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11 Affinity in solution
11.3 Evaluation of affinity in solution

206

Biacore T100 Software Handbook BR-1006-48 Edition AE

Click

Next>

to calculate the results.

The table shows the numerical results for the sample series. Samples that lie
outside the range of the calibration curve are marked as

N/A

(not applicable) in

the column for

Calc.Conc.Beq

.

The plot panel shows the sample measurements with a line fitted according to
the equation for 1:1 affinity (see Section 11.1.2). Right-click on a point to exclude
it from the fitting.

Notes:

The plot of free B against total A is presented by default with a logarithmic
scale on the x-axis. In this form, the equilibrium constant KD is given by
the concentration of A at the inflection point in the fitted curve.

Zero values cannot be plotted on a logarithmic scale. If you have included
a sample with zero concentration of A in the sample series and want to
display this point on the plot, choose

Scale

from the right-click menu in

the plot panel and set a linear scale for the x-axis.

The intercept of the fitted curve on the y-axis represents a fitted value for
the parameter

ConcB

. This value should be the same as or close to the

value entered for the variable

ConcB

in the method.

The calculated K

D

value is shown in the panel below the table.

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Summary of Contents for Biacore T100

Page 1: ...GE Healthcare Biacore T100 Software Handbook ...

Page 2: ......

Page 3: ... 21 2 3 6 Tools menu 21 2 3 7 Right click menus 22 2 4 File storage 23 2 4 1 Wizard templates and methods 23 2 4 2 Result files 23 3 Manual run 3 1 Preparing for a manual run 25 3 1 1 Instrument preparations 25 3 2 Starting a manual run 26 3 3 Controlling a manual run 27 3 4 Ending a manual run 29 4 Application wizards 4 1 Wizard templates 31 4 1 1 Creating and editing wizard templates 31 4 1 2 Ru...

Page 4: ...periments 74 5 Methods 5 1 Opening methods 77 5 2 Method structure 78 5 3 Method overview 79 5 4 General settings 80 5 5 Assay steps 81 5 5 1 Base settings 82 5 5 2 Number of replicates 84 5 5 3 Recurrence 84 5 5 4 Assay step preparations 85 5 6 Cycle types 85 5 6 1 Commands 86 5 6 2 Variables 92 5 6 3 Report points 95 5 7 Variable settings 96 5 8 Verification 97 5 9 Setup Run 97 5 9 1 Detection 9...

Page 5: ...7 2 When solvent correction should be used 116 6 7 3 How solvent correction works 116 6 7 4 Applying solvent correction 117 6 8 Evaluation methods 119 6 8 1 Creating evaluation methods 119 6 8 2 Applying evaluation methods 120 7 Data presentation tools 7 1 Sensorgram items 121 7 1 1 Selecting sensorgrams for display 122 7 1 2 Removing data 122 7 1 3 Sensorgram adjustment 123 7 1 4 Markers 124 7 2 ...

Page 6: ...cedure 155 9 2 2 Multiple ligand densities 162 9 3 Batch mode evaluation 163 9 4 Quality assessment for kinetics evaluation 164 9 4 1 The Quality Control tab 164 9 4 2 Statistical parameters 168 9 4 3 Components of the fit 170 9 4 4 Check kinetic data 170 9 5 Quality assessment for affinity evaluation 172 9 6 Summarizing kinetics and affinity results 173 9 6 1 Creating kinetic summaries 173 9 6 2 ...

Page 7: ... solution 205 Appendix A Data import and export A 1 Exporting data 209 A 1 1 Export functions 209 A 2 Importing data 210 A 2 1 Control Software 210 A 2 2 Evaluation Software 213 Appendix B Method examples and recommendations B 1 Affinity in solution 215 B 2 Calibration free concentration analysis 216 B 2 1 Assay steps and general settings 216 B 2 2 Cycle types 216 B 2 3 Variable settings 217 B 2 4...

Page 8: ...8 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 9: ...up to four flow cells connected in series The flow cells are arranged in pairs Fc1 2 and Fc3 4 with minimum dead volume between the flow cells in a pair to provide accurate reference subtraction The sample compartment accommodates one microplate 96 or 384 well regular or deep well capacity and one reagent rack for reagent vials A combined sample and reagent rack can be used in place of the separat...

Page 10: ...s and documentation describing the technology are available from GE Healthcare Information may also be found on the Internet at www biacore com 1 4 Biacore terminology Biacore monitors the interaction between two molecules of which one is attached to the sensor surface and the other is free in solution The following terms are used in the context of work with Biacore systems see Figure 1 1 The part...

Page 11: ...ample passes over two or more flow cells in series where one flow cell usually the first serves as a reference while ligand is attached in the other flow cell s Surfaces with ligand are referred to as active blank surfaces used for reference purposes are reference A particular sensorgram is referred to as a curve in several contexts in the software This terminology is used to distinguish between d...

Page 12: ... Biacore terminology 12 Biacore T100 Software Handbook BR 1006 48 Edition AE Figure 1 2 Schematic illustration of a sensorgram The bars below the sensorgram curve indicate the solutions that pass over the sensor surface ...

Page 13: ...Biacore T100 Software Handbook BR 1006 48 Edition AE 13 Control Software ...

Page 14: ...14 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 15: ...ination or measurement of kinetic constants Each wizard consists of an ordered series of dialog boxes ensuring that the essential features of the application setup are correctly defined Methods provide greater flexibility and conversely less guidance in setting up applications allowing customized applications that are not covered by wizards Methods are defined in a graphical interface called Metho...

Page 16: ...point table lists report points for the currently displayed cycle Report points record the response at a set time and are defined automatically custom report points can also be added in methods or after the run in either the Control Software or the Evaluation Software The keyword table lists keywords for the currently displayed cycle Keywords are defined automatically in wizard runs or in the meth...

Page 17: ...urve in the cycle is current in the display Options in the View menu Section 2 3 4 control which curves are displayed in the sensorgram window 2 3 2 File menu The Open New options for wizard templates and methods create new wizard templates and methods and open existing templates and methods for editing or for starting a run Open opens result files Most result files just display the sensorgrams an...

Page 18: ... still switched on you may choose to shut down the instrument for a shorter or longer period if required See the Biacore T100 Instrument Handbook or the on line help for more details None No sensorgrams will be printed Current cycle The current cycle will be printed with the View Show setting and scale as shown on the screen Range and All cycles Multiple cycles will be printed For Range enter a ra...

Page 19: ... cycles Report points created in the Control Software cannot be edited in the Evaluation Software The Evaluation Software offers functions for creating and editing custom report points that can be applied to all cycles in the run in a single operation This is usually preferable to adding report points in the Control Software 2 3 4 View menu Chip Properties opens a dialog box that displays the prop...

Page 20: ...Adjust Scale sets the scale to the full data range This will not affect the Auto scale setting in the Scale dialog Adjust Scale overrides but does not turn off the Lock scale setting To scale the sensorgram display interactively drag with the cursor over the area to be scaled Double clicking in the display or choosing View Unzoom restores the previous zoom setting Reference line toggles display of...

Page 21: ...tools the Wizard Results option opens a window showing the results of the run All other runs are evaluated in the Evaluation Software Sensorgram Markers controls display of report point and event markers and labels in the sensorgram window 2 3 5 Run menu The options in the Run menu are used to start the different types of runs see Chapter 3 and Sections 4 1 2 and 5 9 7 2 3 6 Tools menu Options in ...

Page 22: ...acore T100 Instrument Handbook Preferences controls aspects of file storage and data import see Section 2 4 More Tools provides access to maintenance test and service tools Details are given in Appendix B of the Biacore T100 Instrument Handbook 2 3 7 Right click menus Right clicking with the mouse in some windows opens context menus specific for the window Sensorgram window Scale opens the same di...

Page 23: ...le name extension Method Note The extension will not be displayed if the default setting Hide file extensions for known file types is selected in the Windows Explorer folder options Turning this setting off can help you to identify file types in dialog boxes Templates and methods may be saved in any location A folder structure under the default location as specified in Tools Preferences is however...

Page 24: ...2 Control Software general features 2 4 File storage 24 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 25: ...re be prepared using options from the Tools menu 1 Dock the chip that you want to use and immobilize ligand on the surface see Section 4 3 2 if this has not already been done 2 Choose Tools Prime to flush the flow system with fresh buffer 3 Choose Normalize from the Maintenance Tools section of Tools More Tools if the detector has not been normalized since the chip was docked In many cases the det...

Page 26: ... the flow rate at any time during the run You can change the flow path at any time during a cycle the available options are restricted by the choice made when the cycle is started Choose the rack and microplate settings These will apply throughout the run and cannot be changed Click Eject Rack to eject the rack tray so that you can load your samples Click Start to start the run You will be asked t...

Page 27: ... a queue The queue is listed in the left hand panel with commands that have been executed in gray text and those that are pending in black text The command currently being executed is marked with a working icon Right click on a pending command for a menu with options for editing the command inserting a new command before the selected command you choose the command to insert from a dialog box delet...

