High Precision
Streptavidin Biosensor (SAX)
Quantitation and Kinetic Assays
Technical Note
Scope
This Technical Note provides insight into the utilization of Octet®
High Precision Streptavidin (SAX)
Biosensors for analyte quantitationand kinetic characterization.
Keywords or phrases:
Octet®, Bio-Layer Interferometry,
BLI, protein-protein interaction,affinity analysis, quantitation assay,high precision Streptavidin, SAX,
method development, quality
control, ligand binding, drug
discovery, ligand immobilization

Executive Summary

Principle

Ligand binding assays that utilize solid surface chemistries
often deploy streptavidin as the molecule of choice for ligand
immobilization onto the solid-surface. Streptavidin’s biophysical properties enables it to form a high affinity noncovalent bond with biotin allowing for the tight immobilizationof biotinylated ligands. Sartorius’s High Precision Streptavidin
Biosensors (SAX) are developed to be used with Octet®
instruments and are qualified for applications in downstream
drug discovery and regulated environments. They are
QC-tested to meet our precision-controlled coefficient ofvariation (CV) specification of <4% and can used with any
biotinylated molecule for both kinetics and quantitation
assays where stringent precision requirements are in place.

Streptavidin is a 52.8 kDa protein purified from the bacterium
Streptomyces avidinii. Streptavidin homo-tetramers have an
extraordinarily high affinity to biotin. With a dissociation constant (kd) on the order of ~10-6 mol/L, the binding of biotin to
streptavidin is one of the strongest non-covalent interactions
known in nature. High Precision Streptavidin Biosensors aredesigned for direct capture of biotin-labeled proteins forboth kinetic characterization and quantitation measurementson all Octet® systems.

---Materials Required
Octet® instrument with Octet® BLI Discovery Softwareand Octet® Analysis Studio SoftwareHigh Precision Streptavidin (SAX) Biosensors: (Sartorius
part no. 18-5117 [tray]; 18-5118 [pack]; 18-5119 [case])
For all Octet® instruments: 96-well, black, flat bottom,
polypropylene microplate (Greiner Bio-One part no.
655209)Optional for Octet® RH16 instruments:
384-tilted well, black, flat-bottom, polypropylene microplate (Sartorius part no. 18-5080 [pack]; 18-5076 [case])
384-well, black, flat-bottom, polypropylene microplate(Greiner Bio-One part no. 781209)

Equilibration

Streptavidin

Loading

Biotin-Quantitation assays: Sample Diluent (Sartorius part no.
18-5028) for dilution of all samples. If undiluted crude
samples will be quantitated, a matching blank matrix is
required.
Kinetic assays: The SAX Biosensor is compatible witha wide range of buffers, although 1X Kinetics Buffer isrecommended. Dilute 10X Kinetics Buffer (Sartorius part
no. 18-5032) 1:10 with PBS, pH 7.4). Best results are obtained
when all matrices are matched as closely as possible.

Baseline

Anti-PSA Antibody

Association

Dissociation

PSA

Figure 1: Binding kinetics experiment using SAX Biosensors and PSA model binding pair. After an initial equilibration step in buffer, biosensors are dipped
into the solution of biotinylated anti-PSA antibody. A buffer baseline step is performed followed by association and dissociation of PSA protein analyte.

-

Tips for Optimal Performance2

Fully equilibrate all reagents, samples and calibrators(for quantitation assays) to room temperature prior to
sample preparation. Thaw frozen samples completely andmix thoroughly prior to use.
Hydrate the biosensors for a minimum of 10 minutes prior
to use. Hydrating the biosensors in a buffer consistent
with the buffer used throughout the assay is recommended.
Ensure that the Octet® instrument is turned on and thelamp is warmed to room temperature for at least 40 minutes
prior to starting the assay.
Set the sample plate temperature in Octet® BLI Discovery
Software by selecting File > Experiment >Set Plate Temperature…. Enter the desired temperature.
Sartorius recommends running assays at 30°C, using othertemperatures may require modifying the assay times discussed in this protocol. Set the default startup temperature(software versions 6.4 and later) by selecting File > Options.
Enter the desired temperature under “Startup”.

