Dip and Readâ¢ Residual Protein A Detection Kit - ForteBio

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Dip and Read Residual Protein A Detection KitF Consolidated Assay ProtocolEquilibrate all reagents and samples to room temperature prior touse (at least 30 minutes). Preheat the heating block to 100 °C.Sample and Calibrator Preparation (in Tubes)1 Prepare the Recombinant Protein A standard curve in SampleDiluent. The recommended concentrations for the RecombinantProtein A Calibrator are 25, 10, 5, 2.5, 1, 0.5, 0.25, 0.1, and0 ng/mL.2 Dilute the samples with Sample Diluent to fall within the rangeof the standard curve.3 Add Sample Denaturing Buffer to the samples at a 2:1 ratio. Forexample, add 80 µL denaturing buffer to 160 µL of sample.4 Mix thoroughly by vortexing.5 Incubate at room temperature for 5 minutes.6 Place the tubes in the heating block for 1minute.7 Remove the tubes from the heating block and vortex briefly.8 Centrifuge the tubes for 5 minutes at 14,000g.9 Transfer 180 µL of supernatant from each tube into a well of a96-well black microplate.10 Add 18 µL of Neutralization Buffer to each well containingsample and mix thoroughly.If desired, the assay can be paused at this point and stored overnight.1 Seal the microplate.2 Store at 2–8 °C. Do not store for more than 24 hours.Assay ProcedureIf samples were stored at 2–8 °C, equilibrate all reagents andsamples to room temperature prior to use (at least 30 minutes).1 Incubate the Residual Protein A biosensors with the samples.If using a Sidekick shaker:aPlace the 96-well sample microplate on the shaker.b Remove the Residual Protein A biosensors from theirpouch and then remove the tray of biosensors from theblue bottom tray.cCarefully lower the tray of biosensors into the sample wells.d Incubate for 1 hour at 30 °C at 1000rpm.If using a standard microplate shaker or incubating overnightwithout shaking:b Remove the Residual Protein A biosensors from their pouchand then remove the tray of biosensors from the blue bottomtray.b Place the 96-well sample microplate on the blue biosensortray, aligning well A1 on both trays.cIncubate for 2–3 hours with moderate shaking (someoptimization may be necessary) or without shaking on thebenchtop overnight.2 Prepare a 96-well black microplate containing SampleDiluent+PEG buffer according to the sample plate layout.3 When incubation is complete:If using a Sidekick shaker:aRemove the biosensor tray and place the microplatecontaining only Sample Diluent+PEG buffer on the shaker.Lower the biosensor tray onto the microplate on the shakerand shake for 5 minutes.If using a standard plate shaker or incubating overnightwithout shaking:aRemove the biosensor tray assembly from the shaker,if using. Place the microplate containing only SampleDiluent+PEG buffer in the blue biosensor tray bottom andlower the tray of incubated biosensors into it. Place thetray assembly on the biosensor stage in the Octet instrument.Allow the biosensors to incubate without shakingfor 10 minutes.4 Prepare a 96-well black microplate with one column of SampleDiluent+PEG and one column Protein A Detection Reagent.For example: column 11 can contain the Diluent + PEG andcolumn 12 can contain the detection reagent. Place the microplateon the sample stage in the Octet instrument.5 From the Experiment Wizard (ctrl+N) choose the AdvancedQuantitation option.6 In tab 1 of the Octet Data Acquisition software:aClick the Modify button and choose the Residual Protein ADetection method from the list on the left (must be createdbefore the assay can be run on an instrument, see instructionsabove). The settings in this list are default settings. Tomodify the settings use the up or down arrows.b Choose the Sample Plate radio button and right-click onthe Sample Plate map to open the contextual menu todefine the location of the samples, controls, and calibratorsin the sample plate (i.e., the microplate used during theoffline biosensor incubation step).cEnter Sample IDs, concentrations and dilution factors asdesired into the Sample Plate table.d Choose the Reagent Plate radio button and right-click onthe Reagent Plate map to open the contextual menu todefine the locations of the buffer (Sample Diluent+PEG)and the detection reagent (diluted Detection Reagent).6
Technical Note 187 In tab 2:aDefine the locations of the biosensors. Locations can bechanged by highlighting the biosensors and using theRemove, Fill or Fill Plate buttons.b Choose Residual Protein A from the Sensor Type dropdownmenu (must be created before the assay can be runon an instrument, see instructions above).cAdd biosensor information in the table on the right (ifdesired).8 In tab 3:aEnter the location and folder name in which the datashould be saved.b Enter a delay if needed to complete the 5-minute minimumsample equilibration time.9 Start the assay by clicking the Go button. The default settingswill ensure the data, method and runtime charts are savedautomatically.DATA ANALYSIS1 Start the Data Analysis software (version 6.1 or later).2 In tab 1:aLoad data by locating the data folder in the lower explorertree, right click on it, and choose Load Folder.b Select data to analyze by clicking on the data folder in theQuantitation section of the Loaded Data tree. Multiple datasets can be selected for analysis. All selected data sets willbe analyzed as a group.cOnce the data is loaded, sample ID, group, concentrationand dilution factors can be modified in the table if needed.3 In tab 2:aSelect the desired standard curve.b Select R equilibrium as the binding rate equation. Thisequation will fit the binding curve generated during theexperiment and calculate a response at equilibrium as theoutput signal.cClick Calculate Binding Rate. Results will be displayed automaticallyin the table.4 Click the Save Report button or select File > Save Report togenerate a Microsoft® Excel formatted report.7
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