Page 28: ...t up dialog Make sure that the chosen position contains enough solution for the injection The required volume for the specified contact time is indicated in the dialog box Check High viscosity solution if your regeneration solution has a relative viscosity higher than about 3 corresponding to about 35 glycerol or 40 ethylene glycol at 20 C This will adjust the injection procedure to ensure correct...

Page 29: ... paused Resume Run Resumes a run that is paused Add report point Adds a report point to the sensorgram Help Displays help for the manual run 3 4 Ending a manual run To end a manual run 1 Issue a Stop Run command The command will normally be placed at the end of the queue If you want to stop the run before the queue is completed use the right click menu in the queue panel to delete commands from th...

Page 30: ...3 Manual run 3 4 Ending a manual run 30 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 31: ...mplete assay definition including sample details if desired If a wizard sequence is closed before reaching the last step however you are given an opportunity to save the template which will then contain settings as far as they have been defined 4 1 1 Creating and editing wizard templates To create a new wizard template or edit an existing template choose File Open New Wizard Template and select th...

Page 32: ...sed for the run are stored in the result file and can be saved as a new wizard template from the completed run 4 2 Common wizard components Several dialogs are common to a number of wizards with equivalent functions and only minor differences These dialogs are described in the present section Any wizard specific variations in these common components are described in the section on the appropriate ...

Page 33: ...p for example selecting Sensor Chip NTA will automatically check the Capture option and include an injection of nickel before the ligand capture step and will suggest 0 35 M EDTA as a conditioning solution Choose chip type Custom if you are using a chip type that is not listed Injections Check the injections that you want to include The illustration panel shows the sequence of included injections ...

Page 34: ...In some assays conditioning may be required to prepare the ligand in which case the solution is specific to the particular ligand Conditioning cycles are usually not necessary for sensor chips where the ligand or capturing molecule has been attached in an immobilization step For these chips Sensor Chip C1 CM series and SA the option to run conditioning cycles is unchecked by default You may want t...

Page 35: ...few cycles and start up cycles allow the response to stabilize before the first analysis cycle is performed Three start up cycles are generally recommended for most assay purposes to ensure a stable response in the analysis Start up cycles are treated separately from analysis cycles in the evaluation software Start up cycles are run at the beginning of the experiment after the conditioning cycle a...

Page 36: ...ilization time may be used after the last injection instead of regeneration for systems where analyte dissociates completely from the surface Exposure of the surface to regeneration solution can often lead to transient changes in the baseline Inclusion of a stabilization time after regeneration helps to ensure a stable baseline for the next cycle Sample injection Normally the injected sample solut...

Page 37: ...asis of the number of samples that will be run Control samples are required for some wizards e g Concentration Analysis and optional for others e g Binding Analysis Where control samples are optional the Control Sample table is accessed through a button in the Samples step Sample details can be imported from an external file if this option is enabled under Tools Preferences See Appendix A for deta...

Page 38: ...re is the temperature at the flow cell If the specified value differs from the current temperature the system will wait at the beginning of the run until the analysis temperature is stable at the new value You can choose to ignore temperature instability but the response will drift as the temperature stabilizes The absolute response decreases by about 150 RU for a 1 C increase in temperature The S...

Page 39: ...dentifier for each cycle Other keywords may also be generated according to the wizard purpose Keyword information is used in the Evaluation Software 4 2 6 Rack positions This dialog box shows where samples and reagents are placed in the microplate and or rack Positions are color coded by region according to sample and reagent categories you can change the color coding in the Automatic positioning ...

Page 40: ...t will be used Note The volumes listed in the table are minimum volumes Use somewhat larger volumes if material is available to allow for slight variations in the dead volume in microplates and vials You can change sample and reagent positions manually in two ways Click on a sample or reagent in the sample plate and rack illustration and drag it to a new empty position You cannot drag to a positio...

Page 41: ...en saved in the wizard template Automatic Positioning This option controls the way samples and reagents are positioned automatically Samples and reagents are placed by region and regions are kept contiguous as far as possible Region This column lists the sample and reagent regions Color This option controls the display color for the region Orientation This column determines whether samples are arr...

Page 42: ...d to the Rack Positions table replacing the existing positioning information See Appendix A for more details Simple Position Import Imports positioning details from an external file Details of the import settings and file format are described in Appendix A Export Positions Exports the data in the positioning table to a tab separated text file See Appendix A for details of the exported file format ...

Page 43: ...ilder based runs Section 5 4 buffer names are shown in the Prepare Run Protocol dialog Notes The estimated run time is a minimum time that does not include any time that cannot be predicted when the wizard is set up This includes time for temperature equilibration at the beginning of the run or between cycles for ligand injection in immobilization runs using Aim for immobilized level Section 4 3 2...

Page 44: ...outing is fixed Step 1 Setup Choose the flow path for the pH scouting Immobilization pH scouting is restricted to a single flow cell within a run The sensor surface in the flow cell should be unmodified Enter the buffers and pH values to be used for scouting The default list covers sodium acetate buffers in the pH range 4 to 5 5 available as ready to use solutions from GE Healthcare Buffers will b...

Page 45: ...usually fairly insensitive to temperature Step 4 Rack positions See Section 4 2 6 Immobilization pH scouting requires one position for ligand solution at each pH tested and one for the surface wash solution Accept or change the rack positions for the various solutions required see Section 4 2 6 Step 5 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 7 This text will b...

Page 46: ... necessarily the value that gives the highest ligand binding Beware of conditions that give irregular sensorgrams with incomplete dissociation this behavior often indicates aggregation or denaturation of the ligand 4 3 2 Immobilization The Immobilization wizard supports immobilization of ligand in any combination of the four flow cells in one run Immobilization in each flow cell is performed indep...

Page 47: ...to create and save an immobilization wizard template but you must dock a corresponding chip type before the immobilization can be performed Check the flow cells where you want to perform immobilization For each flow cell set the parameters as follows Choose the immobilization method Predefined methods are provided for standard immobilization chemistries Customized methods can be defined by clickin...

Page 48: ...vel injects a pulse of ligand over the unactivated surface in order to estimate the rate of preconcentration The surface is washed to remove traces of ligand and then activated The procedure then uses ligand contact times based on the preconcentration estimate to attempt to reach the specified target level If preconcentration is either too fast or too slow to permit the target level to be reached ...

Page 49: ...e method You cannot delete the predefined methods marked with a icon For a new method enter a name in the Method name field Construct the sequence of injections for the immobilization method using the buttons to the right of the main panel The ligand injection is created automatically and cannot be deleted solution and contact time for the ligand injection are specified in the main wizard dialog A...

Page 50: ...y contain one Pre conc injection The Pre conc injection should always be placed before surface activation it will usually be first in the method although it may be preceded by a surface conditioning injection if required If you place the Pre conc injection after the surface activation it will be executed there and the ligand will be immobilized on the activated surface After the Pre conc injection...

Page 51: ...ftware The summary lists the procedure and method the name of the ligand and whether the target was reached with Aim for immobilized level Two response values are reported one directly after the ligand immobilization and one after the deactivation injection The difference between these values is an indication of the amount of non covalently bound ligand that is washed from the surface by the deact...

Page 52: ...icates the extent of regeneration Each condition should be tested for at least 3 cycles in sequence recommended number 5 in order to detect trends in the regeneration behavior with the given condition When testing multiple conditions start with the mildest conditions to minimize the risk of losing ligand activity at the beginning of the scouting series Step 1 Injection sequence Choose the detectio...

Page 53: ... kind of regeneration conditions e g different pH values or different concentrations of ethylene glycol within the same run Results are most easily interpreted if you use a separate wizard run with a new flow cell or sensor chip for each kind of regeneration condition that you test so that the outcome with one kind of condition is not affected by the history of exposing the ligand to another You m...

Page 54: ...n scouting results Regeneration scouting results are presented in the Control Software when the run is completed Note This result presentation is not shown if the run is opened in the Evaluation Software The Trend chart tab shows the results as a plot of baseline and sample response for each cycle in the run grouped by regeneration conditions Conditions are identified in tool tips for the data poi...

Page 55: ...m lock to keep the scale fixed when the choice of sensorgrams is changed Result files from regeneration scouting can also be opened in the Evaluation Software if you want to prepare other sensorgram displays or plots see Chapter 7 See the Biacore Sensor Surface Handbook for a discussion of how to interpret the results of regeneration scouting 4 4 2 Buffer scouting The Buffer Scouting wizard helps ...

Page 56: ... you want to test You can enter up to 4 different buffers The buffers will be tested in the order given Note If you use less than 4 buffers positions A B and C will be used in order If you enter buffer names on non consecutive rows the table will be adjusted when you leave the dialog box Step 3 Injection parameters Specify the injection parameters for each injection in the cycle see Section 4 2 3 ...

