Presentation on theme: "Scott Zhou, North China FAS MB:15810470035, Mar.20 th, 2013 Octet Training Part III: Quantitation on the Octet."— Presentation transcript:
Scott Zhou, North China FAS MB: , Mar.20 th, 2013 Octet Training Part III: Quantitation on the Octet
Agenda BLI Quantitation Workflow BLI Quantitation Applications
BLI Quantitation Workflow
Sandwich ELISA Assay Procedure Bottom of well (1)Plate is coated with a capture antibody (2)Sample is added, and any antigen present binds to capture antibody (3)Detecting antibody is added, and binds to antigen (4)Enzyme-linked secondary antibody is added, and binds to detecting antibody (5)Chromogen/substrate is added, and is converted by enzyme to detectable form Typical read out could be fluorescence or luminescence Each step involves incubation time and wash steps in between. Manual ELISA can be an all day assay for just a few plates. Multiple Steps add to higher CV 5-25%.
Distance between the two reflecting surfaces = ℓ Intensity λ = ƒ( λ, ℓ ) Relative Intensity Wavelength (nm) 100% 0 nm shift Time 可实时检测到两个反射表面间距的改变 Biolayer Interferometry （ BLI ）
Biosensor selection According to application & sample type etc. 新开发了四种传感器 1.Anti-GST Biosensor: 用于含 GST 标签的蛋白 2.NTA Biosensor ：用于含 His 标签的蛋白 3.Anti-human Fab-CH1 ：用于人 Fab, F(ab’)2 及 Ab1~4 4.Anti-Flag biosensor ：用于含 Flag 标签的蛋白
Octet Workflow for Quantitation Anti-human IgG (Fc specific), anti-murine IgG (Fab’ specific), or Protein A sensors Standard samples of known concentration used to generate a standard curve Unknown samples
Octet Automated Workflow for Quantitation Anti-Human IgG, Anti-Murine or Protein A Biosensors Calibrants Test Samples Binding (nm) Time Binding Rate Concentration Calibrants used to plot a binding rate vs conc. calibration curve The binding rates of test samples are then measured and plotted on the calibration curve to determine their concentration One sensor per one sample well; one step assay with pre-made sensors.
Octet Workflow for Quantitation with Regeneration Standards Test Samples Binding (nm) Time (sec) Binding Rate Concentration The binding rates of test samples are measured and interpolated from the standard curve to determine concentration 96 samples analyzed in minutes Reuse of standard curve is optional Octet Biosensors 120 Buffer/Neut. Regen
Lab Work Example – Quantitation Experiment Set up the plate shown below: Analyze using Protein A Biosensors and running the standard Protein A Protocol Refer to Quick Start Q Assay pdf for further details of running Quantitation Assays Generate a Quantitation Report Calculate CVs of each of the 8 replicates of calibrators A ……Glycine pH2.0SD B ……Glycine pH2.0SD C ……Glycine pH2.0SD D ……Glycine pH2.0SD E ……Glycine pH2.0SD F ……Glycine pH2.0SD G ……Glycine pH2.0SD H ……Glycine pH2.0SD SamplesCalibrators Regeneration Neutralization
Octet Binding Curve = rate of increase in optical thickness as the sample binds to the sensor. Different protein concentrations result in different binding curves Quantitation – Real-time binding curves Rate of binding correlates to concentration Rate of binding is proportional to concentration 700 ug/mL 500 ug/mL 300 ug/mL 100 ug/mL 30 ug/mL 10 ug/mL 3 ug/mL 1 ug/mL
BLI Quantitation Applications
Fc-fused protein Quantitation Overview Samples: 14 hFc-fused proteins in supernatant 1 Standards: Fc-fused proteins with original conc. of 92.09mg/ml Biosensors : Protein A biosensor Octet platform: RED96 Other reagents & consumables : fresh medium, supernatant 2, PBS, 10mM pH1.5 glycine, Greiner 96-well micro-plate, pipettes, tips and etc. Goal: CV% & Re%, throughput and etc. as to ELISA
Workflow for the Fc-fused protein quantitation Test for dilution factors Dilute standards and unknowns with diluted supernatant Enter sample information into software Bind Fc-fused protein to protein A biosensor Generate standard curve & Regenerate biosensors Bind known concentrations of Fc-fused proteins to regenerated sensor Bind unknown samples & Regenerate biosensors Interpolate samples from standard curve to determine active concentration
Determine dilution factor A-H: PBS, 0-, 10- and 100-fold diluted fresh medium with PBS and 0-,10-,100- &1000-fold diluted. PBS as control while fresh medium and blank sup1 was diluted with PBS, and finally 100-fold diluted sup 1 was chosen based on a balance of matrix effect and sensitivity as showed above. Red curve was 100 fold diluted sup1.