---

Quantitation assays only:
Match the matrix of the samples, standards, references,and hydration solution as closely as possible.
Perform a dilution study and a dynamic range study as
outlined in the Assay Optimization section.
Use a blank negative control in a matching matrix forreference subtraction. This is especially important when
optimizing accuracy and detecting low-concentration
analytes.

Quantitation Assays
Assay Optimization
High Precision Streptavidin (SAX) Biosensors can becustomized with any biotinylated molecule for quantitation
use in a simple and straightforward manner. Such customization
can be done by dipping SAX Biosensors into a solution
containing 20 μg/mL of biotinylated ligand for 300 – 600
seconds with shaking at 1000 rpm (actual optimal biosensor
coating parameters might vary and can be adjusted by user
depending on material or time requirements). The following
assays are recommended each time a new matrix or new
protein is analyzed.

---

Overview
Determine the minimal required dilution (MRD) factor
to achieve the targeted assay dynamic range.
Perform a spike | recovery study to determine assay
recovery.
Determine data analysis parameters.
Apply the finalized protocol and data analysis parametersin routine assays.
Dilution factor determination for matrix
Differences between matrices can potentially influence assay
performance. Diluting the sample matrix using Sartorius’
Sample Diluent is a convenient and often effective means ofminimizing matrix effects. It is therefore recommended
that the minimum required dilution factor be determinedusing Sample Diluent to achieve the desired assay performance.
1. Prepare 2 mL of sample matrix diluted both two-foldand 10-fold in Sample Diluent. General guidelines fordilutions are described in Table 1.

2. Set the assay temperature to run at 30°C by selecting
File > Experiment > Set plate temperature.
3. Prepare a spiked sample of the protein to be quantitatedin: Sample Diluent, neat matrix, two-fold diluted matrix
and 10-fold diluted matrix by mixing the minimum volume of analyte and 0.5 mL of each matrix (four samples
total). The final concentration of the protein should be in
the middle of the desired quantitation range.
4. Transfer each sample to a 96 or 384-well sample platein duplicate (eight wells total).
5. Hydrate biosensors in the sample matrix that matches
each sample type (e.g., biosensors to be used in wellswith 10-fold diluted matrix should be hydrated in 10-folddiluted matrix). Place the sample plate and the hydrated
biosensors into the Octet® instrument. Recommended
sample plate warm-up in the instrument and biosensor
hydration time is 10 minutes. The delay timer can beused in Octet® software to automatically start the assay
after 600 seconds.
6. Set up a Basic Quantitation assay according to theOctet® BLI Discovery Software User Guide.
7. Run the assay.
8. Data will be displayed in real time during the assay.
Data files, method files and assay pictures (.jpgs) will besaved automatically.
9. Load data into Octet® Analysis Studio Software.

Table 1: Recommended minimum dilution for common sample types.
In all cases, the matrix for the diluted samples, standards and biosensor
hydration solution should be matched as closely as possible.

10. Visually inspect the real-time binding traces and determine the dilution required to:Sample type

Minimum recommended
dilution in sample diluent

a. Minimize non-specific binding from the matrix.

Purified proteins

Dilute into assay range

Samples from column eluents

Dilute into assay range

b. Show equivalent analyte binding in the matrix-spiked
sample and the Sample Diluent control.

Serum-free cell culture supernatants media

Neat or 10-foldSerum-containing cell culture supernatants

Neat

Bacterial cell pellet lysed by sonication

10-fold

Bacterial cell pellet lysed by B-PER

20-foldc. Use this dilution factor for routine assays.