Page 57: ...change the rack positions for the various solutions required see Section 4 2 6 Step 7 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 7 This text will be displayed at the start of a run when the wizard template is used Save the wizard template if required and start the run Buffer scouting results When the wizard run is completed the results are opened automatically i...

Page 58: ...o confirm that the regeneration conditions that you identified in regeneration scouting hold good for an extended number of cycles Step 1 Injection sequence Choose the detection settings chip type and injection sequence for the surface performance test cycle see Section 4 2 1 Step 2 Setup Specify conditioning and start up cycles see Section 4 2 2 Set the number of repetitions of the analysis cycle...

Page 59: ...roughout the run 4 5 Assay wizards 4 5 1 Binding analysis The Binding Analysis wizard supports injection of up to four samples in series in addition to ligand capture enhancement and regeneration steps This wizard is suitable for analysis of applications like multi component complex formation and pair wise epitope mapping as well as simple applications like screening for binding partners to an imm...

Page 60: ...ashing procedures Step 4 Samples The sample table contains one column for each sample injection in the injection sequence New rows are created as you enter data in the table The illustration above shows how the wizard could be used to set up a pair wise epitope mapping experiment Click Import to import the sample data from an external file Import of sample information must be enabled in Tools Pref...

Page 61: ...ons step to verify where control samples will be run during the assay Step 5 System preparations Check the System preparations options as required see Section 4 2 5 Step 6 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 6 Step 7 Prepare run protocol Edit the Prepare Run Protocol text if desired Section 4 2 7 This text will be displayed at the s...

Page 62: ...nalysis see Section 4 2 1 Step 2 Setup Specify the conditioning and start up cycles see Section 4 2 2 Start up cycles are recommended for concentration assays to ensure that the initial response drift that may occur with a new chip does not interfere with the first measurements Step 3 Injection parameters Specify the injection parameters for each injection in the cycle see Section 4 2 3 The sample...

Page 63: ...ion curve at intervals during the assay The calibration curve will be run at the beginning of the assay and at the specified interval Thus specifying a repeat every 15 sample cycles and running 35 samples will result in calibration curves at the beginning and after samples 15 and 30 If the Repeat calibration box is not checked calibration will be run once at the beginning of the assay Enter the co...

Page 64: ...trol samples the controls will be run once only at the start of the assay If you check Repeat control samples every the controls will be run after the first calibration curve and then at the specified interval for sample cycles not counting repeated calibration curves and once at the end of the assay Use the Cycle Run List option in the System Preparations step to verify where control samples will...

Page 65: ...mport the sample data from an external file Import of sample information must be enabled in Tools Preferences to use this function See Appendix A for details of import functions and file formats Step 7 System preparations Check the System preparations options as required see Section 4 2 5 Step 8 Rack positions Accept or change the rack positions for the various solutions required see Section 4 2 6...

Page 66: ...kinetics supported in Method Builder see Section B 9 Step 1 Injection sequence Choose the detection settings chip type and injection sequence for the assay see Section 4 2 1 Only reference subtracted detection using either Fc2 1 or Fc4 3 is available for kinetic analysis The Kinetics wizard supports capture but not enhancement injections A Carry Over injection is also available in the Kinetics Aff...

Page 67: ...for analyses using small organic analytes that require dimethyl sulfoxide DMSO in the sample buffer to maintain solubility The principles and application of solvent correction are described in Section 6 7 Set the required number of injections per cycle and the frequency of solvent correction cycles The default settings use 8 injections per cycle and repeat the solvent correction cycle every 15 sam...

Page 68: ...d When a molecular weight is entered the right hand Concentration column displays the conversion from molar to weight based or vice versa Samples with the same sample name may not be given different molecular weights The samples may be analyzed either in the order entered in the table or sorted in increasing concentration The order displayed in the sample table is not affected by the choice of run...

Page 69: ...y the controls will be run at the start of the assay and then at the specified interval and again at the end of the assay Use the Cycle Run List option in the System Preparations step to verify where control samples will be run during the assay Step 5 System preparations Check the System preparations options as required see Section 4 2 5 Step 6 Rack positions Accept or change the rack positions fo...

Page 70: ...njection sequence Choose the detection settings chip type and injection sequence for the assay see Section 4 2 1 Options for detection settings are the same as for kinetics affinity determinations Section 4 5 3 The thermodynamics wizard supports capture but not enhancement injections The same injection sequence will be used at all temperatures Step 2 Setup Specify the conditioning and start up cyc...

Page 71: ...nterval that the ligand and analyte tolerate Start from the lowest temperature to minimize the time needed for temperature equilibration between measurements increasing the analysis temperature takes less time than decreasing it The system will wait for a stable temperature between each determination As an additional control you may want to include a replicate of the first temperature at the end o...

Page 72: ...yses The Mass transfer control experiment analyses the interaction of one or more analyte concentrations at three different flow rates to establish whether the observed binding rate varies with flow rate A dependence of binding rate on flow rate indicates that the binding is limited to some extent by mass transfer of analyte to the sensor surface If mass transfer limitations are too significant re...

Page 73: ...rol experiment has largely been superseded by functions in the evaluation software The control experiment may however still be useful to confirm suspected mass transfer limitations if desired 4 6 2 Linked reactions control Each sample entered in the Samples step will be analyzed at a flow rate of 10 µl min with fixed contact times of 0 5 3 and 10 minutes and a dissociation time of 10 minutes Use o...

Page 74: ... to zero response and time at the baseline report point Compare the observed binding rates at the different flow rates If the observed binding rate during sample injection varies with flow rates there is some degree of mass transfer limitation in the data Note that it may still be possible to obtain kinetic information from the sensorgrams see Chapter 9 This example shows a clear dependence of bin...

Page 75: ...ero time at the end of the sample injection Compare the observed dissociation rates at the different contact times Variation of the observed dissociation rate after sample injection with contact time indicates linked reactions in the interaction model This example shows a clear dependence of dissociation rate on contact time indicating linked reactions in the interaction model ...

Page 76: ...4 Application wizards 4 6 Control experiment wizards 76 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 77: ...k New to create a new method Predefined methods for common applications are provided in the folder Biacore Methods If you make changes to a predefined method you must save your changed method under a new name Check Show importable wizard templates to display wizard templates that can be opened in Method Builder Opening a wizard template imports all wizard settings into a method and allows you to a...

Page 78: ...nalysis Analysis temperature and buffer can be set individually for each assay step Cycle types Cycle types define the details of how assay steps will be performed in terms of sample and reagent injections Each assay step is linked to one cycle type but the same cycle type can be used in multiple assay steps For example sample and control sample analysis are two assay steps that will typically use...

Page 79: ...table for the intended purpose Each aspect of Method Builder is described in detail in the following sections 5 3 Method overview This screen provides a summary of the method The main panel shows the assay steps in the method see Section 5 5 Click on an assay step to show the settings for the step and the details of the cycle definition see Section 5 6 in the panels to the right The cycle definiti...

Page 80: ...on 5 6 1 Use the setting Multi if you are not sure what you need this will provide maximum flexibility for the injection settings Sample compartment temperature This is the temperature in the sample compartment not the analysis temperature at the flow cell which is set for each assay step Check the Vary with analysis temperature box to set the sample compartment temperature automatically to the sa...

Page 81: ...temperature to set the analysis temperature when the run is completed The rack temperature will also be reset if the Vary with analysis temperature box is checked This setting provides automated control of the chip and detector environment after the completion of a run for example in preparation for another run at a different temperature 5 5 Assay steps This screen determines the main structure of...

Page 82: ...hand panel The cycle list for the run as currently defined is shown in the right hand panel 5 5 1 Base settings Name This is the name of the assay step Each step in a method must have a unique name New steps are by default named Assay Step n where n is a serial number change the name to something that describes the context or intent of the step to make the method easier to follow Purpose Assay ste...

Page 83: ...step so that the control sample and sample analyses are performed in the same way Set Control sample to recurring within Sample to repeat the control sample analysis at intervals through the assay Sample Used for sample analysis in all applications At least one sample step is required for application specific evaluation Solvent correction Used for solvent correction cycles This step should be conn...

Page 84: ...ed interval within the context of another step The recurrence can be specified as Every n cycles so that the number of occurrences will depend on the number of cycles in the assay step or Distribute n occurrences evenly in which case the number of occurrences is fixed and they are distributed as evenly as possible among the cycles in the assay step In addition the recurring step can be specified t...

Page 85: ...erature at the start of an assay step does not match the setting for the step the system will wait until the setting is reached Buffer Select the running buffer to be used for the assay step The default buffer is A corresponding to buffer bottle and tubing A on the instrument 5 6 Cycle types Cycle types define the detailed sequence of operations to be performed in each assay step Top level step Nu...

Page 86: ...pe are configured in the lower part of the work area The settings are divided into Commands and Report Points accessed on the respective tabs 5 6 1 Commands The commands in a cycle definition correspond to different kinds of injection of liquid over the sensor surface To add a new command to the cycle definition choose the command type from the pull down list and click Insert The command will be i...

