4 Sandwich ELISA Assay Procedure Typical read out could be fluorescence or luminescenceBottom of wellPlate is coated with a capture antibodySample is added, and any antigen present binds to capture antibodyDetecting antibody is added, and binds to antigenEnzyme-linked secondary antibody is added, and binds to detecting antibodyChromogen/substrate is added, and is converted by enzyme to detectable formEach 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%.
5 Intensity λ =ƒ(λ, ℓ) Biolayer Interferometry（BLI） 可实时检测到两个反射表面间距的改变 Relative IntensityWavelength (nm)100%Distance between the two reflecting surfaces = ℓThe Octet will monitor change in wavelength shift over time. This real-time binding measurement can be used to calculate on and off rates, and ultimately concentration by plotting rates against concentration.Octet可以实时检测由于表面厚度变化带来的波长偏移，这种实时检测到的变化可以用来计算结合及解离的速度，并且最终得到相应的浓度变化nm shiftIntensity λ =ƒ(λ, ℓ)Time
6 Biosensor selection According to application & sample type etc. 新开发了四种传感器Anti-GST Biosensor: 用于含GST标签的蛋白NTA Biosensor：用于含His标签的蛋白Anti-human Fab-CH1：用于人Fab, F(ab’)2及Ab1~4Anti-Flag biosensor：用于含Flag标签的蛋白
7 Octet Workflow for Quantitation Anti-human IgG (Fc specific), anti-murine IgG (Fab’ specific), or Protein A sensorsStandard samples of known concentration used to generate a standard curveUnknown samplesHow does it work? This is the tip of a fiber. We coat the tip end with a special optical layer. The capturing molecule is then attached to the tip. The tip is dipped into the sample containing target molecule. The target molecule binds to the capture molecule, and two form a molecular layer. A white light is directed into the fiber. Two beams will be reflected to the back end. The first beam comes from the tip as a reference. The second light comes from the molecular layer. The difference of two beams will cause a spectrum color pattern as shown here. The phase is a function of the molecular layer thickness and corresponding to the number of molecules on the tip surface.
8 Octet Automated Workflow for Quantitation Anti-Human IgG, Anti-Murine or Protein A Biosensors CalibrantsBinding (nm)Test SamplesTimeCalibrants used to plot a binding rate vs conc. calibration curveThe binding rates of test samples are then measured and plotted on the calibration curve to determine their concentrationOne sensor per one sample well; one step assay with pre-made sensors.Binding RateConcentration
9 Octet Workflow for Quantitation with Regeneration Octet BiosensorsStandardsBinding (nm)Test SamplesTime (sec)120RegenBuffer/Neut.The binding rates of test samples are measured and interpolated from the standard curve to determine concentration96 samples analyzed in minutesReuse of standard curve is optionalBinding RateConcentration9
10 Lab Work Example – Quantitation Experiment Set up the plate shown below:Analyze using Protein A Biosensors and running the standard Protein A ProtocolRefer to Quick Start Q Assay pdf for further details of running Quantitation AssaysGenerate a Quantitation ReportCalculate CVs of each of the 8 replicates of calibrators123456789101112A700500…Glycine pH2.0SDBC300D30100EFGHCalibratorsSamplesRegenerationNeutralization
11 Quantitation – Real-time binding curves Rate of binding correlates to concentration Octet Binding Curve = rate of increase in optical thickness as the sample binds to the sensor.Different protein concentrations result in different binding curvesRate of binding is proportional to concentration700 ug/mL500 ug/mL300 ug/mL100 ug/mL30 ug/mL10 ug/mL3 ug/mL1 ug/mLThe amount of IgG in a sample is determined by the initial rate of binding of the antibody to either the anti-human IgG or Protein A biosensor.The graph here shows the rate of association of IgG from 1ug/ml to 700ug/ml
13 Fc-fused protein Quantitation Overview Samples: 14 hFc-fused proteins in supernatant 1Standards: Fc-fused proteins with original conc. of 92.09mg/mlBiosensors : Protein A biosensorOctet platform: RED96Other 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
14 Workflow for the Fc-fused protein quantitation Test for dilution factorsDilute standards and unknowns with diluted supernatantEnter sample information into softwareBind Fc-fused protein to protein A biosensorGenerate standard curve & Regenerate biosensorsBind known concentrations of Fc-fused proteins to regenerated sensorBind unknown samples & Regenerate biosensorsInterpolate samples from standard curve to determine active concentration