3

Spike Recovery Assay
To determine the dynamic range and data analysis
parameters suitable for a specific analyte, establish astandard curve and spike recovery as described below.
1. Prepare standards in the matrix using the dilution factor
determined in the prior dilution factor determination for
matrix experiment. The minimum volume needed ineach well varies with the plate used:
200 μL/well in a 96-well microplate(all Octet® instruments)
80 μL/well in a 384-well microplate(Octet® RH16 instruments)
40 μL/well in a 384-well, tilted-bottom microplate(Octet® RH16 instruments only)--

2. Using the same sample matrix from Step 1, prepare 700 μL
of two unknown samples. The concentration of thesesamples should be within the assay dynamic range.
3. Transfer triplicates of the standards and the unknowns
to a sample plate. It is recommended to organize samples in columns, from A – H. Fill at least one well withblank diluted matrix for reference subtraction during
data analysis. An example plate map is shown in Figure 2.
4. Hydrate biosensors in the matrix that matches the blankdiluted matrix. Place the sample plate and the hydrated
biosensors in the Octet® instrument. The recommended
sample plate warm-up in the instrument and biosensor
hydration time is 10 minutes. The delay timer can beused to automatically start the assay after 600 seconds.

11. Exclude data points for the standard curve that lie outside
the defined dynamic range.
12. Iteratively adjust the following processing parameters
and re-calculate the binding rate:
a. Adjust the read time window if necessary (typically120 seconds).
b. Adjust the zero concentration threshold if necessary(recommended 0.0001).
c. Adjust the low concentration threshold to 0.001(recommended).
d. Select the appropriate standard curve equation.
13. Evaluate the calculated concentration value of theunknowns by defining acceptable values of % recovery(accuracy) and % CV (precision). ± 15% recovery with a
10% CV are frequently used threshold values but mayvary depending on the requirements of each assay.
1

2

3

4

5

6

7

8

9

10

11

12

A

Standard

B

ReferenceC

UnknownD
E
F
G
H

5. Set up a Basic Quantitation assay using the assay
parameters used in the prior Dilution Factor Determination
for Matrix experiment. Availability of the pre-loaded
assay parameters will be software version-dependent.
For details on how to set up an assay, see the Octet®
BLI Discovery Software User Guide.
6. Run the assay. Data will be displayed in real time during
the assay. Data files, method files and assay pictures (.jpgs)
will be saved automatically.
7. Load the data into Octet® Analysis Studio Software.
8. If blank matrix was included as a reference, use the reference subtraction option to correct the data as appropriate.
9. Calculate the binding rate.
10. Define a dynamic range by selecting acceptable % CV
values for the lower and upper concentration limits. 10%
is routinely used as a threshold, but may vary dependingon the requirements of each assay.
4

Figure 2: Example plate layout for a routine assay run in a 96-well microplate.

Assay Protocol
1. Pre-load the biosensors with custom biotinylated ligand
which specifically recognizes the molecule to be analyzed.
2. Prepare samples, calibration standards and hydration
solutions according to the information in Table 1. The
minimum volume needed in each well varies with theplate used:
200 μL/well in a 96-well microplate(all Octet® instruments)
80 μL/well in a 384-well microplate(Octet® RH16 instruments)
40 μL/well in a 384-well, tilted-bottom microplate(Octet® RH16 instruments only)--3. Pipette standards, controls and samples into a black
polypropylene microplate (see Figure 2 for sample platelayout).
4. Pipette biosensor hydration solution into the wells of a
96-well, black, flat-bottom microplate corresponding to
the number and position of the biosensors to be used.
5. Place the biosensor tray with the hydration plate in the
Octet® instrument. Place the sample plate in the Octet®
instrument. Warm the sample plate in the instrument
and hydrate the biosensors for 10 minutes prior to starting
the experiment. The delay timer can be used to automatically start the assay after 10 minutes (600 seconds).