Page 87: ...4 If then command This command allows construction of conditional methods where commands are executed or skipped depending on the outcome of certain conditions The illustration below shows a cycle which will perform an additional regeneration if the relative response after the first regeneration exceeds a specified value Predip Check this box to dip the needle in a separate position before aspirat...

Page 88: ...ce exact equality is an unpredictable condition in view of noise in the SPR response To construct an equality condition combine one Greater than and one Less than condition so that a window of tolerance is created For example the combined condition A greater than B 1 AND A less than B 1 is equivalent to A equals B with a tolerance of 1 3 Choose the actions to be taken when the condition is met and...

Page 89: ...ties The injected solution contact time and flow rate can be set as variables Evaluation variables can also be defined for General commands see Section 5 6 2 Inject and recover command This command recovers analyte that is bound to the sensor surface and is intended for use in applications where the bound material is analyzed further Several features of the command are designed specifically for in...

Page 90: ... flow cells to maximize the amount of analyte that binds to the surface 3 The flow system is washed with the specified Wash solution Distilled water or an MS compatible buffer should be used as washing solution 4 A small volume approximately 2 µl of Recovery solution separated from the surrounding buffer by air segments is injected into the flow cells The flow is stopped for the specified Incubati...

Page 91: ...me and flow rate can be set as variables Sample command This command is intended for injection of sample containing analyte Only Sample commands are recognized as analyte injections in the Evaluation Software for kinetics affinity and concentration evaluation The injected solution contact time dissociation time and flow rate can be set as variables Evaluation variables can also be defined for the ...

Page 92: ... 5 6 2 Variables Parameters for many of the commands in a cycle definition may be set as variables Values for variables are entered in either the Variable Settings in the method or the Setup Run step when the method is run Sections 5 7 and 5 9 2 and determine the number of cycles that will be performed in the run Variables fall into two broad classes Method variables such as sample name or contact...

Page 93: ...les and check the variables you want to use Click Add to set up user defined variables Note For specific assay purposes you should generally check all suggested variables If you leave some variables unchecked the assay specific evaluation may not work Predefined evaluation variables for different assay purposes are described in the table below see also Section 5 10 Evaluation purpose General Kinet...

Page 94: ...ples left blank for unknowns Dilution Dilution factor used for unknown samples to calculate original concentrations Evaluation purpose Calibration free concentration analysis MW Analyte molecular weight used in calculation of concentration from binding rates empty for blank injections D 20 C Diffusion coefficient of the analyte at 20 C empty for blank injections Blank Identifies blank cycles for b...

Page 95: ...f the last injection will not be created Note Do not position report points far away from events so that they lose their relevance to the event or so close to an event so that the report point window overlaps the event itself Enter the report point details as follows Name The report point name must be unique within the cycle type Choose a name that reflects the function of the report point Sec Ent...

Page 96: ...red independently for the different assay steps even if the assay steps use the same cycle type If you choose to specify values for all variables in the method the values are entered in this screen For each assay step one row of variable values represents one analysis cycle the cycle may be repeated if the Repeat property is set in the Assay Step screen Each row in the variables table corresponds ...

Page 97: ...es will be used for all cycles the number of cycles is determined by the number of rows of variable values in Setup Run Depending on how the method is defined there may be variable tables for several assay steps Variable handling must be defined for all steps before the method will pass verification 5 8 Verification This step checks that the method is correctly and completely defined A method that...

Page 98: ...ed according to commands in the cycle type definition Use the right mouse button in the variables table to access functions for copying and pasting cell contents and for inserting and removing rows The columns in the table are listed in the order they are defined in the method see Section 5 6 2 Click Import to import the variables table from an external file See Appendix A for details of the impor...

Page 99: ...e summary of the cycles that will be performed in the run This view is for information only and cannot be edited Check through the cycle list to confirm that the variable tables are correctly filled in Click Overview to display the method overview Section 5 3 5 9 4 System preparations Choose which preparation steps should be executed before the method starts ...

Page 100: ...a file name for the results 5 10 Requirements for assay specific evaluation This section describes the requirements and recommendations if assay specific evaluation is to be applied to method based runs 5 10 1 Concentration analysis See Chapter 8 for a description of concentration evaluation Using calibration At least one assay step is required with purpose Calibration and one with purpose Sample ...

Page 101: ...t least one of the non zero concentrations should be measured in duplicate Although kinetic and affinity evaluation can be applied to runs with fewer sensorgrams the results will generally be less reliable if these recommendations are not followed Five sample concentrations are recommended for single cycle kinetics and affinity injected in order of increasing concentration Duplicate cycles are rec...

Page 102: ...tting These samples should contain only component B Samples in the Sample step must have concentrations specified in the variables ConcB fix and ConcA variable At least 3 samples with the same concentration of component B mixed with different concentrations of component A are required 5 10 5 Other requirements Application of solvent correction see Section 6 7 requires an assay step with purpose So...

Page 103: ...Biacore T100 Software Handbook BR 1006 48 Edition AE 103 Evaluation Software ...

Page 104: ...104 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 105: ...from analyses with low molecular weight analytes that give low response levels and require organic solvents e g dimethyl sulfoxide DMSO to maintain solubility This chapter describes the organization of the evaluation software The various evaluation functions are described in detail in the following chapters 6 1 User interface 6 1 1 Organization The Biacore T100 Evaluation Software screen is divide...

Page 106: ...it selected items using the Remove and Edit buttons at the top of the explorer panel You can hide the Evaluation Explorer panel to increase the useful size of the work area by clicking on the pin icon in the explorer title bar If the panel is hidden it will automatically reappear when you move the mouse over the left hand edge of the evaluation window 6 2 Opening files To open a file in the evalua...

Page 107: ...caled automatically if the scale has been set to specified values 6 3 2 Right click menus Right click in display windows for options relating to the display The available options vary according to the type of window and also depending on whether you right click on a point a curve or elsewhere in the window Labels Displays a label on each point in a plot window showing cycle number flow cell and sa...

Page 108: ...lso access this function by double clicking on either the x or y axis in the display Uncheck Auto and enter minimum and maximum values to specify a scale Copy graph Copies the current display as it appears on the screen to the Windows clipboard as a graphic object from where it can be pasted into third party software such as word processing or presentation programs Copy table Copies the table data...

Page 109: ...ed by assay step 6 4 2 Plots Plots are created for most wizards if the appropriate report point is present in the results Common predefined plots are listed below Separate plots will be created if there are multiple injections with similar report points for example baseline for capture and sample injections The Plot Settings cannot be changed but the plot can be modified using the selectors and th...

Page 110: ...number for the carry over injection only for reference subtracted curves Controls binding Relative response for the report point binding against cycle number for control samples only for reference subtracted curves Controls stability Relative response for the report point stability against cycle number for control samples Binding levels Relative response for the report point binding against cycle ...

Page 111: ...ion AE 111 Evaluation software general features 6 6 5 1 Adding report points Click Add to add a new report point Enter the id for the report point maximum 30 characters in the Id field The report point id must be unique within an evaluation session ...

Page 112: ...d select the required report point if you want to calculate the responses at the custom report point relative to another report point If the box is not checked the closest preceding baseline report point will be used for calculating relative response values You can apply custom report points either to cycles with selected assay step purposes or to cycles selected by cycle number Choose the appropr...

Page 113: ... keywords in the evaluation software and edit the contents of certain keywords Choose Tools Keyword Table to open the keyword table When you save changes to the keyword table all user defined items in the evaluation session will be deleted Save the session before editing the keyword table if you do not want to lose your work Click Cancel in the Keyword Table dialog to close the dialog without appl...

Page 114: ...cts only predefined concentration keywords Numerical user defined keywords are simply numbers and will not be re interpreted when you change the concentration unit even if they are intended to hold concentration information Click Add Keyword to create a new keyword in the table You can choose between predefined keywords and user defined keywords see Section 5 6 2 If there are multiple Sample or Ge...

Page 115: ...e surface is slightly smaller than that on the reference surface Figure 6 1 Figure 6 1 Bulk solution is excluded from the volume occupied by ligand molecules on the ligand surface so the bulk contribution to the relative response is smaller than on the reference surface As long as the refractive index of the samples is constant this excluded volume effect introduces a constant error in reference s...

Page 116: ...t the procedure is intended to correct 6 7 3 How solvent correction works Solvent correction factors are determined by injecting a series of blank samples containing a range of solvent concentrations over the active and reference surfaces and plotting the difference in relative response between the surfaces as a function of the relative response on the reference surface Each sample measurement is ...

Page 117: ...ction cycle there is one correction curve for each set of reference subtracted sensorgrams All cycles are shown by default in an overlay plot Select specific rows in the cycle list to display the corresponding solvent correction curves Clear the checkmark in the Included box to exclude cycles from the correction calculation You must include at least one solvent correction cycle for each curve Samp...

Page 118: ...cessarily indicating poor quality The range of report point values that are candidates for solvent correction in the assay data is indicated by vertical red lines in the window If report points lie outside the correction range these values cannot be properly corrected Some small extrapolation of the correction plots may be permissible Use the Extrapolate button to extend the correction range The s...