15 Determine dilution factor Red curve was 100 fold diluted sup1.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.
16 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 1Octet settings: 400rpm, 300s reading, 3-cycle regeneration with pH1.5 glycine
18 Standard curve 1 in 100-fold diluted sup 1 Std.curveSpiked Std.Theo.con.DetectedCalculatedMeanSDCV%Re%20002081.62029.42005.21891.5500504.1493.6501.2501.6125122.6124.8599.88125.2124.612731.2531.331.299.84317.81259.269.3751209.479.31.95312.762.7352.772.632.780.24410.07440.14770.09480.1240.0610.15670.1974UndefinedSpiked std.Theo.con.DetectedCalculatedMeanSDCV%Re%ug/ml Fc-pro0.97661.481.51251.581.430.9766ug/ml Fc-pro1.563.9063ug/ml Fc-pro3.90635.685.6755.735.645.6515.625ug/ml Fc-pro15.62517.917.75113.617.817.562.5ug/ml Fc-pro62.561.460.696.9658.56161.5250ug/ml Fc-pro250294.3117.95297.5293.72941000ug/ml Fc-pro10001124.611181137.81168.1
19 Re% as compared to ELISA Unknowns interpolated from standard curve 1 with 15s reading compared to ELISASampleELISABLI DetectedBLI CalculatedRe% as compared to ELISA1-100872.89.829821726.42-100122011.411402700.13-100unknown10100031168.14-10070411.311304791.15-1001060.812.312305807.66-10013.713706823.37-100111100156.257819.58-10011.611608851.39-1001280.814.414409779.810-10010.71070720.411-10011.2112012-100110815-1003.2832816-1003.363361-100~ means 100-fold diluted samples while original samples 1-16 were tested meanwhile.
20 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 1Octet settings: 400rpm, 300s reading, 3-cycle regeneration with pH1.5 glycine
21 Standard curve 2 determined in 100-fold diluted sup 1 Analysis model: Linear point-to-pointEffective 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
22 Standard curve 2 in 100-fold diluted sup 1 Theo.con.DetectedCalc.con.MeanSDCV%Re%62.563.80.88710062.261.831.2530.131.2250.86699.9231.4220.127.116.11515.90.31015.815.615.27.81257.237.77250.37099.4887.918.067.893.953.90750.0483.933.843.911.851.95250.08099.96822.031.930.9950.97810.0390.96830.92911.020.44080.0320.50360.50070.50790.24560.24440.0160.25210.22110.25880.08040.03727.3880.14410.16430.14430.06390.0140.04430.03890.06720.02980.0210.02640.06510.01640.03330.0090.01520.01480.0242Standard curve 2 in 100-fold diluted sup 1Std.curveSpiked Std.Spiked std.Theo.con.DetectedCalculatedMeanSDCV%Re%50ug/ml Fc-pro5059.259.15118.359.112.5ug/ml Fc-pro12.514.213.914.43.125ug/ml Fc-pro3.1253.483.313.49ug/ml Fc-pro0.85710.88180.9034ug/ml Fc-pro0.23420.31340.279
23 Re% as compared to ELISA Unknowns interpolated from standard curve 2 with 15s reading compared to ELISASampleELISABLI DetectedBLI CalculatedRe% as compared to ELISA1-100872.89.729722-100122010.310303-100unknown9.319314-10070411.111105-1001060.811.311306-10010.910907-10010.810808-10012.29-1001280.810-10011-10010.5105012-100110810.1101015-1003.2932916-1002.742741-100~ means 100-fold diluted samples.
24 SummaryMethodELISAStd. curve 1Std. curve 2Analysis model?4PL5PLLinersample1872.8982945972212201140110010303unknown1000967931470411301090111051060.81230118061370133071060108081160112091280.81440139010107011105012110810101532830832916336316274Test Conditions400rpm,15s400rpm,120sDynamic Range2000ug/ml ug/ml62.5ug/ml ug/mlA 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.
25 ConclusionA 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.
26 Crude sample detection: quantitation Case 1 Object: quantitate pro in supernatantSolution : Pro A sensor with regeneration steps.(1000rpm, ReadTime 120s)Matrix: supernatant from CHO cells without centrifugeOutcome: good data.