Standard curve 1 in 100-fold diluted sup 1 Sensors regenerated 10 times. Dynamic range setup: 2000ug/ml-0.061ug/ml(4-fold dilution series) Spiked standard: 1000ug/ml ug/ml (4-fold dilution series) Unknowns: Dilute unknowns with diluted supernatant 1 Octet settings: 400rpm, 300s reading, 3-cycle regeneration with pH1.5 glycine
Unknowns interpolated from standard curve 1 with 15s reading compared to ELISA SampleELISA BLI Detected BLI CalculatedRe% as compared to ELISA unknown unknown unknown unknown unknown ~ means 100-fold diluted samples while original samples 1-16 were tested meanwhile.
Standard curve 2 in 100-fold diluted sup 1 Sensors regenerated 10 times. Dynamic range setup: 62.5ug/ml ug/ml(2-fold dilution series) Spiked standard: 50ug/ml ug/ml (4-fold dilution series) Unknowns: Dilute unknowns with diluted supernatant 1 Octet settings: 400rpm, 300s reading, 3-cycle regeneration with pH1.5 glycine
Standard curve 2 determined in 100-fold diluted sup 1 Analysis model: Linear point-to-point Effective Dynamic range setup: 62.5ug/ml ug/ml(2-fold dilution series) Spiked standard: 50ug/ml ug/ml (4-fold dilution series) Octet settings: 400rpm, 120s reading
Unknowns interpolated from standard curve 2 with 15s reading compared to ELISA 1-100~ means 100-fold diluted samples. SampleELISABLI DetectedBLI CalculatedRe% as compared to ELISA unknown unknown unknown unknown unknown
MethodELISAStd. curve 1Std. curve 2 Analysis model?4PL5PLLiner sample unknown unknown unknown unknown unknown Test Conditions?400rpm,15s400rpm,120s Dynamic Range?2000ug/ml ug/ml62.5ug/ml ug/ml Summary A std curve for high concentration sample detection was determined as std curve 1 as above. A std curve for low concentration sample detection was determined as std curve 2 as above.
Conclusion A dilution factor of 100 for sup 1(samples in sup1) was determined using PBS as control due to severe matrix effect of blank sup 1 as well as fresh medium. A std curve with dynamic range 2000ug/ml ug/ml (4-fold dilution series, unweighted 4PL/5PL analysis) was developed for high concentration samples under 400 rpm with 15s reading, and samples were interpolated. More sensitive method was developed under 400 rpm with 120s- 300s reading and the dynamic range was 62.5ug/ml ug/ml(2- fold dilution series, Linear point-to-point analysis). Sensors could be regenerated well in 10mM pH1.5 glycine buffer.
Crude sample detection: quantitation Case 1 Object: quantitate pro in supernatant Solution : Pro A sensor with regeneration steps.(1000rpm, ReadTime 120s) Matrix: supernatant from CHO cells without centrifuge Outcome: good data.