Page 119: ... changes made in the custom report points or keyword table see Sections 6 5 and 6 6 6 8 1 Creating evaluation methods To create an evaluation method choose File Save Evaluation Method As and specify a file name and location The method is saved with file extension evalmethod An evaluation method may only contain one definition of a kinetics affinity evaluation item If there are more than one item i...

Page 120: ...ds To apply an evaluation method to the contents of an evaluation session choose File Apply Evaluation Method and choose the method A preview of the method is shown so that you can check the method contents Click Apply to apply the method Items in the method will be created where possible Items that cannot be created will be reported in a dialog box ...

Page 121: ... more cycles in the results A sensorgram item showing all sensorgrams for the first reference subtracted curve in the results or first active curve if reference subtraction is not used is created automatically when the result file is opened You can change the display settings in this item or create additional sensorgram items if required Click Sensorgram in the toolbar or choose Add sensorgram fro...

Page 122: ...he second file and so on For each option click the browse buttons to browse backwards or forwards through the list one item at a time Click the selector button to open the list for selecting one or more items Drag with the mouse or use shift click to select contiguous multiple items Use control click to select non contiguous multiple items To accept a selection click anywhere outside the list or p...

Page 123: ... the cycle Y adjustment Choose to set the zero response point to either a report point or an injection event If this setting is Off the actual response values will be shown If you check Enable Second Y Adjustment you can select a report point or injection event where the response value will be set to 100 Each sensorgram will then be normalized separately to the first and second adjustment point so...

Page 124: ...tems are not affected Note Subtracting a blank sensorgram is not the same as using reference subtracted data Reference subtraction gives the difference between active and reference values for each cycle separately whereas blank subtraction subtracts one curve from all others in the result set 7 1 4 Markers You can choose to display markers and or labels for report points and events in the cycle wi...

Page 125: ...n the selected report point will not be represented in the plot Response type may be response absolute or relative response or relative response adjusted for molecular weight if the keyword MW is defined or sensorgram slope at the selected report point Adjustment for molecular weight is performed by dividing the response in RU by the molecular weight in Da Points for which the molecular weight val...

Page 126: ...urve name lists the type of sensorgram from which the points are taken active reference reference subtracted and solvent corrected where applicable Assay Step Purpose filters the points according to the assay step purpose The third selector lists the variable values represented on the x axis This option is not available for plots of report point against report point Selection operates in the same ...

Page 127: ...ion Select rows in the table to highlight the corresponding points in the plot If you select a single row the highlight is augmented with lines drawn to the plot axes By default the table shows x and y values and is sorted in ascending order of x values Click on the header row to select the sort value and to change the sort order Sorting the table does not have any effect on the plot display Choos...

Page 128: ...e table column header Section 7 2 2 Note By default the table associated with a sorted plot retains a column labeled X Value This is the value of the variable originally defined for the plot and does not correspond to the x axis as displayed in the sorted plot Choose Sort As Defined to restore the x axis to the originally defined variable value Plots of one report point against another cannot be s...

Page 129: ...fit is reported for linear fitting as the correlation coefficient R2 and for 4 parameter fitting as the chi squared value see Section 9 7 1 Curve fitting cannot be applied to plots that have been sorted where y and x are the plot coordinates Rhi and Rlo are fitting parameters that correspond to the maximum and minimum response levels respectively A1 and A2 are additional fitting parameters y Rhi R...

Page 130: ...e control You can also select a sample for the negative control Select whether the adjustment should be made using a linear or polynomial second degree fitting function The display panels in the dialog show a plot of the response against cycle number before and after adjustment Click OK to apply the adjustment Adjustment normalizes the sample responses relative to the positive and negative control...

Page 131: ...rve or below the x axis if no negative control is selected Any points that lie in such regions will be excluded from the adjusted plot Adjustment for controls applies only to the current plot and does not affect any other evaluation item Note Beware of using a polynomial fitting function with less than 4 control samples The parabolic curve created by the function can deviate greatly from the point...

Page 132: ...s are specified in RU Boundaries are shown as horizontal red lines in the plot labeled with the boundary value The classification of the points is recorded in the table Ranking results are independent for each plot item Note Editing the definition of a plot does not affect ranking boundaries If you for example change the y axis parameter of a ranked plot from relative response to absolute response...

Page 133: ...splay in the lower left hand panel cycles in the center panel and report points in the right hand panel In each panel you can select multiple rows by dragging with the mouse or using Shift click for contiguous rows or Control click for non contiguous rows Click on the column header in the table of cycles in the center panel to sort the table by the contents of the column to simplify selection of r...

Page 134: ... Color by list to color the bars in the chart according to the parameter value A Color key will be added to the table in the appropriate panel Show column labels Use this option to display labels identifying the bars in the chart The bars are also identified by tool tips regardless of whether labels are displayed or not 7 4 Report point table Report points are automatically created for all wizard ...

Page 135: ... when the evaluation session includes more than one file the cycle number is prefixed with a file number Choose File Properties to display the mapping of source files to file numbers Cycle Cycle number within the file Fc The curve to which the report point applies identified as the flow cell Report point Report point id Time s Report point time in seconds from the start of the cycle Window s Repor...

Page 136: ... n number of points and y response in RU Slope RU s Slope during time window in RU s 1 calculated as LRSD Alignment of slope to a straight line regression coefficient calculated as where Baseline Yes for report points defined as baseline Otherwise No RelResp RU Relative response difference in absolute response from the baseline in RU N A if no baseline has been set AssayStep Purpose CycleType Iden...

Page 137: ... column will be displayed You can apply multiple filters to the table at the same time To remove a filter choose All from the list of column values in the filter setting Copying report point table contents To copy the selected contents of the report point table select cells by dragging with the mouse and press Ctrl C or choose Copy from the right click menu Choose Copy Table to copy the whole tabl...

Page 138: ...7 Data presentation tools 7 4 Report point table 138 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 139: ...the method must be constructed as described in Section 5 10 1 if necessary the keyword table can be edited so that the conditions are met in full see Section 6 6 Note however that the command name cannot be edited in the keyword table Refer to Chapter 5 for details of how to construct methods in Method Builder Evaluation of unknown samples is based on the nearest preceding calibration curve If the...

Page 140: ...pectively Choose the appropriate settings for the calibration curves and click Finish to complete the evaluation 8 2 1 Calibration curves A calibration curve is constructed from the cycles in each calibration step If two calibration steps are run in direct succession so that there are no other cycles between the steps they will be combined in a single calibration curve Check Use average calibratio...

Page 141: ...e name will be the same for all calibration cycles but different names can be introduced either in method based runs or by editing the keyword table Changing sample names in calibration cycles only changes the title in the calibration curve panel and has no effect on the curve itself Flow cell Concentration analysis may be performed without a refer ence cell since the unknown samples are determine...

Page 142: ...ion against cycle number on the right Select a row in the table to highlight the corresponding point on the plot The table lists the expected concentration as entered for the control samples the response and calculated concentration the calculated concentration as a percentage of expected and the calibration curve used to calculate the concentration Replicate control samples are summarized with av...

Page 143: ...s of the calibration Note The limits of calibration are defined by the concentrations corresponding to the response values on the fitted curve for the highest and lowest calibration samples Depending on how well the curve fits the experimental points these limits may not coincide exactly with the actual concentrations in the highest and lowest samples The right hand panel shows the calibration cur...

Page 144: ...ned using similar principles as equation models for kinetics and affinity see Section 9 9 3 8 2 5 Evaluating combined result sets If you use the Append File function to combine result sets from separate runs concentration analysis can be evaluated provided that the conditions specified in Section 5 10 are fulfilled in the combined set The software does not check the validity of any evaluation appl...

Page 145: ...ted on the basis of calibration curves from another file In such cases it is important to ensure that the calibration curves and sample analyses in the different files refer to the same analyte and are performed under as far as possible identical condition 8 3 Evaluating calibration free measurements Calibration free concentration analysis calculates the analyte concentration from the measured mas...

Page 146: ...all cycles to collapse the cycle details and show only samples in the table Notes Selecting a row in the cycle list does not display the corresponding cycle in the sensorgram panel Use the selector bar in the sensorgram panel to view different samples The value shown for the diffusion coefficient D is adjusted to the analysis temperature and may not be the same as the value for 20 C as entered whe...

Page 147: ...e cube root of the flow rate so the quotient Q has a value of 1 When there is no mass transport limitation the binding rate is independent of the flow rate so Q has a value equal to the cube root of the flow rate ratio The range of possible theoretical values for Q will thus depend on the flow rates used for flow rates of 5 and 100 µl min the value is 0 37 The QC ratio is calculated from the measu...

Page 148: ...the corresponding blank sensorgrams shown in gray Note The evaluation will always use blank subtracted data where possible even if this first dialog shows unsubtracted data Blank subtraction Blank cycles are identified by the value y or yes in the Blank keyword Section B 2 and must have the same flow rate and contact time as the sample cycles from which they are subtracted If there is no preceding...

