CHO Host Cell Protein Detection

Technical Note
Keywords and phrases
Host Cell Proteins, CHO Cells,
CHO HCP detection, HCP
contaminants, Impurities, Octet
Biosensors, Octet® Anti-CHO
HCP Kit

Abstract
The Octet® Anti-CHO HCP Kit has been designed for a rapid high-throughput quantitation of residual CHO Host Cell
Proteins (HCPs) in bioprocessing samples during production and development of biopharmaceuticals. The kit utilizes
the industry-standard Cygnus 3G Anti-CHO HCP antibody and enables assays with broad HCP recognition, high precision(5-10 %CV) and high sensitivity (detection limit as low as 0.5 ng/mL). These features, combined the Octet® platform’s
real‑time, label-free high-throughput analysis capability, make the Octet® Anti-CHO HCP kit a superior alternative to
ELISA for HCP detection. The Octet® RH96 system allows for a completely hands-off, walk-away HCP analysis of
96 samples in less than one hour, which is three times faster than the manual and labor-intensive ELISA platform.

Find out more: www.sartorius.com

Contents
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Assay Accuracy and Specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Materials Required. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Storage and Stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Assay Matrix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Octet® RH96 System HCP
Assay Protocol: A Completely Walk‑Away Assay . . . . . . . . . . . .  4
Prepare Samples and Detection Plates . . . . . . . . . . . . . . . . . . .  4
Run the Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Analyze Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Octet® RH16 and R8 System HCP Assay Protocol with
Octet® AS Station. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Process samples on the Octet® AS Station. . . . . . . . . . . . . . . .  6
Example Data from a Routine Assay . . . . . . . . . . . . . . . . . . . . . .  6
Run the Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Analyze Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Example Data from a Routine Assay . . . . . . . . . . . . . . . . . . . . . .  9
Assay Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10

Overview
Host cell proteins (HCPs) are contaminants found inbiopharmaceuticals expressed in bacterial, yeast or
mammalian production cell lines. Among protein
expression cell lines, Chinese hamster ovary (CHO)
cells are the most commonly used mammalian hosts
for industrial production of recombinant protein
therapeutics. However, manufacturing and production
processes of biopharmaceuticals often leave behindcontaminating HCPs from CHO cells. Such residual
HCPs carry substantial risk of decreasing efficacy of the
drug and causing adverse immunogenic reactions inpatients. Hence, the detection of residual host cell
protein contaminants and methods that reduce themto the lowest acceptable levels have become criticalaspects of drug safety and qualification.
HCP analyses can be broadly classified into two
categories, generic assays and process-specific assays.
Commercially available generic assays are intended todetect HCPs that might contaminate a productindependent of downstream purification processes.
These assays normally use HCPs obtained from far

2

upstream (e.g., conditioned media) or after some minimallyselective purification step (e.g., clarification or filtration) as
an antigen to generate broadly-reactive polyclonal
antibodies for detection. They are very useful when most
HCPs are conserved among related strains and processes.
On the other hand, process-specific assays are thosederived from an immunogen which is specific to a defined
purification process. These assays are tailor-made for an
established process and thus have the potential to be more
specific to the panel of HCPs produced within suchprocesses. However, high specificity for typical HCPs
occurring within the process carries the risk that any
atypical HCPs arising due to unintended and undetected
deviations in a purification process may go undetected —potentially leading to substantial setbacks in the drug
development timeline.
The vast majority of process-specific assays are developed
only after a process is set and defined, which typicallyoccurs at the later stages of drug development. Detection
antibodies for process-specific assays also require
substantial time and cost to develop. Since the FDA
generally does not require process-specific HCP analyses
until phase III trials, generic assays are typically employedfor overall HCP detection until a drug candidate survives
the initial phases of clinical trials. Even after a processspecific assay is developed and implemented, genericassays can still be used routinely to complement processspecific testing to maximize detection of any potential
atypical HCPs. Because of all these factors, generic assays
will always remain the most easily assessable and highly
useful tools in any drug development program.

Anti-CHO HCP Biosensor
Cygnus 3G anti-HCP

HCP
Fluorescein-tagged
anti-HCP

HRP anti-tag conjugate
HRP

HRP

HRP

HRP

HRP
Metal DAB

Precipitation

Figure 1: Biosensor-based assay format for the detection of CHO HCP.