Method development with Pro A sensor Std. curve in buffer with spike Std. Curve obtained in buffer Original (ug/ml) Calculated (ug/ml,4 replicates) mean (ug/ml) SDCV%Re% Std. Spike Original (ug/ml) Calculated (ug/ml) Mean (ug/ml) SDCV%Re% (4 replicates)
Method development with Pro A sensor Std. curve in medium with spike Std. Curve obtained in medium Original (ug/ml) Calculated (ug/ml,4 replicates) mean (ug/ml) SDCV%Re% Std. Spike in medium Original (ug/ml) Calculated (ug/ml) Mean (ug/ml) SDCV%Re% 4 replicates
Method development with Pro A sensor regeneration Samples calculated with loaded Std. curve obtained in medium Regeneration Original (ug/ml) Calculated (ug/ml) Mean (ug/ml) SDCV%Re%Plate No. (duplicates) 10 times times times
Samples calculated with loaded Std. curve obtained in medium Regeneration Original (ug/ml) Calculated (ug/ml) Mean (ug/ml) SDCV%Re%Plate No. (duplicates) 40 times times '(22) times '(33) Method development with Pro A sensor regeneration
Crude sample detection: quantitation Case 2 Object: quantitate pro X in milk Solution : AHC with regeneration steps. Matrix: 100 fold dilution milk Outcome: good data.
RED96,R 2 = ELISA, R 2 =0.98 BLI vs ELISA Standard curve obtained in milk with Octet RED96 OriginalCaculatedMean SDCV%Re% ug/ml Sensor type: AHC with regeneration Matrix: 100 fold diluted milk 数据来自中国农业大学。
Sample ELISA （ mg/ml ） SD CV% Fortebio(mg/ml) SD CV% beestings month months months months Std. curve: ELISA R 2 =0.98; Fortebio R 2 = CV<10% ； Re% ： % BLI vs ELISA Sensor type: AHC with regeneration Matrix: 100 fold diluted milk 数据来自中国农业大学。
Assay Protocols for Increasing Sensitivity on the Octet ||-Capture : Analyte : Ab-Enz : 1) Substrate High Speed Mixing 2) anti-Ab-Mass Longer Incubation 2nd reagent PPT Substrate Last steps are measured on-line to obtain signal Longer incubation at the 1 st step allows signal amplification 1 st and 2 nd steps can be done off-line to shorten the steps ||-Capture : Analyte High Speed Mixing Longer Incubation sensor 1-Step ||-Capture : Analyte : Ab High Speed Mixing Longer Incubation 2nd reagent sensor2-Step sensor 3-Step 1 st Step : 2 nd Step : 3 rd Step Amplification Special Conjugate
Qualifying Concentration – Which Assay Method Fits Customer Need
Higher sensitivity Example : Clone Screening Using Sandwich Assay 2 nd reagent format does NOT require wash step simple and easy protocol YY Y YYY YYY 2: Anti-hIgG Ab 1: hIgG Anti-hIgG (fc) biosensor Anti-hIgG Ab 2 min incubation YYY hIgG; on Octet or offline longer incubation
2-Step Clone Selection assay using Octet QK (sensitivity down to 156 pg/mL of HIgG) ng/mL Run Condition - O/N rpm - 2 nd Ab = Special Conj.
ELISA Conversion Flow Chart Obtain Assay Requirements and Existing ELISA Format (customer input) Select Sensor Type (immobilization mode) Select Assay Format (sensitivity & throughput) Validating Assay Format Managing NSB & Matrix Effect Optimize Reagent Formulation Further optimization if not meeting the spec. (modifying configuration to increase specific signal and reduce NSB)
Summary Workflow Test for dilution factors Dilute standards and unknowns with diluted supernatant Enter sample information into software Bind Standard to pre-wetted biosensor Generate standard curve & Regenerate biosensors Bind known concentration samples to regenerated sensor Regenerate biosensors Bind unknown samples Interpolate samples from standard curve(different models & different time- windows) to determine active concentration Calculate CV%, Re% of standard curve and spiked samples and determine proper standard curve Optimization Shaking Speed( much higher more sensitive) Detection time(longer more sensitive) Regeneration pH(sometimes need scouting) Data Models(try different models)
Pall ForteBio 解决方案 Label-free Real time Fluidics-free Fast ， Accurate ， Easy.