27 Std. Curve obtained in buffer Method development with Pro A sensorStd. curve in buffer with spikeStd. SpikeOriginal (ug/ml)Calculated (ug/ml)Mean (ug/ml)SDCV%Re%(4 replicates)10.83820.840.010.7783.720.84170.82780.841254.224.180.040.9083.604.134.19201717.680.502.8288.3818.117.618Std. Curve obtained in bufferOriginal (ug/ml)Calculated(ug/ml,4 replicates)mean (ug/ml)SDCV%Re%100.00100.4100.610198.1100.031.3133.3333.418.104.22.16899.9811.1111.211.310.9122.214.171.1243.703.73.743.693.710.020.64126.96.36.199.261.190.032.52100.410.410.42010.41580.39730.41480.012.44100.920.140.13750.13770.1360.000.58100.13
28 Std. Curve obtained in medium Method development with Pro A sensorStd. curve in medium with spikeStd. Spike in mediumOriginal (ug/ml)Calculated (ug/ml)Mean (ug/ml)SDCV%Re%4 replicates10.880.870.011.1586.810.8654.434.470.071.5489.404.464.424.572018.6018.580.291.5592.8818.2018.90Std. Curve obtained in mediumOriginal (ug/ml)Calculated(ug/ml,4 replicates)mean (ug/ml)SDCV%Re%100.00101.90100.6097.501.8533.3332.9033.6033.7033.2033.350.371.11100.0611.1111.1011.2011.130.050.45100.233.703.693.730.020.51100.071.241.211.231.261.69100.410.410.400.420.012.69101.610.140.130.001.56100.15
29 Samples calculated with loaded Std. curve obtained in medium Method development with Pro A sensorregenerationSamples calculated with loaded Std. curve obtained in mediumRegenerationOriginal (ug/ml)Calculated (ug/ml)Mean (ug/ml)SDCV%Re%Plate No.(duplicates)10 times33.3032.2032.050.210.6696.25131.9011.1010.5010.651.9995.9510.803.333.260.0097.901.111.091.100.010.6598.6520 times31.8031.500.421.3594.59431.2010.3010.550.353.3595.053.290.041.2998.803.321.130.63101.351.1230 times31.250.782.4993.84230.700.282.6910.703.233.250.030.8797.603.2799.10
30 Samples calculated with loaded Std. curve obtained in medium Method development with Pro A sensorregenerationSamples calculated with loaded Std. curve obtained in mediumRegenerationOriginal(ug/ml)Calculated (ug/ml)Mean (ug/ml)SDCV%Re%Plate No.(duplicates)40 times33.3031.5030.751.063.4592.34330.0011.1010.1010.350.353.4293.2410.603.333.183.220.051.5496.5188.8.131.52.010.63100.4550 times30.9030.300.852.8090.992'(22)29.709.8510.080.323.1690.7710.300.020.6795.353.191.071.080.6696.8560 times29.600.993.3488.893'(33)28.909.699.950.363.6389.5910.203.113.151.5794.441.101.2999.101.09
31 Crude sample detection: quantitation Case 2 Object: quantitate pro X in milkSolution : AHC with regeneration steps.Matrix: 100 fold dilution milkOutcome: good data.
32 Standard curve obtained in milk with Octet RED96 BLI vs ELISAELISA, R2=0.98RED96,R2=Standard curve obtained in milk with Octet RED96OriginalCaculatedMeanSDCV%Re%ug/ml10099.5100.5100.000.715052.148.750.402.404.77100.802525.724.325.000.993.9612.513.811.412.601.703.476.2184.108.40.206.3220.127.116.110.247.681.561.821.371.600.329.95102.24Sensor type: AHC with regenerationMatrix: 100 fold diluted milk数据来自中国农业大学。
33 BLI vs ELISA Sample ELISA（mg/ml） SD CV% Fortebio(mg/ml) SD CV% beestingsmonthmonthsmonthsmonthsStd. curve: ELISA R2=0.98; Fortebio R2=CV<10%；Re%：84-118%Sensor type: AHC with regenerationMatrix: 100 fold diluted milk数据来自中国农业大学。
34 Assay Protocols for Increasing Sensitivity on the Octet 1st Step : 2nd Step : 3rd Step Amplification1-Stepsensor||-Capture : Analyte High Speed MixingLonger Incubation2-Stepsensor||-Capture : Analyte : Ab High Speed MixingLonger Incubation2nd reagent3-Stepsensor||-Capture : Analyte : Ab-Enz : 1) Substrate High Speed Mixing2) anti-Ab-Mass Longer Incubation2nd reagentPPT SubstrateLast steps are measured on-line to obtain signalLonger incubation at the 1st step allows signal amplification1st and 2nd steps can be done off-line to shorten the stepsSpecial Conjugate
35 Qualifying Concentration – Which Assay Method Fits Customer Need
36 Example : Clone Screening Using Sandwich Assay Higher sensitivityExample : Clone Screening Using Sandwich AssayY2: Anti-hIgG Ab1: hIgGAnti-hIgG (fc) biosensorAnti-hIgG Ab 2 min incubationhIgG; on Octet or offline longer incubation2nd reagent format does NOT require wash step simple and easy protocol36
37 2-Step Clone Selection assay using Octet QK (sensitivity down to 156 pg/mL of HIgG) Run ConditionO/N500 rpm2nd Ab = Special Conj.ng/mL1052.51.250.6250.3130.156
38 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 FormatManaging NSB & Matrix EffectOptimize Reagent FormulationFurther optimization if not meeting the spec.(modifying configuration to increase specific signal and reduce NSB)
39 Summary Workflow Test for dilution factors Dilute standards and unknowns with diluted supernatantEnter sample information into softwareBind Standard to pre-wetted biosensorGenerate standard curve & Regenerate biosensorsBind known concentration samples to regenerated sensorRegenerate biosensorsBind unknown samplesInterpolate samples from standard curve(different models & different time-windows) to determine active concentrationCalculate CV%, Re% of standard curve and spiked samples and determine proper standard curveOptimizationShaking Speed( much higher more sensitive)Detection time(longer more sensitive)Regeneration pH(sometimes need scouting)Data Models(try different models)
40 Label-free Real time Fluidics-free Fast，Accurate，Easy. Pall ForteBio解决方案Label-freeReal timeFluidics-freeFast，Accurate，Easy.