Principle
Among existing HCP analytical methods, ELISA is perhaps
the more commonly used analytical method. Western blots
and SDS-PAGE are also used, but are limited by their qualitative nature and lack of quantitation sensitivity.
There are several inherent problems with ELISA, stemmingfrom its reliance on highly manual processing steps that
introduce variability in measurement, multiple timeconsuming incubation steps, and reliance on colorimetricor fluorescent probes that can yield false positive signals.
Sartorius’ Octet® Platform provides a superior alternative to
ELISA with improved precision in measurements, better or
equivalent sensitivity and dynamic range, low userintervention, rapid assay development enabled by real-timemonitoring, and much faster time-to-results. The platform isused for generic CHO HCP assays in early phases of clinicalproduct development, and process-specific CHO HCP
assays can also be constructed using the same assay format
if a process-specific antibody has been developed.
This Technical Note outlines a protocol for using theOctet® Anti-CHO HCP Kit in developing and routine
running of process-independent assays on the Octet®
platform. The measurement involves a sandwich-type assay
on an Anti‑CHO HCP Biosensor that is pre-coated with thegold‑standard 3G Anti-CHO HCP antibody from Cygnus
Technologies (Figure 1). A completely hands-off, walk-away
HCP assay analyzing 96 samples can be set up to run
automatically on an Octet® RH96 instrument with results
obtained in one hour. The assay can also be run on other
8‑and 16-channel Octet® instruments together with theOctet® AS Station with time-to-results of 75 and90 minutes, respectively.

Assay Accuracy and Specificity
In certain cases, the drug products themselves orcomponents in the formulation buffer may interfere withthe assay’s ability to detect HCPs. Also, different lots of thesame anti-CHO HCP antibody might give results that are
2-4X different, whereas different generations of the same
antibody might produce results that are 10-20X different.
The expected trend of results in accordance with theprocess stage (decreasing HCP readings with further
purification) is considered more important than theabsolute HCP values. Factors such as extremes in pH,
detergents, organic solvents, high protein concentration,and high buffer salt concentrations are all potential

interference factors. Also, different lots of the same antiCHO HCP antibody might give results that are 2-4X
different, whereas different generations of the same
antibody might produce results that are 10-20X different.
The expected trend of results in accordance with theprocess stage (decreasing HCP readings with further
purification) is considered more important than theabsolute HCP values. It is therefore necessary to validateby established experimental procedures (i.e. ICH and FDA
guidelines) that the assay results will be accurate. We
recommend users perform two critical experiments inestablishing assay accuracy and specificity: spike recovery
and dilutional linearity (please also refer to “Assay Matrix” onpage 4). If it is determined that there is significant
product or matrix interference in the assay, further dilution
or buffer exchange of the product to render it into a moreassay compatible buffer might be necessary. The same
diluent used to prepare the kit standards is ideally thepreferred material for dilution or buffer exchange of yoursamples. For each sample type to be tested, users should
demonstrate that the assay can recover added HCP orother contaminants spiked into that sample matrix. Thiscan be performed by spiking the highest standard
provided with the kit into your sample types and thentesting in the assay.

---------

Materials Required
Octet® RH16, RH96 or R8 instrument with Octet® BLI
Discovery and Analysis Studio Software version 8.1 or later.
Octet® AS Station for high throughput assays if using
Octet® RH16 or R8 instrument (not needed with theOctet® RH96 instrument).
Black polypropylene 96-well or 384-well microplates(Greiner Bio-One part no. 655209 or 781209).
Volume of samples to be analyzed (including positive andnegative controls): 80 µL (384-well microplate) or 200 µL(96-well microplate).
Octet® Anti-CHO HCP Kit (PN 18-5141), containing
the following:
Part 1: Biosensors for Host Cell Protein Detection
Part 2: Reagents A for Host Cell Protein Detection
Octet® CHO Standard, 48 µL, 20 µg/mL
Octet® Fluorescein anti-CHO, 240 µL, 100X concentrate
Octet® Anti-FITC HRP, 480 µL, 50X concentrate
Octet® Sample Dilution Buffer, 3 x 50 mL
Part 3: Reagents B for Host Cell Protein Detection
Octet® DAB/Metal Concentrate, 2.4 mL,
10X concentrate
Octet® Peroxide Substrate Buffer (1X), 46 mL

3

-

Storage and Stability
Anti-CHO HCP Reagents A should be stored at 4°C. The
Octet® CHO Standard can be stored short-term up to one
month at 4°C and long-term at -20°C. More than threefreeze-thaw cycles should be avoided.
Anti-CHO HCP Reagents B - Upon arrival, Octet®
Peroxide Substrate Buffer should be stored at 4°C
and Octet® DAB/Metal Concentrate should be stored
at -20°C.
Anti-CHO HCP biosensors should be stored atroom temperature.