Page 149: ...he selected data will be removed from all sample series or only from a selected series in the Settings panel In Single sample series mode use the selector bar to choose which sample series you want to work with You cannot remove data selectively from individual sensorgrams in a sample series Note Evaluation is performed using data from 3 s after injection start to 3 s before injection stop You do ...

Page 150: ...hich is the value calculated from the fitting and Calc Conc which is obtained by multiplying the Measured Conc by the dilution factor to give the concentration in the original sample You can change the units for the reported concentration in the header for the Measured Conc column Note The diffusion coefficient D listed in the evaluation results is the value at the analysis temperature calculated ...

Page 151: ... 10 with caution Examine the values in the Measured Conc column The operating range of the method is approximately 0 05 5 µg ml values outside this range are likely to be unreliable 8 3 5 Fitting model Evaluation of calibration free concentration relies on fitting the data to a model for 1 1 interaction see Section 9 8 1 where the mass transport coefficient is provided through calculation from the...

Page 152: ...8 Concentration analysis 8 3 Evaluating calibration free measurements 152 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 153: ... and summarized in the separate Biacore T100 Kinetic Summary software Section 9 6 This chapter describes how to evaluate surface bound kinetics and affinity If there are multiple sample series in the same data set evaluation can be performed either in single mode where each sample series is evaluated separately with interactive control over several aspects of the evaluation or in batch mode where ...

Page 154: ...c If the concentration is not given in molar based units the keyword MW must also be included with a value for the molecular weight For method based runs the method must be correctly constructed as described in Section 5 10 2 if necessary the keyword table can be edited so that the conditions are met in full see Section 6 6 Note however that the command type cannot be edited in the keyword table R...

Page 155: ...oose the curves included in the evaluation A concentration series is defined by a set of curves with the same sample name analysis temperature and curve identity Select the concentration series you want to work with in the respective pull down lists Note For runs with immobilized ligand there will be only one choice for Ligand name Multiple choices may be available if the ligand is captured and va...

Page 156: ...m the other curves when you proceed to the next step If you do not want to perform blank subtraction exclude the zero concentration sensorgrams from the data set You can also choose to use blanks from other concentration series for blank subtraction these are listed at the bottom of the table and are excluded by default Only blank sensorgrams with the same contact and dissociation times as the sam...

Page 157: ...g the injection start is seldom necessary Drag the vertical reference line to adjust the injection start point You can adjust the start by 10 s from zero The same adjustment is applied to all curves in the data set whether they are currently included for evaluation or not Click Next when you have selected the curves to be evaluated 2 The second dialog shows the blank subtracted curve set and allow...

Page 158: ...d for editing are shown in light color Note that all curves will be evaluated whether they are selected for editing or not removing the Edit checkmark does not exclude a curve from the data set for evaluation Click Affinity for steady state affinity evaluation or Kinetics for kinetics evaluation when you are satisfied with the curves 3 Affinity only If you choose to evaluate steady state affinity ...

Page 159: ...he starting values or scope of any of the parameters see Section 9 7 2 for details then click Fit to perform the fitting During the fitting procedure the fitted curves are shown in black overlaid on the experimental data Fitting progress is indicated in the sensorgram window by display of the iteration number the current chi squared value and the relative change in the parameter that was changed m...

Page 160: ...the item in the evaluation explorer panel You can click Back to review the choice of data for the evaluation however if you make any changes to the data e g remove additional sections from a curve or exclude a curve from the set all current fits will be deleted Current fits are also deleted if you switch between kinetics and affinity evaluation Kinetics results When the fit is completed the result...

Page 161: ... equations Affinity results For affinity determination the reported KD value is marked on the plot as a vertical line for a 1 1 interaction KD is the same as the analyte concentration at a response equal to half Rmax If the reported value is higher than half the highest concentration used this line will be shown broken in red as a warning that the value may be unreliable because the plot does not ...

Page 162: ...ure that the experimental conditions are comparable as far as possible To set up evaluation of the same sample series over multiple ligand densities click Multiple Rmax in the first dialog box for kinetics and affinity evaluation A panel for data subsets representing different Rmax values opens to the left of the curve table Click Add to add a new data subset If you have multiple sets of the curre...

Page 163: ...g with a common ligand or measurement of the same interaction at different temperatures automatically You select the series to be evaluated and the fitting model to be used and evaluation is carried to completion without further intervention To perform evaluation in batch mode start the kinetics affinity evaluation Section 9 2 1 and check the Batch mode option in the first dialog CAUTION Make sure...

Page 164: ...e created for each sample series Notes A data set for batch evaluation is defined as a set of cycles with the same ligand sample and analysis temperature Blank cycles will be subtracted within each data set Evaluation in batch mode does not support multiple ligand densities adjustment of injection start selection of blank cycles or removal of selected data Individual evaluation items created durin...

Page 165: ...uch parameters are said to be correlated One example is the pair of kinetic rate constants ka and kd that are related through the affinity constant KD KD kd ka Parameter uniqueness is assessed by testing correlation between pairs of the parameters ka kd and Rmax If significant correlation is found this will be reported as a warning that parameters cannot be uniquely determined Note This test does ...

Page 166: ...as neutral since the RI parameter was not evaluated Sensorgram curvature You should check that the sensorgrams have sufficient curvature for kinetic determination Ideally the sensorgrams for at least the one or two highest concentrations should show measurable binding rates at the beginning of the sample injection and approach a steady state towards the end of the injection Sensorgrams that approx...

Page 167: ...s an aid in judging the residuals guidelines are drawn on the residual plot to indicate the range of acceptability Most of the residuals should be within the inner green limits Figure 9 3 The residuals for a good fit left scatter around 0 ideally in a random distribution representing the noise in the sensorgrams For a poor fit right the residual curves show a definite shape and deviate farther fro...

Page 168: ...ared residual per data point If the model fits the experimental data precisely chi squared represents the mean square of the signal noise Chi squared is listed on the Report tab Standard error and T value The significance of parameter values is indicated by the standard error SE or T value listed on the Parameters tab in the fitting results This is a statistical indication of the significance of a...

Page 169: ... the interaction and mass transport processes to the closeness of fit by examining the results of correlated changes whereas the standard error is a mathematical assessment of the significance of a single parameter If the Check Kinetic Data tool indicates that the rate constants are not significant the standard error for the constants may be expected to be high However the converse is not always t...

Page 170: ... only significant if the observed binding is not seriously limited by mass transport of analyte to the surface see Section 9 8 For 1 1 fitting results you can check whether mass transport is limiting or not using the Check Kinetic Data function Choose Tools Check Kinetic Data to open a dialog that displays simulated sensorgrams based on the fitting results with the interaction rate constants ka an...

Page 171: ...m the original curves the fitting is sensitive to changes in the rate constants and the curves probably contain significant kinetic information If on the other hand the divergence is negligible the values of the rate constants do not matter because the binding is fully limited by mass transfer Mass transfer places an upper limit on the rate constants that can be measured on the borderline the fitt...

Page 172: ...fit is reported as a chi squared value calculated in the same way as for kinetics Note however that the number of points in the steady state affinity plot is very much lower than for kinetic evaluation so that chi squared is a more sensitive indicator of fitting quality The plot of Req against C approaches a limiting value equivalent to Rmax at very high concentrations Robust evaluation of the dat...

Page 173: ...ion Software 9 6 1 Creating kinetic summaries To create a kinetic summary of the results in a single evaluation file simply open the evaluation file file type bme in the Kinetics Summary software The Kinetics Summary software can also be started from the Tools menu in the Evaluation Software To create a summary of the results in multiple files you can either select multiple files within the same f...

Page 174: ...l display settings Choose Display Settings from the View menu or the right click menu for display settings for the thumbnails Small Provides an overview of many thumbnails for general comparison Details will not be legible in the thumbnails Individual thumbnails are identified in a tool tip Standard Shows the thumbnails with identification legible axis and a summary of fitting model and kinetic or...

Page 175: ...e Thumbnails tab always sorts the thumbnails and table rows in ascending order Sort the rows on the Table tab if you want to use a descending sort order Excluding items To exclude an evaluation from the summary right click on the item thumbnail and choose Exclude thumbnail or remove the checkmark from the Included column on the Table tab You can also exclude kinetic evaluation items using the righ...

Page 176: ...ctions in the summary by plotting the association rate constant ka against the dissociation rate constant kd both on logarithmic scales Since the affinity constant KD is the ratio of kd to ka interactions that have the same affinity will appear on diagonal lines representing the KD value The diagonals are shown as broken lines on the plot with the KD value indicated Points that lie widely separate...

Page 177: ...e model equations and it is wise always to examine the results obtained for reasonableness of the values obtained In addition any mechanistic conclusions drawn for the interaction from fitting results e g concerning multiple interaction sites or conformational changes should ideally be tested using independent techniques 9 7 1 Fitting procedure Kinetic parameters are extracted from experimental da...

