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1、Biacore application guidesAntibody screening with Biacore systems2Antibody screening with Biacore systemsIn the search for therapeutic or analytical antibodies the selection of appropriate kinetics/affinity,specificity and biophysical properties is essential.Antibody screening aims to identify cell

2、clones that produce antibodies appropriate for the purpose,by challenging the antibodies with known antigen.Sample matrices are often complex,e.g.hybridoma culture supernatants or phage display preparations.Obtaining kinetic information early in the screening process is often a significant advantage

3、.Information that is generally sought includes:Specificity which clones produce antibodies with the desired specificity?Binding characteristics which clones produce antibodies with suitable kinetic and/or affinity properties?Expression level which clones produce sufficient amounts of antibodies?Scre

4、ening can be performed on full size antibodies or parts thereof,such as Fab fragments or scFvs.This Application guide deals with screening of complete monoclonal IgG antibodies.Assay conditions and prerequisites might differ for other antibody types and fragments.3Assay formatAntibody screening can

5、be run using two different assay formats:Format DescriptionAntibody as ligand Antigen as analyteSamples to be screened are injected over a generic antibody-capturing surface.Antigen is then injected as analyte.The surface is regenerated by removing captured antibody and bound antigen.Antigen as liga

6、nd Antibody as analyteAntigen is immobilized or captured on the surface.Samples to be screened are then injected as analyte.The surface is regenerated by removing the bound antibody.(If reversible antigen capture is used,the surface may be regenerated by removing captured antigen and bound antibody)

7、.In some Biacore systems,the analyte injection is termed sample.Do not confuse this terminology with the generic use of sample to refer to the variable element in the assay,in thiscase the captured ligand.Screening typesInformation from antibody screens using Biacore systems may be obtained at diffe

8、rent levels as summarized below.Screening typeDescriptionReport point-based screeningProvides information on specificity and expression level from single point binding level measurements.Off-rate rankingProvides comparative information on the dissociation rate of the antibody-antigen complex.Kinetic

9、 screeningProvides information on both the association and dissociation rates of antibody-antigen binding.Capturing the antibody as ligand is generally more straightforward with respect to surface preparation and assay development,but requires injection of antigen for each cycle in the screen.Using

10、the antigen as ligand can involve additional work to establish immobilization and regeneration conditions.Screening using antigen as ligand is only recommended for assessment of specificity and/or expression level,for several reasons:Antibody concentrations are usually not known,and are required for

11、 kinetic screening Complete antibodies are usually bivalent,making evaluation of off-rate ranking and kinetic screening more difficultThe two approaches differ in experimental setup and evaluation,and are considered separately in the following sections.Frequently,report point-based screening is used

12、 as an initial screen to eliminate non-producing clones.Off-rate ranking and kinetic screening are then used for more detailed characterization of the clones that produce potentially interesting antibodies.4Tips for antibody screening Choose the assay format depending on your needs and preferences.C

13、hoose antibody as ligand if your aim is to obtain kinetic information.When using antigen as ligand,estimate how much you need to immobilize in order to obtain high enough responses from the injected antibody Check for non-specific binding from the sample matrix by injecting blank samples(sample matr

14、ix without antibody)and check for binding to both the active and reference surfaces A data collection rate of 1 Hz is usually sufficient.Using higher data collection rates increases file sizes without providing additional information.Establish that the assay is suitable for purpose using a few sampl

15、es and controls before you start extended runs with many samples If you are using pooled sample positions in the microplate,use Predip with the injection to minimize sample dilution For off-rate ranking and kinetic screening,consider the use of blank cycles.For best performance include one blank cyc

16、le per sample.To increase throughput and reduce antigen consumption a blank cycle can be included at selected intervals and used for all samples.Note that this approach is generally less precise.General considerationsSurface preparationResponse levels in antibody screening are usually quite high,and

17、 it is not necessary to use high ligand levels.Specific considerations for screening approaches with antibody as ligand and antigen as ligand are given in the respective sections below(see Surfaces for antibody capture,on page 5 and Preparing the antigen surface,on page 8 respectively).BuffersHBS-EP

