Biacore T200 Getting Started - Duke University
GE HealthcareBiacore T200Getting Started
ContentsBiacore T200 Getting StartedIntroduction . 5Background informationBiacore terminology . 9Sensor surface properties . 10Immobilization . 11Interaction analysis . 16Regeneration . 17ExercisesDescription of the experimental set-up . 23Exercise 1. Start up . 24Exercise 2. Effect of buffer on pre-concentration . 30Exercise 3. Immobilization . 34Exercise 4. Interaction analysis . 39Exercise 5. Interaction analysis – multiple binding . 48Methods . 54Exercise 6. Kinetic analysis . 56Biacore T200 routine maintenanceDaily maintenance . 69Weekly maintenance. 69Monthly maintenance . 70When necessary . 70Biacore T200 Getting Started 28-9840-98 Edition AB3
4Biacore T200 Getting Started 28-9840-98 Edition AB
Biacore T200 Getting StartedBiacore T200 Getting StartedIntroductionThis Getting Started handbook is designed as a self-study guide to introduce youto the basic operations of BiacoreTM T200, Biacore T200 Control Software andBiacore T200 Evaluation Software.The handbook, together with the associated reagent kit, will take you throughthe basic steps in a Biacore experiment. The sections of this handbook markedwith a shaded border provide important background information that will helpyou carry out the exercises described in the later sections. There you will alsofind useful information regarding basic assay development.The reagents in the Getting Started Kit are for training purposes only and aresupplied in quantities allowing for two repeated exercise sessions, following theinstructions in this handbook. Biacore AB can accept no responsibility for resultsobtained with these reagents in any other context.The reagents should be stored at between 4 and 8 C and should be usedwithin one week of opening.RequirementsThe following are required for completing the Getting Started exercises: Time: approximately one day Biacore T200 instrument with Reagent Rack 2 and Sample and ReagentRack Familiarity with PC and Windows Getting Started Kit Series S Sensor Chip CM5 (not included within the Getting Started Kit) Amine Coupling Kit (not included within the Getting Started Kit) Micropipettes; 2-10 µl, 10-100 µl and 100-1000 µl and tipsReferencesFor further details on the topics discussed in this booklet, refer to: Biacore T200 Instrument Handbook Biacore T200 Software HandbookBiacore T200 Getting Started 28-9840-98 Edition AB5
Biacore T200 Getting StartedIntroduction Biacore Advisor Tutorial (CD-ROM) Sensor Surface HandbookContents of the Getting Started KitThe contents of the Getting Started Kit are listed in Table 1. All solutions, exceptfor the 10X HBS-EP buffer, ligand, analyte and enhancement molecule areready for use. The 10X HBS-EP buffer, ligand, analyte and enhancementmolecule should be diluted as described in the exercises and used immediatelyafter dilution.Table 1. Contents of the Getting Started Kit. For in vitro use only. Storage: 4 to 8 C forall solutions.6Reagent/Item, quantitySpecification10X Running buffer (50 ml)10 HBS-EP buffer; 0.1 M HEPES pH 7.4,1.5 M NaCl, 30 mM EDTA, 0.5% (v/v)Surfactant P20Ligand (50 µl)Monoclonal mouse-anti-human 2microglobulinAnalyte (50 µl)Human 2-microglobulinEnhancement molecule (50 µl)Polyclonal rabbit-anti-human 2microglobulinImmobilization buffer (1 ml),2 pcs10 mM sodium acetate, pH 5.0Regeneration solution (1 ml),2 pcs10 mM glycine-HCl, pH 2.5BIAnormalizing solution 70%(1.5 ml)70% (w/w) glycerolPlastic vials, 40 pcs0.8 ml polypropylene microvials, diam.7 mm.Caps for 7 mm plastic vials,40 pcsVentilated kraton G (SEBS) rubber caps,diam. 7 mm.Plastic screw top vials andcaps, 6 pcs of each2.0 ml polypropylene screw top vials,diam. 10.8 mm and non-penetrablescrew caps with o-ring seal(for Biacore T200, Biacore T100, Biacore3000 and Biacore X)Biacore T200 Getting Started 9840-98 Edition AB
Biacore T200 Getting StartedOrdering informationOrdering information is given in Table 2. For further information, please visitwww.biacore.com or contact your local Biacore system representative.Table 2. Ordering information.ItemCode No.10X HBS-EP buffer (1000 ml)BR-1006-69Series S Sensor Chip CM5BR-1006-68(package of 3 chips)Amine Coupling KitBR-1000-50BIAmaintenance KitBR-1006-51Biacore T200 Instrument Handbook28-9768-63Biacore T200 Software Handbook28-9768-78Biacore Advisor Tutorial, CD-ROMBR-1005-44Sensor Surface HandbookBR-1005-71Plastic vials, 7 mm (package of 1000 vials)BR-1002-12Plastic screw top vials and caps 10.8 mm(package of 1000 vials and 1000 caps)BR-1002-14Caps for 7 mm vials (package of 600 caps)BR-1005-02Biacore T200 Getting Started 28-9840-98 Edition AB7
Biacore T200 Getting StartedIntroduction8Biacore T200 Getting Started 28-9840-98 Edition AB