Assay Matrix
Differences between matrices can potentially influence
assay performance. Diluting the sample matrix using Octet®
Sample Dilution Buffer with Kathon is an effective way tominimize matrix effects. Prior to running the assay, we recommend an optimization step where samples are diluted
with varying amounts of Sample Diluent in order to determine the minimum dilution factor required for optimal assay
performance. It is also important to test the dilution linearityof the sample to ensure no interference from matrix.

with all local, state, and federal guidelines. Proper personalsafety measures should also be taken when handling hazardous materials.
1.

Equilibrate the samples, reagents and buffers to room
temperature and mix thoroughly prior to use. Metal
Enhanced DAB concentrate should remain at -20°C
until immediately before use.
2. Prepare each concentration of HCP calibration
standard in Octet® Sample Dilution Buffer. The
calibration standards selected should cover the HCP
assay range from 0.5–200 ng/mL, and therecommended calibrator concentrations are 0.5, 1, 2, 8,
25, 75, and 200 ng/mL. It is also recommended to run
buffer-only controls as references. A calibration curveshould be included in each run.
A

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

B
C
D
E
F
G
H
I
J
K
L

Octet® RH96 System HCP
Assay Protocol: A Completely
Walk‑Away Assay

M
N
O
P

Standards and samples prepared in Octet® Sample Dilution Buﬀer
Fluorescein anti-CHO prepared 1:100 in Octet® Sample Dilution Buﬀer
Buﬀer: Octet® Sample Dilution Buﬀer

The entire CHO HCP assay can be set up on the Octet®
RH96 system to enable high throughput, walk-away assays.
This automated assay format eliminates any potentialuser-related data variation that can be caused by manual
processing and helps achieve the most streamlined andefficient laboratory workflow. A full assay can be performed
and data obtained in about one hour with excellent assay
precision and run-to-run consistency.

Prepare Samples and Detection Plates

Figure 2: Sample Plate layout for 96 biosensor mode in 384-wellplate for Octet® RH96 system.

A

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

B
C
D
E
F
G
H
I
J

Notes:KL

All buffers and diluents used in this assay should be
azide‑free. The presence of azide can inhibit the activityof the HRP enzyme.

M
N
O
P

Enzyme: Anti-FITC HRP 1:50 in Octet® Sample Dilution Buﬀer

Protect Octet Fluorescein anti-CHO, Anti-FITC HRP,
and Metal Enhanced DAB reagents from light.
®

Please refer to the materials safety data sheet (MSDS) for
safety information on the Metal Enhanced DAB concentrate. Dispose of unused and used reagent in accordance4

Detection reagent: Metal DAB 1:10 in Octet® Peroxide Substrate Buﬀer
2nd Buﬀer: Octet® Peroxide Substrate Buﬀer
Unused wells

Figure 3: Detection Plate layout for 96 biosensor mode for Octet®
RH96 system.

3. Prepare the Sample Plate (example plate layout shownin Figure 2):
a. Pipette 80 µL of standard and unknown samples
prepared in Octet® Sample Dilution Buffer into thewells of a 384-well microplate.
b. Pipette 80 µL of Fluorescein anti-CHO prepared at
1:100 dilution in Octet® Sample Dilution Buffer into
the wells of the 384-well microplate.
c. Pipette 80 µL of Octet® Sample Dilution Buffer into
the wells of the 384-well microplate.
4. Prepare the Detection Plate (example plate layout
shown in Figure 3):
a. Pipette 80 µL of Anti-FITC HRP prepared at 1:50
dilution in Octet® Sample Dilution Buffer into thewells of a 384-well microplate.
b. Pipette 80 µL of Octet® Peroxide Substrate Buffer
into the wells of the 384-well microplate.
c. Pipette 80 µL of Metal Enhanced DAB prepared at
1:10 dilution in Octet® Peroxide Substrate Buffer into
the wells of the 384-well microplate.

5. Prepare the Biosensor Tray. Biosensor locations should
correspond to filled wells in the Sample Plate (one biosensor for each filled well in the Sample Plate).
6. Prepare a Hydration Plate by pipetting 200 µL of
Octet® Sample Dilution Buffer into each well of a
96-well polypropylene plate. Well locations filled with
Sample Diluent in the Hydration Plate should correspond to biosensor locations in Biosensor Tray.

Run the Assay
1.

Place the Sample and Detection Plates in the
Octet® RH96 instrument on the defined plate stations.
2. Launch Octet® BLI Discoveryand choose theAdvanced Quantitation option in the ExperimentWizard. In the Assay Settings window, click Modify to
set up the assay parameters as shown in Figure 4. The
96 biosensor mode settings are shown in the left andright panels. Note that the long sample incubation andFluorescein anti-CHO incubation steps (run in

Figure 4: Settings for Advanced Quantitation assays for HCP detection on the Octet® RH96 system. The left panel shows the Sample Plateconfiguration and assay settings in 96 biosensor mode which contains a sample incubation step, an Fluorescein anti-CHO incubation step, and twowash steps with Sample Diluent. The right panel shows the Detection Plate configuration and assay settings for detection using 96 biosensor mode.
5

5.
6.
7.