18、+(HEPES-buffered saline with 0.3 mM EDTA and 0.05%Surfactant P20,available from Cytiva)or similar is recommended as running buffer for antibody screening.Samples may be diluted if required using the same buffer.Precise matching of sample and running buffer is neither needed nor practicable for antib

19、ody screening work,since the report points used are placed after the sample injections.Sample preparationSamples for antibody screening are typically clarified material such as hybridoma culture supernatants or phage display preparations,used without further purification.To verify that there is no b

20、inding of the sample matrix to the surface a control experiment is recommended in which only the sample matrix(cell culture supernatant or corresponding matrix)is injected over the sensor surface.If there is non-specific binding from the sample matrix this can be reduced by diluting the samples with

21、 running buffer(typically 1:1).Addition of NSB Reducer(available from Cytiva)at 1 mg/mL can also help to reduce non-specific binding.5Screening using antibody as ligandThe general approach to screening with antibody as captured ligand is summarized below.StepAction1Dock a pre-immobilized sensor chip

22、 or immobilize the capturing molecule.2Run at least 3 startup cycles using buffer and regeneration to equilibrate the system.3Inject sample containing antibodies(for example,hybridoma culture supernatants or phage display preparations)over the active surface to capture antibodies from the sample.Cru

23、de samples can be injected directly,without pre-treatment.4Inject antigen as analyte over both the active and reference surfaces.5Evaluate the results.The experimental setup differs slightly according to whether the results will be evaluated from single report points or as off-rate or kinetic rankin

24、g.Details are given below.Surfaces for antibody captureReady-to-use kits for preparing surfaces for capture of mouse and human antibodies and human antibody fragments are available from Cytiva(see the table below).Custom capturing molecules may also be immobilized on the sensor chip if required.In g

25、eneral,aim to immobilize about 5000 RU or less of the capturing molecule,depending on the expected antibody expression levels and the size of the antigen.Using higher levels can lead to non-specific binding from complex samples.Sensor chips preimmobilized with Protein A(MabSelect SuRe),Protein G and

26、 Protein L are also available from Cytiva.Protein A and Protein G bind selectively to antibodies from different species and subclasses.Protein L binds specifically to antibodies containing kappa light chains,with a broader selectivity than Protein A and Protein G.Ordering information may be found on

27、 the Products pages at nameIntended forMouse Antibody Capture KitMouse IgG,IgA,and IgM antibodiesHuman Antibody Capture KitHuman IgG antibodiesHuman Fab Capture KitHuman Fab fragments(kappa and lambda)Sensor Chip Protein AAntibodies according to the binding profile of Protein ASensor Chip Protein GA

28、ntibodies according to the binding profile of Protein GSensor Chip Protein LAntibodies according to the binding profile of Protein L6Conditions for report point-based screeningRecommendations for report point based screening are listed below.Use the predefined screening method if one is provided wit

29、h your Biacore system.ParameterRecommended valueCommentsSample injection(ligand capture)Flow rate10 L/minHigher flow rates consume more sample.Contact time60 to 180 sLong enough to give confidently measurable response levels without compromising throughput.PoolingNot applicableMolecular weightNot re

30、quiredConcentrationNot requiredAntigen injection(analyte)Flow rateNot lower than 30 L/minAvoid mass transport limitations that can obscure differences in ligand levels.Injection typeLow sample consumptionContact time60 sUse longer contact times if antigen binding is slow.Dissociation time120 sInclud

31、e a dissociation time to provide a rough indication of antigen binding stability.PoolingOptionalAnalyte may be pooled to conserve microplate space and reduce antigen consumption.Molecular weightNot requiredConcentrationNot requiredUse the same concentration in all cycles.7Conditions for off-rate ran

32、king and kinetic screeningRecommendations for off-rate ranking and kinetic screening are listed below.ParameterRecommended valueCommentsSample injection(ligand capture)Flow rate10 L/minHigher flow rates consume more sample.Contact time60 to 180 sLong enough to give confidently measurable response le