11. Define a dynamic range by selecting acceptable % CV
values for the lower and upper concentration limits. 10%
is routinely used as a threshold, but may vary dependingon the requirements of each assay.
12. Exclude data points for the standard curve that lie outside
the defined dynamic range.
13. Iteratively adjust the following processing parameters
and re-calculate the binding rate:
a. Adjust the read time window if necessary (typically120 seconds).
b. Adjust the zero concentration threshold if necessary(recommended 0.0001).

6. Set up a Basic Quantitation assay according to theOctet® BLI Discovery Software User Guide.
An example plate map is shown in Figure 2.
7. Run the assay. Data will be displayed in real time during
the assay. Data files, method files and assay pictures (.jpgs)
will be saved automatically.
8. Load data into Octet® Analysis Studio Software.
9. If blank matrix was included as a reference, use the reference subtraction option to correct the data as appropriate.
10. Calculate the binding rate.

A.

c. Adjust the low concentrations threshold to 0.001(recommended).
14. To export the analyzed data, use the Save Report button
to generate a Microsoft® Excel® report.
Representative Data
Figure 3 shows detection of a hIgG standard using High
Precision Streptavidin (SAX) Biosensors on the Octet®
RH16 system with assay parameters (1000 rpm for
2 minutes; 15 minutes run time) for a standard dynamic
range. Figure 3A shows the hIgG dose response andFigure 3B the resulting calibration curves. Sample diluent
was used as a matrix for all samples. See Table 2 for thestatistical analysis of data from Figure 3.

B.

3.0

25

20

Calc Conc.

Binding Rate (nm)

2.5

2.0

1.5

10

1.0

5

0.5

0

15

500

600

700

800

900

1000

Time (sec)

1100

1200

1300

0.02

0.04

0.06

0.08

Binding Rate

Figure 3: Detection of a hIgG standard using High Precision Streptavidin biosensors (SAX) on the Octet® RH16 system with assay parameters(1000 rpm, 2 min; as shown 15 min) for a standard dynamic range. A) hIgG dose response. B) Representative resulting calibration curves from A).
Sample diluent was used as a matrix for all samples.

5

4. Place the biosensor tray with the hydration plate in the
Octet® instrument. Place the sample plate in the Octet®
instrument. Warm the sample plate in the instrument
and hydrate the biosensors for 10 minutes prior to starting
the experiment. The delay timer can be used to automatically start the assay after 10 minutes (600 seconds).

Table 2: Average calculated concentration and % CVs for hIgG (n=15).
hIgG
Expected conc.
(μg/mL)

Avg. conc. μg/mL(N=15)% CV(N=15)

24

24.3

6.0%

12

12.1

5.8%

6

6.0

4.1%

3

3.0

4.5%

1.5

1.5

2.8%

0.75

0.75

1.6%

5. Set up a Basic Quantitation assay according to theOctet® BLI Discovery Software User Guide. See Table 3
for assay setup parameters, and Figure 4 for example
plate maps.
6. Run the assay. Data will be displayed in real time during
the assay. Data files, method files and assay pictures (.jpgs)
will be saved automatically.

Assay Protocol-Precision
1. Prepare biotin-Protein A conjugate (1 μg/mL), buffer,hIgG (6 μg/mL), and hydration solutions according to
the information in Table 3.

7. Load data into Octet® Analysis Studio Software.
8. Calculate the nm shift of biotin-Protein A and hIgG.
Representative Data
Experimental raw data showing 96 replicates of the biotinProtein A conjugate quantitative precision assay are shownin Figure 5.

2. Pipette standards, controls and samples into a black
polypropylene microplate (see Figure 4 for a sample
plate layout).
3. Pipette biosensor hydration solution into the wells of a
96-well black flat-bottom microplate corresponding to
the number and position of the biosensors to be used.
Table 3: Precision assay setup parameters.
Step

Step name

Time (s)SampleShake speed

Step type1

Equilibration

120

3

1000

Custom2

Loading

300

1

1000

Loading3

Baseline

60

3

1000

Baseline

4

Association

900

2

1000

Association

1

2

3

4

5

6

7

8

9

10

11

12

1

A

A

B

B

C

C

D

D

E

E

F

F

G

G

H

H

2

3

Figure 4: Sample and biosensor plate layout (left and right respectively) for the precision assay.