Page 178: ...data set Constants have a fixed value that is not changed in the fitting procedure An example is the analyte concentration Constants may also be local separate values for each curve or global one value for the whole data set The local global status of parameters can be changed through the Parameters button in the fitting dialog step 4 in Section 9 2 without making any changes to the model Evaluati...

Page 179: ...ot totally limiting see Section 9 8 The rate of mass transfer of analyte to the surface under the conditions of non turbulent laminar flow that prevail in the Biacore flow cell is characterized by the mass transfer coefficient km units m s 1 One form used in fitting models in Biacore T100 is referred to as the mass transfer constant kt units RU M 1 m s 1 obtained by adjusting the mass transfer coe...

Page 180: ...yte concentration M Provided as input tc Flow rate independent component of the mass transfer constant Fitted f Flow rate µl min Provided as input tOn Sample injection start time s Provided as input tOff Sample injection end time s Provided as input RI Bulk refractive index contribution in the sample Fitted Report parameters Calculated as ka Association rate constant M 1 s 1 ka kd Dissociation rat...

Page 181: ...irst site interaction at the second site does not contribute to the SPR response For this reason the association rate constant for the second interaction is reported in units of RU 1 s 1 and can only be obtained in M 1 s 1 if a conversion factor between RU and M is available Similarly a value for the overall affinity or avidity constant is not reported Model parameters Obtained from ka1 Associatio...

Page 182: ...rams Concentrations and molecular weights are required for both analytes If absolute molecular weights are not known relative values can be entered without affecting the outcome of the fitting The model cannot evaluate interactions where the proportions and relative sizes of the analytes are unknown A1 B A1B A2 B A2B Report parameters Calculated as ka1 Association rate constant for the first site ...

Page 183: ...s input tc1 tc2 Flow rate independent component of the mass transfer constant for the first and second analytes Fitted Rmax1 Analyte binding capacity of the surface for the first analyte RU Fitted Rmax2 Analyte binding capacity of the surface for the second analyte RU Fitted rcf Response correction factor allowing for different refractive index contributions for the two analytes This factor is def...

Page 184: ...ound to give the best fit to the observed sensorgrams further experimental efforts to reduce the heterogeneity are recommended where possible A B1 AB1 A B2 AB2 Report parameters Calculated as ka1 ka2 Association rate constant for the first and second analytes M 1 s 1 ka1 ka2 kd1 kd2 Dissociation rate constant for the first and second analytes s 1 kd1 kd2 KD1 KD2 Equilibrium dissociation constant f...

Page 185: ...and RU Fitted Rmax2 Analyte binding capacity of the second ligand RU Fitted Conc Analyte concentration M Provided as input tc Flow rate independent component of the mass transfer constant Fitted f Flow rate µl min Provided as input tOn Sample injection start time s Provided as input tOff Sample injection end time s Provided as input RI Bulk refractive index contribution in the sample Fitted Report...

Page 186: ...ted using other techniques e g spectroscopy or NMR rather than direct evidence that a conformational change is taking place Rmax2 Analyte binding capacity of the second ligand RU Rmax2 Conc Analyte concentration M Conc tc Flow rate independent component of the mass transfer constant tc Flow Flow rate µl min f kt Mass transfer constant tc f1 3 RI Bulk refractive index contribution in the sample RI ...

Page 187: ...nalyte binding M 1 s 1 ka1 kd1 Dissociation rate constant for analyte from the complex s 1 kd1 ka2 Forward rate constant for the conformational change s 1 ka2 kd2 Reverse rate constant for the conformational change s 1 kd2 KD Overall equilibrium dissociation constant M kd1 ka1 kd2 kd2 ka2 Rmax Analyte binding capacity of the surface RU Rmax Conc Analyte concentration M Conc tc Flow rate independen...

Page 188: ... editing models To create your own models for kinetics of affinity evaluation choose Tools Models from the main menu and select the type of model you want to work with You can use existing models as templates Choose an existing model from the list and click New answer Yes in the following dialog to create a new model based on the chosen template or No to create a blank model For kinetic models you...

Page 189: ...odel or edit an existing definition 1 On the Interaction tab click New to add new reactants For each reactant choose whether it is analyte ligand or complex see below and enter an identifier for the reactant Enter parameter names or expressions for the reactant properties Note Numbers are used as part of the identifier not in the conventional chemical sense of stoichiometry Thus a complex named AB...

Page 190: ...stant Molecular weight Check this box and enter a molecular weight if required This information is used to calculate relative response contributions for heterogeneous analyte models it is not used for conversion of weight based to molar concentration units this conversion is performed if necessary in the sample table Number of blocked sites Check this box and enter a number if binding of one analy...

Page 191: ...a result of the sample injection Check the Drift box and enter an expression describing the drift most commonly a linear function of time to account for baseline drift 2 Enter the reaction scheme in the Reaction panel using the pull down list for each reactant Enter parameter names for the forward and backward rate constants for each line in the reaction scheme The terms k forward and k backward a...

Page 192: ...tively choose Attach to and select a parameter from the list If you attach a parameter to Keyword the initial parameter value will be set to the value of the keyword with the same name as the parameter Check Allow negative value if the parameter can be below zero Enter a description of the parameter for ease of identification If you have only used single parameter names as opposed to expressions f...

Page 193: ...ysis 9 4 Click Rate Equations to display the equations generated by the software You can select the equations in the display and click Copy to copy the equations to the Windows clipboard Use this function and paste the equations in to e g Wordpad to print a copy of the rate equations ...

Page 194: ...aluation can also be entered as an expression defining response as a function of time t To create an equation model choose New in the kinetics models dialog then choose to create the new model without using the currently selected model as a template Select Equation model in the subsequent dialog Parameters and report parameters are defined in the same way as for kinetic models ...

Page 195: ...efining Req as a function of concentration Conc Parameters and report parameters are defined in the same way as for kinetic models Note Beware of trying to define and use complex models for steady state affinity Because of the relatively few points available for fitting to steady state affinity models typically about five concentrations in duplicate complex models tend to give unstable fitting beh...

Page 196: ...9 Kinetics and affinity analysis 9 9 Creating and editing models 196 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 197: ...uting in the expression and rearranging gives A plot of ln KD against 1 T should thus be a straight line with slope ΔH R and intercept on the y axis ΔS R This simplified relationship does not hold if the heat capacities of reagents and products differ since different amounts of energy will be required to raise the temperature by the same amount on the two sides of the reaction In such cases the pl...

Page 198: ...stants for the forward and backward reactions can be obtained from plots of ln ka T and ln kd T respectively against 1 T Note that the Eyring equation does not have a corresponding non linear form that takes account of the heat capacity change for transition state formation Non linear fitting to obtain values for ΔCP can only be applied to equilibrium thermodynamic analysis where ΔCP is the heat c...

Page 199: ...ly choose one fitting model Options for the model are 1 1 kinetics steady state affinity recommended or All If you choose All it is possible to combine data from different fitting models in the same evaluation however values for thermodynamic constants are in all likelihood meaningless if the data is obtained from a mixture of different models Check the rows for data that you want to use in the th...

Page 200: ... the data to be included The results are displayed first as plots of affinity and rate constants against temperature Click Next to display the van t Hoff and Eyring plots together with a table of thermodynamic constants for the equilibrium and transition state formation In any of the plots right click on a point to exclude the point from the line fitting ...

Page 201: ... data in the thermodynamic evaluation the van t Hoff plot will show all affinity values but the Eyring plots will be empty because the steady state data lacks values for the rate constants Plots of kinetic and affinity constants against temperature show temperature values in C while van t Hoff and Eyring plots use absolute temperature values K Click on Finish to finalize the thermodynamic analysis...

Page 202: ...10 Thermodynamic analysis 10 2 Performing thermodynamic analysis 202 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 203: ...nts in affinity in solution determination are denoted A and B A B AB Experiments are set up so that a fixed concentration of B is mixed with variable concentrations of A and allowed to reach equilibrium The free concentration of B is then determined by injecting the sample over a ligand that binds B but not A or the complex AB the interactant A or a derivative thereof is usually suitable as ligand...

Page 204: ...equirements for affinity in solution Affinity in solution experiments are run using a method The method must be correctly constructed as described in Section 5 10 4 if necessary the keyword table can be edited so that the conditions are met in full see Section 6 6 Note however that the command type cannot be edited in the keyword table Refer to Chapter 5 for details of how to construct methods in ...

Page 205: ... Sample If you have run multiple calibration curves in the experiment choose the curve to use in the Calibration curve list A calibration curve is defined as measurements from assay step s with purpose Calibration regardless of the sample name If two or more Calibration assay steps are run contiguously with no intervening steps with a different purpose they will be combined into a single calibrati...

Page 206: ...of free B against total A is presented by default with a logarithmic scale on the x axis In this form the equilibrium constant KD is given by the concentration of A at the inflection point in the fitted curve Zero values cannot be plotted on a logarithmic scale If you have included a sample with zero concentration of A in the sample series and want to display this point on the plot choose Scale fr...

