concentration
Time
1
2
1
2Response
Figure 7-5. Longer contact times allow measurement at lower analyte concentrations. The inset shows the sensorgrams from which the calibration curves are derived.
-
8/19/2019 Conc Handbook
69/106
___________________________________________ Developing and running concentration assays
7.3.2 Inhibition assays
•
Response
log[concentration]
Increasing affinity
Increasing concentrationof detecting molecule
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.
•
-
8/19/2019 Conc Handbook
70/106
Developing and running concentration assays ____________________________________________
Response
log[concentration]
Decreasing concentrationof detecting molecule
•
-
8/19/2019 Conc Handbook
71/106
___________________________________________ Developing and running concentration assays
7.4.1 Unwanted binding in direct binding assays
7.4.2 Unwanted binding in inhibition and surface competition assays
7.4 Unwanted binding of analyte or detecting molecule
-
8/19/2019 Conc Handbook
72/106
Developing and running concentration assays ____________________________________________
Response
log[concentration]
Figure 7-7. Unwanted binding of either analyte or detecting molecule to the
dextran matrix in an inhibition assay will introduce a background response level, reducing the response range of the assay.
7.4.3 Testing for unwanted binding
7.4.4 Dealing with unwanted binding
•
-
8/19/2019 Conc Handbook
73/106
___________________________________________ Developing and running concentration assays
•
•
7.5.1 Measuring specificity
7.5 Specificity and cross-reactivity
-
8/19/2019 Conc Handbook
74/106
Developing and running concentration assays ____________________________________________
100
50
0
1 100
Response/MW
Concentration
Compound A
Compound B
Figure 7-8. Cross-reactivity in direct assays is determined from calibration
curves of molecular weight-corrected response against concentration. In this illustration, compound B shows 1% cross-reactivity with compound A.
7.5.2 Optimizing specificity
•
-
8/19/2019 Conc Handbook
75/106
___________________________________________ Developing and running concentration assays
•
•
•
7.6.1 Non-specific binding to the sensor surface
7.6 Matrix interference
-
8/19/2019 Conc Handbook
76/106
Developing and running concentration assays ____________________________________________
•
•
•
7.6.2 Other matrix interference effects
•
•
-
8/19/2019 Conc Handbook
77/106
___________________________________________ Developing and running concentration assays
•
•
-
8/19/2019 Conc Handbook
78/106
Developing and running concentration assays ____________________________________________
•
•
•
•
7.7.1 Testing assay stability
7.7.2 Dealing with assay stability issues
•
7.7
Assay stability
-
8/19/2019 Conc Handbook
79/106
___________________________________________ Developing and running concentration assays
•
•
•
•
7.8 Running routine assays
-
8/19/2019 Conc Handbook
80/106
Developing and running concentration assays ____________________________________________
•
•
•
-
8/19/2019 Conc Handbook
81/106
___________________________________________Measuring concentration in GxP environments
8.
Measuring concentration in
GxP environments
8.1.1 System performance
8.1
Setting up GxP assays
-
8/19/2019 Conc Handbook
82/106
-
8/19/2019 Conc Handbook
83/106
___________________________________________Measuring concentration in GxP environments
8.2.1 Specificity
•
•
•
8.2.2 Accuracy
.2 Validating GxP assays
-
8/19/2019 Conc Handbook
84/106
-
8/19/2019 Conc Handbook
85/106
___________________________________________Measuring concentration in GxP environments
8.2.5 Range
8.2.6
Robustness
8.3.1 Document the run settings
8.3.2 Data storage
.3 Running GxP assays
-
8/19/2019 Conc Handbook
86/106
Measuring concentration in GxP environments ___________________________________________
8.3.3 Audit trails
-
8/19/2019 Conc Handbook
87/106
__________________________________________________________The SPR detection principle
A.
The SPR detection principle
Incidentlight
Sensor chip
Flow cell
Reflectedlight
SPR angle
Reflected lightintensity
Angle of reflectionSPR angle
Figure A-1. The SPR detection principle.
A.1 Surface plasmon resonance
-
8/19/2019 Conc Handbook
88/106
The SPR detection principle __________________________________________________________
A.2 What SPR measures
A.3 Biacore configuration
-
8/19/2019 Conc Handbook
89/106
__________________________________________________________The SPR detection principle
-
8/19/2019 Conc Handbook
90/106
The SPR detection principle __________________________________________________________
-
8/19/2019 Conc Handbook
91/106
____________________________________________Using binding rates to measure concentration
B.
Using binding rates to measure
concentration
B.1.1
Biochemical interaction rates
[ ][ ][ ] [ ]
−=
B.1 Factors determining binding rates
-
8/19/2019 Conc Handbook
92/106
Using binding rates to measure concentration ____________________________________________
( )
−−=
B.1.2 Mass transport processes
B.1.3 What limits the observed binding?
-
8/19/2019 Conc Handbook
93/106
____________________________________________Using binding rates to measure concentration
Response
Time
Mass transport Interaction
Sensorgram is linear ifmass transport is limiting
Figure B-1. In a partially mass transport-limited situation, mass transport dominates at the beginning of the injection and interaction rate dominates late in the injection.
B.2.1 Advantages of affinity-independent assays
•
•
•
B.2
Affinity-independent assays
-
8/19/2019 Conc Handbook
94/106
Using binding rates to measure concentration ____________________________________________
•
B.2.2 Affinity-independent assays in practice
•
•
•
•
•
-
8/19/2019 Conc Handbook
95/106
____________________________________________Using binding rates to measure concentration
•
Figure B-2. Mass transport-limited binding proceeds at a constant rate (left panel), while interaction-limited binding displays curvature in the sensorgrams (simulated sensorgram data).
•
•
-
8/19/2019 Conc Handbook
96/106
Using binding rates to measure concentration ____________________________________________
B.3 Calibration-independent assays
-
8/19/2019 Conc Handbook
97/106
____________________________________________________________________________Index
Index
-
8/19/2019 Conc Handbook
98/106
Index____________________________________________________________________________
-
8/19/2019 Conc Handbook
99/106
____________________________________________________________________________Index
-
8/19/2019 Conc Handbook
100/106
Index____________________________________________________________________________
-
8/19/2019 Conc Handbook
101/106
____________________________________________________________________________Index
-
8/19/2019 Conc Handbook
102/106
Index____________________________________________________________________________
-
8/19/2019 Conc Handbook
103/106
____________________________________________________________________________Index
-
8/19/2019 Conc Handbook
104/106
-
8/19/2019 Conc Handbook
105/106
-
8/19/2019 Conc Handbook
106/106