Background informationBackground informationThis chapter is intended to provide the basis for a more detailed understandingof the main steps in a Biacore assay. The information goes beyond what isexplicitly covered in the exercises and is, therefore, a complement to theexercises.Biacore terminology When molecular interactions are studied in Biacore, one of theinteractants is immobilized on the sensor surface while the other is passedover that surface in solution. In Biacore terminology, ligand refers to theimmobilized component and the interactant present in the sampleinjected over the surface is referred to as the analyte (Figure 1).Figure 1. “Ligand” refers to the immobilized interactant and “analyte” refers to theother interactant present in the sample injected over the sensor surface. The response is measured in resonance units (RU) and is proportional tothe molecular mass on the surface. For an interactant of a given mass,therefore, the response is proportional to the number of molecules at thesurface. A sensorgram is a plot of response against time, showing the progress ofthe interaction. The sensorgram is displayed on the computer screenduring the course of an analysis.Biacore T200 Getting Started 28-9840-98 Edition AB9
Background informationSensor surface propertiesThere are three major steps in a Biacore assay. These are:1Immobilization: The process by which the ligand is attached to the sensorchip surface.2Interaction analysis: The analyte is injected over the sensor chip surfaceand the interaction between the analyte and the immobilized ligand ismonitored.3Regeneration: The process of removing bound analyte from the ligand onthe surface.Sensor surface propertiesSeries S Sensor chip CM5, which is used in the Getting Started Kit, is a glass slidecoated with a thin layer of gold, to which a matrix of carboxymethylated dextranis covalently attached (Figure 2). The gold is required for generation of thesurface plasmon resonance (SPR) response. The dextran matrix allows covalentimmobilization of biomolecules using well-characterized chemistry andprovides a hydrophilic environment suitable for a wide variety of proteininteractions.Figure 2. Schematic illustration of the structure of the surface of Sensor Chip CM5.In addition to Series S Sensor Chip CM5, which is the most versatile chip, GEHealthcare offers a range of sensor chips with different properties that allowimmobilization of biomolecules with different characteristics. Refer towww.biacore.com for further information.10Biacore T200 Getting Started 28-9840-98 Edition AB
Background informationImmobilizationThere are different ways of immobilizing substances to the sensor surface. Thechoice of immobilization method depends on the properties of the substance.The immobilization approaches may be directed towards amine, carboxyl, thiolor hydroxyl groups on the ligand, or may use specific tags introduced into theligand.Amine couplingAmine coupling chemistry is the most widely applicable approach for covalentlyattaching biomolecules to the sensor chip surface and is suitable for the ligandincluded in the Getting Started Kit. With this method, the dextran matrix on thesensor chip surface is first activated with a mixture of 1-ethyl-3- (3dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) togive reactive succinimide esters. Ligand is then passed over the surface and theesters react spontaneously with amino groups or other nucleophilic groups tolink the ligand covalently to the dextran (Figure 3). After the injection of ligand,ethanolamine is passed over the sensor surface to deactivate remaining activeesters.Figure 3. Amine coupling of ligands to the sensor chip surface.Conditions for immobilizationAt the concentrations of ligand commonly used for immobilization (20-200µg/ml), the expected levels of immobilized ligand would be low in the absenceof a mechanism that attract the ligand molecules to the sensor surface. Themain mechanism for this concentration process is electrostatic attraction of theligand to the surface. This attraction is referred to as pre-concentration and canresult in a several thousand-fold concentration of ligand on the surface.The carboxymethylated dextran matrix of the sensor chip carries a net negativecharge at pH values above about 3.5. The pH of the immobilization buffershould, therefore, be higher than 3.5 and lower than the isoelectric point of theligand in order to achieve efficient pre-concentration. For many proteins,coupling in 10 mM sodium acetate buffer (pH 4.5) works well, although thechoice of pH can be a critical parameter in determining the success ofBiacore T200 Getting Started 28-9840-98 Edition AB11