33、vels without compromising throughput.PoolingNot applicableMolecular weightNot requiredConcentrationNot requiredAntigen injection(analyte)Flow rateNot lower than 30 L/minAvoid mass transport limitations.Injection typeHigh performanceContact time60 to 120 sUse longer contact times if antigen binding i

34、s slow.Dissociation time180 sUse a dissociation time that is long enough to allow estimation of dissociation rates.PoolingOptionalAnalyte may be pooled to conserve microplate space and reduce antigen wastage.Molecular weightNot requiredConcentrationNot required for off-rate rankingMolar concentratio

35、n required for kinetic evaluationUse the same concentration in all cycles.Kinetic screening is performed with a single antigen concentration.Blank cyclesBlank cycles are not necessary for report point-based screening.Include blank cycles for off-rate ranking and kinetics screening.Blank cycles consi

36、st of antibody capture followed by injection of buffer instead of antigen.Ideally,there should be a blank cycle for each sample.Throughput can be improved by using a general blank cycle at intervals(for example,every 5 or 10 sample cycles),using a representative clone for the antibody capture.This a

37、pproach requires that the antibody capture cycles are fairly similar throughout the assay with respect to parameters such as non-specific binding and drift.Screening using antigen as ligandAntibody screening with antigen as ligand is performed with single injections of each antibody clone over antig

38、en on the surface.The antigen may be covalently immobilized or captured.No control samples are included.A typical antibody screening experiment with immobilized antigen includes the following steps:StepAction1Immobilize or capture the antigen on the sensor surface.Assay development work may be neces

39、sary to establish suitable immobilization conditions.2Run at least 3 startup cycles using buffer and regeneration to equilibrate the system.3Inject samples containing antibodies(for example,hybridoma culture supernatants or phage display preparations)over both the active and reference surfaces.Crude

40、 samples can be injected directly,without pre-treatment.4Evaluate the results.8Preparing the antigen surfaceIf the antigen is to be immobilized covalently on the surface,assay development work will probably be required to establish immobilization chemistry and conditions as well as regeneration cond

41、itions.It is important that the immobilization and regeneration do not interfere with antibody binding.Scouting strategies for immobilization and regeneration conditions are described in the Biacore Sensor Surface Handbook.Antigens that carry a suitable tag can be captured on the sensor surface.For

42、most tags,regeneration removes both bound antibody and antigen from the surface,so that fresh antigen is captured for each cycle.Capture of biotinylated antigens on streptavidin surfaces however involves such a high affinity interaction that regeneration can remove the bound antibody but leave the a

43、ntigen intact on the surface.With this approach,antigen is captured once at the beginning of the screen.Ready-to-use kits and pre-immobilized sensor surfaces for capturing tagged antigens are available from Cytiva.Ordering information may be found on the Products pages at nameIntended forHis Capture

44、 KitHistidine-tagged antigensSensor Chip NTAHistidine-tagged antigensBiotin CAPture KitReversible capture of biotinylated antigensSensor Chip SAPermanent capture of biotinylated antigensConditions for screeningRecommended injection conditions for antibody samples are listed below.ParameterRecommende

45、d valueCommentsFlow rate10 L/minHigher flow rates consume more sample.Contact time60 to 180 sLong enough to give confidently measurable response levels without compromising throughput.Dissociation time0A dissociation time is not needed since off-rate evaluation is not used.9Evaluation tools and opti

46、onsAntibody screens can be evaluated with a range of options for displaying the screening results,according to requirements and to some extent personal preferences.Several of the options described in this section are implemented automatically by predefined evaluation methods in the system software.T

47、he table below lists the information provided by commonly used options.Display optionAntibody as ligandAntigen as ligandSensorgram displayQuality controlQuality controlPlot of capture_level against cycleExpression levelNot generally applicable.However,for screening using capture on CAP,NTA or anti-H

48、is surfaces,the capture level may provide a check on surface performance throughout the assay.Plot of stability_early against cycleExpression level/antigen binding capacityExpression level/antigen binding capacityPlot of stability_early against capture_levelAntigen binding capacity adjusted for expr