6

4

5

6

7

8

9

10

11

12

A.

B.

4.5
4.0

1.6
1.4

Binding Rate (nm)Binding Rate (nm)

3.5
3.0
2.5
2.0
1.5

1.0
0.8
0.6
0.4

1.0

0.2

0.5

0

0
0

200

400

600

800

1000

1200

150

200

250

300

350

400

Time (sec)Time (sec)

C.

1.2

2.5

Binding Rate (nm)

2.0

1.5

1.0

0.5

0

500

600

700

800

900

1000

1100

1200

1300

Time (sec)Figure 5: (A) Full experimental raw data showing the same steps measured on 96 replicates. (B) 96 replicates of the second step in the measurement,
biotinylated Protein A loading onto 96 SAX Biosensors. (C) 96 replicates of the 4th step in the measurement, hIgG binding to Protein A.

Table 4: Biotin-Protein A and hIgG precision % CV (n=96).
n=96Biotin-Protein A (1 μg/mL)

hIgG (6 μg/mL)

Mean

1.53

2.58

SD

0.04

0.05

% CV

2.6%

1.8%

7

Kinetic Assays------Assay Protocol
Overview
Prepare assay solutions
Prepare the sample plate
Equilibrate both the hydrated biosensor assembly andthe assay plate for 10 minutes on the Octet® instrument
Run the assay
Process and analyze the data
Save the results-

Prepare the Samples and the Calibration Standards
Equilibrate reagents and samples to room temperature
prior to preparation and mix thoroughly.
The PSA model binding pair provides a reliable, consistentantibody-antigen model binding pair for use in binding
kinetics assays on the Octet® platform. This pair consistsof an anti-human PSA mouse monoclonal antibody andhuman PSA (Prostate Specific Antigen, molecular weight
30 kDa). The affinity of the interaction between the two
proteins is in the low nanomolar range and follows a 1:1
binding kinetics model. This binding pair can be used with
any Octet® instrument and the new High Precision
Streptavidin (SAX) Biosensor.
Biotinylate ligand that specifically binds to the analytethat will be analyzed. Biotinylate according to the protocol
outlined in Technical Note 6, Biotinylation of Protein for
Immobilization onto Streptavidin Biosensors using either
biotin-LC-LC-NHS (Pierce, part no. 21343) or biotin-PEO4NHS (Pierce, part no. A39259). The long linkers in these
two reagents typically produce good quality assays.
Dilute the biotin-anti-PSA antibody ligand to 50 μg/mL
by adding 70 μL of the stock antibody solution to 1330 μL
of 10X Kinetics Buffer (Sartorius part no 18-5032).
Prepare 400 μL of the PSA antigen solution. Add 4 μL of
the PSA antigen to 396 μL 10X Kinetics Buffer to make a
200 nM solution. Mix gently. This solution will be the top
concentration in a dilution series of PSA antigen.

Perform four serial two-fold dilutions of the PSA antigen
solution: add 200 μL of 200 nM PSA antigen solution
from Step 2 to 200 μL 10X Kinetics Buffer to make a 100 nM
solution, mixing gently. Repeat this dilution to make 50 nM,
25 nM, and 12.5 nM PSA solutions.

Running the Assay
1. Ensure that the Octet® instrument is turned on and thelamp has been warmed to room temperature for at least
40 minutes prior to starting the assay.

-

2. Pipette 200 μL/well of biosensor hydration solution
into the wells of a 96-well black, flat-bottom microplate
corresponding to the number and the positions ofbiosensors to be used.
1

2

3

4

5

6

7

8

9

10

11

12

A

200

Biotin-αPSA 50 µg/mL

B

100

PSA (nM)

C

50

10X Kinetics buﬀer

D

25

Reference sample

E

12.5

Empty

F

0

G
H

Figure 6: Sample kinetic assay plate setup.