Background informationImmobilizationimmobilization in some cases. If you have to use other conditions, bear thefollowing considerations in mind: The ionic strength should be low (10 mM monovalent cationsrecommended) for the electrostatic attraction to occur. Buffer components containing primary amine groups and other strongnucleophilic groups (e.g. Tris or sodium azide) must be avoided for aminecoupling, as these will compete with the ligand for active esters on thesensor chip surface.Many proteins show limited stability in low ionic strength solutions and at lowpH. The ligand solutions should, therefore, be prepared directly before use.Example of immobilization pH-scoutingThe experimental procedure of finding the appropriate immobilization pH isreferred to as pH-scouting. The example below shows how to set up animmobilization pH-scouting and how to assess the results.Example 1In this example, the ligand immobilized was the monoclonal anti- 2microglobulin antibody included in the Getting Started Kit. The ligand wasdiluted in sodium acetate buffers with different pH (pH 5.5, pH 5.0, pH 4.5 andpH 4.0) to a final concentration of 30 µg/ml in each sample. The flow rate was10 µl/min and the contact time was two minutes. If your sample availability isrestricted and you have to minimize ligand consumption, the flow rate may be setto 5 µl/min and/or a lower ligand concentration may be used.After the last ligand injection, a wash solution was injected to remove anyremaining ligand molecules. A short pulse of 1 M ethanolamine-HCl pH 8.5(included in the Amine Coupling Kit) or 50 mM NaOH is commonly used forwashing surfaces that have been used for pre-concentration/pH-scoutingexperiments.12Biacore T200 Getting Started 28-9840-98 Edition AB
Background informationAs shown in Figure 4, the attraction of the protein into the dextran matrix on thesensor chip surface increased with decreasing pH of the buffers. This isexplained by the fact that the protein is less positively charged at pH 5.5 ascompared to pH 4.0, which affects the attraction to the negatively chargeddextran matrix. pH 5.5, 5.0 and 4.5 all gave a satisfactory pre-concentrationeffect. At pH 4.0, the sensorgram approached a plateau at the end of theinjection, which indicates that the maximum level of attraction was soon to bereached at that pH.It should be pointed out, however, that pH-scouting does not provide a conclusiveanswer regarding suitable immobilization conditions. It is well known that aminecoupling is less efficient at low pH and in order to fully optimize the conditions andverify the choice of pH, the entire immobilization procedure should be performed.Figure 4. Sensorgram showing the SPR responses generated by the ligand anti- 2microglobulin injected in immobilization buffer pH 5.5, pH 5.0, pH 4.5, pH 4.0, respectively.Injection of wash solution after each ligand injection removes any remaining ligand fromthe sensor chip surface.Biacore T200 Getting Started 28-9840-98 Edition AB13
Background informationImmobilizationIn Figure 5, the sensorgrams from amine couplings of monoclonal anti- 2microglobulin at pH 5.5, 5.0, 4.5 and 4.0 are shown. Although the preconcentration response was very high at pH 4.0 (Figure 4), the finalimmobilization level was lower than at the other pH values tested. This clearlydemonstrates the pH-dependence of the amine coupling chemistry and pH 4.0was, therefore, excluded in this case.As mentioned previously, the pre-concentration effects at pH 5.5, 5.0 and 4.5were all acceptable (Figure 4). Since the immobilization level at pH 5.5 washigher than the immobilization levels at pH 5.0 and 4.5, pH 5.5 could appear tobe the best choice (Figure 5). By looking at the sensorgrams (Figure 5), however,it becomes evident that at pH 5.5 the response after attraction and covalentcoupling has not reached the maximum level. At pH 5.0, the sensorgram beginsto flatten out after the covalent coupling (the maximum level is almost reached),which may contribute to an increase in the robustness of the assay set-up. AtpH 4.5, the plateau is more evident, but at the same time the immobilizationlevel has dropped. Consequently, in this case pH 5.0 should be chosen forimmobilization of monoclonal anti- 2-microglobulin.Figure 5. Sensorgrams showing the immobilization of anti- 2-microglobulin via aminecoupling at pH 5.5, pH 5.0, pH 4.5 and pH 4.0, respectively. For further information onsensorgrams from amine couplings, refer to page 37.14Biacore T200 Getting Started 28-9840-98 Edition AB