49、ession levelNot applicablePlot of stability_early against stability_lateAntigen binding stability together with expression level/antigen binding capacityAntigen binding stability together with expression level/antigen binding capacityOff-rate rankingAntigen binding stabilityNot recommendedKinetic sc

50、reeningAntigen binding kineticsNot recommendedNote:The report points stability_early and stability_late refer to the analyte injection(antigen when antibody is used as ligand and vice versa).SensorgramsSensorgram display can identify any disturbed cycles.Disturbed cycles should be excluded from the

51、evaluation.Disturbances may not be apparent in report point plots.Sensorgrams can be aligned to zero at the baseline before the capture injection or before the antigen injection,to provide different perspectives on the capture and binding behavior.Examples:Sensorgrams from screening with antibody as

52、 ligandSensorgrams from screening with antigen as ligand10Capture_level against cycleA plot of capture_level against cycle number gives a good indication of the relative expression levels of the different clones,on the assumption that non-specific binding of material to the surface can be ignored.Cl

53、ones that do not produce significant amounts of antibody give capture levels close to background.These clones can be excluded from further evaluation if desired.Stability_early against cycleThe report point stability_early is placed immediately after the antigen injection.A plot of stability_early a

54、gainst cycle number provides a combined indication of 2 parameters:Expression level:clones that produce low levels of antibody will show correspondingly low levels of antigen binding Antigen binding properties:antibodies that bind antigen slowly or weakly will show low levels of antigen binding even

55、 if the expression levels are highThe plot of stability_early against cycle number does not distinguish between these causes.However,clones can often be excluded from further work on the basis of low levels of antigen binding,regardless of the cause.Examples:11Stability_early against capture_levelA

56、plot of capture_level against stability_early can distinguish between clones that express low levels of antibody and antibodies that bind antigen weakly.Points for low expression levels lie close to the stability_early axis,while weak binders lie close to the capture_level axis.The same information

57、may be obtained by adjusting the antigen response for the capture level in systems that support this function.Stability_early against stability_lateThe report point stability_late is placed just before the end of the dissociation time after the antigen injection.A plot of stability_early against sta

58、bility_late gives an indication of antigen binding stability,as illustrated to the right.More detailed information can be obtained from off-rate ranking and kinetic screening.Examples:12Off-rate rankingOff-rate ranking is evaluated by fitting the dissociation phase of single-concentration antigen in

59、jections to a 1:1 dissociation model.Blank subtraction should be performed before the data is evaluated.The model describes an exponential process that is independent of the starting concentration,so that antigen concentration is not required.Biacore systems that support off-rate ranking for screeni

60、ng purposes provide both graphical and numerical overview of the results.Example:13Kinetic screeningKinetic screening is evaluated by fitting the association and dissociation phases of single-concentration antigen injections to a 1:1 binding model.Blank subtraction should be performed before the dat

61、a is evaluated.A molar value for antigen concentration is required for the fitting.However,since the same antigen concentration is used for all cycles,an approximate value can be used to obtain relative kinetic parameters if the exact value is not known.Inaccuracy in the molar concentration will be

62、reflected in the reported association rate constants.A plot of association rate constant ka against dissociation rate constant kd(often called an on-off rate chart)is valuable in visualizing the results of a kinetics screen.In this plot,antibody clones with the same affinity lie on the same diagonal

63、.Clones with different rate constants but the same affinity are distributed along the diagonal.(The affinity constant is equal to the ratio of the rate constants for a 1:1 interaction.)Example,showing thumbnails and on-off rate chart: and the Drop logo are trademarks of Global Life Sciences IP Holdc

64、o LLC or an affiliate.Biacore,MabSelect,and MabSelect SuRe are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva.2020 CytivaAll goods and services are sold subject to the terms and conditions of sale of the supplying company operating within the Cytiva business.A copy of those terms and conditions is available on request.Contact your local Cytiva representative for the most current information.For local office contact information,visit