Table 5: Kinetic assay setup parameters.
Step

Step name

Time (s)SampleShake speed

Step type1

Equilibration

60

3

1000

Custom2

Loading

600

1

1000

Loading3

Baseline

120

3

1000

Baseline

4

Association

300

2

1000

Association

5

Dissociation

900

3

1000

Dissociation

8

4. Transfer 200 μL of each assay reagent into the appropriate wells of a black polypropylene microplate. Place thebiosensor hydration assembly in the Octet® instrumenton the left stage. Ensure that both the biosensor tray
and sample plate are securely in place.
5. Equilibrate the plates in the instrument for 10 minutes
prior to starting the experiment. The delay timer can beused to automatically start the assay after 10 minutes(600 seconds).
6. Set up a kinetic assay. For details, see the Octet® BLI
Discovery Software User Guide. Table 5 shows anexample kinetic assay consisting of equilibration, ligand
loading, baseline, association and dissociation steps.
Figure 6 shows an example plate map for a typical kinetic
assay.
7. Run the assay.

A.

4.0
3.5
3.0

Binding Rate (nm)3. Insert the hydration plate into the biosensor tray. Alignthe biosensor rack over the hydration plate and lower
the biosensors into the wells.

Representative Data
Figure 7 shows the kinetic analysis of the interaction
between ligand biotin-anti-PSA (150 kDa) and an analyte
PSA (30 kDa). High Precision Streptavidin Biosensors (SAX)
were hydrated for 10 minutes in 1X Kinetics Buffer prior to
analysis. Assays steps included: 1 minute of equilibration,
10 minutes of biotin-anti-PSA loading (50 μg/mL), 2 minutesof baseline stabilization, 5 minutes of ligand:analyte
association and 15 minutes of ligand:analyte dissociation.
Analyte concentrations were 0, 12.5, 25, 50, 100 and 200 nM.
1X Kinetics Buffer was used as the matrix throughout andthe assay temperature was 30°C. Data was analyzed using
the data using Global (Full) fitting, specifying a 1:1 binding
model. Examples of raw and fitted data are shown in Figure
7A and 7B.

Process and Analyze the Data
1. Load data into the Octet® Analysis Studio Software.

2.5
2.0
1.5
1.0
0.5

2. Process the data by specifying methods for reference
subtraction, y-axis alignment, inter-step correction and
Savitzky-Golay filtering.

4. To export the analyzed data, use the Save Report button
to generate a Microsoft® Excel® report.
Note: For details on processing and analysis parametersand data exporting, refer to the Octet® Analysis Studio
Software User Guide.

0

200

400

600

800

1000

1200

1400

1600

1800

Time (sec)

B.
0.7

0.6

Binding Rate (nm)3. Analyze the data by specifying steps for analysis, fitting
method (local or global) and window of interest.

0

0.5

0.4

0.3
0.2

Table 6: Affinity characterization between biotin anti-PSA and PSA
shown in Figure 7.
n=12

KD (M)kon(1/Ms)kdis(1/s)

Mean

6.08E-09

5.84E+04

3.55E-04

Error

6.10E-12

4.42E+01

2.34E-07

0.1

0

0

50

100

150

200

250

300

350

Time (sec)Figure 7: Kinetic analysis of the interaction between ligand biotin-anti-PSA(150 kDa) and an analyte PSA (30 kDa). 1X Kinetics Buffer was used as
the matrix throughout and the assay temperature was 30°C. Data were
processed and curve fit using a 1:1 binding model. The kinetic results arereported in Table 6 (n=12).

9

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Otto-Brenner-Strasse 20
37079 Goettingen
Phone +49 551 308 0F or further information, visit
www.sartorius.com/octet-support

Specifications subject to change without notice.
Copyright Sartorius Lab Instruments GmbH & Co. KG.
For Research Use Only.
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Phone +1 888 OCTET 75
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