Page 207: ...Biacore T100 Software Handbook BR 1006 48 Edition AE 207 Appendices ...

Page 208: ...208 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 209: ...sheet if it is present when the GxP module is installed see the separate Biacore T100 GxP Handbook Export from the Evaluation software an Excel spreadsheet file extension xls containing separate worksheets for the file properties and for tabulated data for all evaluation items where appropriate i e plot data and evaluation results The worksheets for each item are identified with the item name For ...

Page 210: ...n either ASCII or Unicode format using the Menu Export Positions function in the Rack Positions dialog Section 4 2 6 The file contains two lines identifying the microplate and reagent rack settings followed by the contents of the rack positions table with the columns separated by tabs A 2 Importing data A 2 1 Control Software The Control Software supports data import to sample tables in assay wiza...

Page 211: ...ned samples for example when the user clicks Back and Next in the dialog sequence without changing sample information Sample table import When the sample import function is invoked the contents of the sample table are first exported in Extended Markup Language XML format to a temporary file that is submitted to the specified import program The import program may append new sample data to the file ...

Page 212: ...ill not be imported The position of these two lines in the file does not matter One line specifies the headers for table columns to be imported separated by tabs The headers should correspond to the column headers as they appear in the Rack Positions table with the exception of the Volume column in the table which can be omitted from the import file and is ignored if it is present This line may no...

Page 213: ...le of an exported file for rack positions opened in Microsoft Excel A 2 2 Evaluation Software The Evaluation Software supports import of model definitions for kinetics and affinity evaluation Model files for import should be obtained from GE Healthcare or created by exporting models from another installation ...

Page 214: ...Appendix A Data import and export A 2 Importing data 214 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 215: ...ds Refer to these methods as guidelines in constructing your own methods that exploit similar features B 1 Affinity in solution This method is designed for measurement of affinity in solution as described in Chapter 11 The method includes a Calibration assay step for measurement of component B and a Sample step for measurement of mixtures of components A and B Both these assay steps are connected ...

Page 216: ...llection rate of 10 Hz set in the General workspace Do not change this setting B 2 2 Cycle types The cycle type for sample analysis performs a High performance injection with a contact time of 36 seconds and a dissociation time of 5 seconds The dissociation time is not critical evaluation uses only data from the association phase Do not use shorter contact times than 36 seconds since the actual co...

Page 217: ...006 48 Edition AE 217 Method examples and recommendations Appendix B B 2 3 Variable settings Sample cycles All the variables for assay step Sample are set at run time except for the method variable Blank which is set to No in the method ...

Page 218: ...and may be left blank B 2 4 Setup Run At run time variables need to be assigned for assay steps Sample and Blank Each sample should be run at two or more flow rates values of 5 and 100 µl min are recommended and are entered for one sample Use the same set of flow rates for each sample Values are also required for analyte molecular weight MW diffusion coefficient at 20 C D 20 C and dilution factor ...

Page 219: ...er both flow cells followed by regeneration The sample analysis cycle uses two capture injections one to capture recombinant GST on the reference surface only flow path setting First and the second to capture GST tagged ligand on the active surface only flow path setting Second A stabilization time of 180 seconds is included after capturing the ligand on the active surface to allow for dissociatio...

Page 220: ...on AE This method is set up for kinetic analysis but can be adapted for other assays Note If you change the detection setting to Multi you will need to include additional capture injections for the active surfaces in flow cells 3 and 4 Do not use detection setting Single without removing the first capture injection ...

Page 221: ...rometry and contains two assay steps The first conditions the surface by washing three times with 0 5 trifluoroacetic acid while the second performs the sample injection and recovery operation The cycle for recovery of bound analyte contains just one single InjectAndRecover command Additional commands are usually not needed since the recovery component of the InjectAndRecover command serves as a r...

Page 222: ...cover 222 Biacore T100 Software Handbook BR 1006 48 Edition AE All parameters in the InjectAndRecover command are fixed check the appropriate boxes in the Method Variables list if you want to use variable parameters There are no evaluation variables for this command ...

Page 223: ...variables included for the evaluation purpose Kinetics heterogeneous analyte providing separate concentration and molecular weight variables for two analytes B 6 L1 liposome capture The recommended method for liposome capture on Sensor Chip L1 uses one conditioning cycle consisting of two injections of 40 mM octylglucoside followed by three start up cycles using a dummy sample before the actual sa...

Page 224: ...molecular weight compounds The essential addition to wizard based counterparts is the inclusion of a Solvent Correction assay step This assay step is connected to a cycle type that includes 8 Solvent Correction commands for injection of 8 different solvent concentrations see Section 6 7 Note In Biacore T100 Control Software version 2 0 and later solvent correction is included in the Kinetics Affin...

Page 225: ...affect the response in subsequent injections The Biacore method is designed for screening of low molecular weight compounds and includes a solvent correction step as described in Section B 7 Low molecular weight compounds frequently dissociate readily from their targets and the example method does not include regeneration If you require a regeneration step it is advisable to include regeneration a...

Page 226: ...lies on chelation of Ni2 ions on the surface and regeneration is generally performed by stripping the Ni2 with EDTA Applications that use this approach require two capture injections in each cycle one for Ni2 and one for the poly His tagged ligand The Biacore method provided is designed for kinetic or affinity determinations but may be readily modified for other applications that use Sensor Chip N...

Page 227: ...hod for single cycle kinetics uses the sample command type Single cycle kinetics with 5 sample concentrations per cycle Note that with this command type the evaluation purpose is fixed as Kinetics Affinity and the number of predefined variables for sample concentration corresponds automatically to the number of sample concentrations per cycle S ...

Page 228: ...Appendix B Method examples and recommendations B 9 Single cycle kinetics 228 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 229: ...eps 78 81 analysis temperature 85 buffer change 85 connecting to cycle type 84 recurrence 84 replicates 84 run order 84 auto scale 20 automatic positioning 41 autosampler 63 average calibration curve 139 140 avidity 181 B bar charts 133 display options 134 baseline 96 for report points 96 112 136 in regeneration scouting 54 in sensorgram display 20 plot 109 batch mode 153 163 Biacore methods 77 21...

Page 230: ...tact time 36 48 at low flow rates 36 control experiments 72 results 74 control samples 36 61 83 130 binding analysis 60 concentration analysis 64 142 kinetics affinity 69 plot 110 control software Edit menu 19 File menu 17 Run menu 21 screen regions 16 Tools menu 21 View menu 20 copy graph 22 108 copy table 108 correlated parameters 165 169 correlation coefficient 129 create assay step 81 cycle ty...

Page 231: ...t 17 export curves 22 108 export rack positions 42 export to Excel 209 export to XML 209 exporting data 209 exporting plots 108 exporting rack positions 42 210 exporting the report point table 210 extra wash after injection 87 extrapolating solvent correction curves 118 Eyring equation 198 F File menu control software 17 file name extensions 23 file storage 23 filter keywords 113 filtering report ...

Page 232: ...nd immobilization 46 limit guides 172 linear fit 129 141 linked reactions 72 control experiment 73 control results 75 liposome capture 223 local parameters 161 178 lock scale 20 low molecular weight compounds 224 low sample consumption 89 91 LRSD 136 M main screen control software 16 evaluation software 105 maintenance tools 22 manual run 15 25 initial settings 26 markers 124 mass spectrometry 9 8...

Page 233: ... results 18 printing wizard templates 42 Q QC ratio 147 acceptance 147 150 quality control 160 164 queue 25 27 R rack 41 rack illumination 22 rack positions 39 automatic import 42 default positions 41 export 42 210 import 42 212 rack settings in manual run 26 ranking 132 rate equations 192 193 reactants 189 reagent rack 25 40 41 recovering bound analyte 89 recovery solution 90 recurrent assay step...

Page 234: ...re 105 screening 59 method example 225 SD 136 selecting data for thermodynamic evaluation 199 selector bar 17 122 126 selector button 122 Sensor Chip L1 223 Sensor Chip NTA 226 Sensor Chip SA 50 sensor chip type 33 sensorgram 11 adjustment 123 baseline 20 copy graph 22 curvature 166 display 121 export curves 22 108 in evaluation 121 markers 21 normalizing 123 printing 18 reference line 20 removing...

Page 235: ...d 70 evaluation 197 requirements for evaluation 101 tool tips 121 toolbar 16 Tools menu control software 21 transition state 198 T value 151 168 two state reaction 186 U uniqueness 165 169 unknown samples 139 143 unzoom 20 107 108 user defined caption 107 user defined variables 92 U value 169 V van t Hoff equation 197 variable settings 96 variables 78 92 96 113 125 entering values at run time 98 e...

Page 236: ...236 Biacore T100 Software Handbook BR 1006 48 Edition AE ...

Page 237: ......

Page 238: ...ished Feb 2005 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp ...

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