Background informationImmobilization levelsThe binding capacity of the surface will depend on the levels of immobilizedligand. The term maximum response (Rmax) is often used in connection withBiacore experiments and describes the binding capacity of the surface in termsof the response at saturation. A theoretical Rmax value can be calculated usingthe formula below:analyte MWR max ------------------------------ immobilized amount stoichiometric ratioligand MWA theoretically calculated Rmax is often higher than the experimentally derivedRmax for the same interaction. There are many potential explanations for this,such as that the ligand is not fully active or that there is steric hindrance of theinteraction, for example.Different applications may require different binding capacities. A low Rmax, forexample, is often beneficial in kinetic analyses, while higher levels areadvantageous in concentration measurements.Ligand requirementsThe quality and purity of the ligand have an important effect on determining thespecificity and analyte binding capacity of the surface. The ligand should be asspecific as possible for the analyte as the selectivity of the assay is determinedby specificity. If necessary, an enhancement molecule can be used to increasethe sensitivity and/or specificity of the assay.The purity of the ligand is of vital importance for the experimental results.Impurities may be immobilized on the sensor chip and could affect the analytebinding capacity of the surface. Sensor surfaces immobilized with impure ligandsolutions may give rise to results that are difficult to interpret. Therecommendation is, therefore, to use as pure ligand solutions as possible.Cross-reactivity (i.e. binding of analyte-related molecules to the ligand) and nonspecific binding (i.e. general binding to the ligand) are other factors, which canbe controlled through the choice of ligand. Note that these factors are generallyapplicable to the vast majority of immunoassay formats and are not specific toBiacore assays.Biacore T200 Getting Started 28-9840-98 Edition AB15
Background informationInteraction analysisInteraction analysisDuring injection of the analyte, the binding of analyte to ligand takes place andis monitored in real time on the screen. After the analyte injection, when bufferflows over the surface, the dissociation of the analyte/ligand complex ismonitored. Although analytes in crude sample matrices can be measured andthe need for sample purification is low, a few guidelines on sample requirementsare given below.Sample requirementsThe detection principle in Biacore technology measures changes in refractiveindex (RI) that are related to changes in mass close to the sensor surface. Thedesign of the opto-interface allows for measurements of samples in crudeenvironments, such as serum and cell culture supernatants.Sample environments that differ greatly from the running buffer will give rise toa bulk refractive index (RI) effect that is commonly present during an injection.Bulk refractive index effects do not affect the binding. Our recommendation is,however, that the samples should be diluted in running buffer to minimize bulkshifts.The bulk RI contribution of the sample requires that measurement points (reportpoints) are set before and immediately after an injection, where running bufferflows over the surface. The relative response then reflects the amount bound(Figure dBulkcontributionBaselineTimeFigure 6. Contribution of bulk refractive index to the response.16Biacore T200 Getting Started 28-9840-98 Edition AB
Background informationOne useful feature of Biacore T200 Control Software is that the bulk contributioncan be automatically subtracted. Flow cell 1 and/or 3 can be used as areference cell for in-line reference subtraction, providing direct recording anddisplay of blank-corrected sensorgrams. The reference curve subtractionautomatically corrects for the small time delay between the serial flow cells. Aschematic illustration of the principle of reference subtraction is shown in Figure7.Figure 7. The effect of reference subtraction. The sensorgram from the reference surfaceshows the contribution of the bulk, whereas the sensorgram from the active surfaceshows the actual binding response plus the bulk contribution. In the reference-subtractedsensorgram, only the binding response is shown.Although the reference subtracted sensorgram shows the actual bindingresponse, it is important to inspect the sensorgrams from both the referenceand active surfaces. The sensorgram from the reference surface will, forexample, reveal non-spe
Biacore T200 Getting Started 28-9840-98 Edition AB 5 Biacore T200 Getting Started Biacore T200 Getting Started Introduction This Getting Started handbook is designed as a self-study guide to introduce you to the basic operations of BiacoreTM T200, Biacore T200 Control Software and Biacore T200 Evaluation Software.
Biacore T200 Getting Started Guide, which in conjunction with the Getting Started Reagent Kit and CM5 sensor chip, provides users a self-guided tutorial through the basic steps of a basic Biacore experiment performed using amine-coupling chemistry. Biacore T200 Instrument Handbook, the instruction manual for operating the Biacore T200 instrument
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