8.
9.

Analyze Data
The analysis of the data obtained on the Octet® RH96 system is identical to the analysis of data obtained from other
instruments. To analyze the data:
1. In Octet® BLI Analysis Software, load the data folder tobe analyzed.
2. Select the reference well and perform reference subtraction if needed.
3. Group and Concentration information can be modified
in the table if needed.
4. In the Results tab:a. Select R equilibrium as the binding rate equation.
This equation will fit the binding curve generated
during the experiment and calculate a response atequilibrium as the output signal.
b. Click Calculate Binding Rate. Results will be displayed automatically in the table.
c. Click Save Report or select File > Save Report togenerate a Microsoft® Excel® report file.

Octet® RH16 and R8 System HCP Assay
Protocol with Octet® AS Station
The HCP assay for 8- and 16-channel Octet® instruments is
performed according to the steps shown in Figure 6. Whenusing these systems, the initial incubation steps are processed using the Octet® AS Station. After biosensors are
incubated with HCP samples and Fluorescein anti-CHO
antibody, the remaining steps are performed on the
Octet® instrument.

6

Example Data from a Routine Assay
40

30

Binding Rate (nm)3.
4.

96 biosensor mode) are defined in the Sensor Loadingtab, and the remaining steps are defined in the Plate
Definition tab. Alternatively, users with Octet® BLI
Discovery Software can open pre-set method files bychoosing Experiment from the main menu, thenTemplates, then Quantitation, then Advanced
Quantitation, and choose the appropriate method file.
Click OK when assay parameters have been defined.
Define the Sample Plate layout to correspond to thelayout in Figure 2.
Define the Detection Plate layout to correspond to thelayout in Figure 3.
Enter Sample and Sensor information in the Plate Definition tab and the Sensor Assignment tab as desired.
In the Run Experiment tab, enter a delay time of
600 seconds in order to give the plates at least
10 minutes inside the Octet® instrument to equilibrate
to assay temperature.
Enter a location and file name for saving the data.
Click GO to run the assay.

20

10

0
130

140

150

Time (sec)

160

170

Expectedconc. (ng/mL)

Calculated conc.
(ng/mL)

Recovery

%CV

200.0

200.0

100%

4.2%

75.0

76.3

102%

5.0%

25.0

25.3

101%

4.4%

8.0

8.08

101%

6.2%

2.0

2.01

100%

4.2%

1.0

1.00

100%

5.0%

0.5

0.52

104%

9.8%

180

Figure 5: Example data from a CHO HCP assay on the Octet® RH96
system. The data shows a dose response for the calibration standards(N=12). The calculated concentrations and %CV values resulting fromthe analysis of the data are shown in the accompanying table.

Process samples on the Octet® AS Station
Notes:All buffers and diluents used in this assay should be azidefree. The presence of azide can inhibit the activity of the
HRP enzyme.
Protect Octet® Fluorescein anti-CHO, Anti-FITC HRP,
and Metal Enhanced DAB reagents from light.
Please refer to the safety data sheet for safety informationon the Metal Enhanced DAB concentrate. Dispose ofunused and used reagent in accordance with all local, state,
and federal guidelines. Proper personal safety measures
should also be taken when handling hazardous materials.

1.

Equilibrate the samples, reagents and buffers to room
temperature and mix thoroughly prior to use. Metal
Enhanced DAB concentrate should remain at -20°C
until immediately before use.
2. Prepare each concentration of HCP calibration
standard in Octet® Sample Dilution Buffer. The
calibration standards selected should cover the
HCP assay range from 0.5–200 ng/mL, and therecommended calibrator concentrations are 0.5, 1, 2, 8,
25, 75, and 200 ng/mL. It is also recommended to run
buffer-only controls as references. A calibration curveshould be included in each run.
3. Prepare the Sample Plate (example plate layout shownin Figure 7):
a. Pipette 200 µL of each standard into the wells of a96‑well microplate.
b. Pipette 200 µL of each unknown sample into thewells in the remainder of the microplate.
4. Prepare the Biosensor Tray. Biosensor locations
should correspond to filled wells in the Sample Plate(one biosensor for each filled well in the Sample Plate).
5. Prepare one Hydration Plate and two additional Wash
Plates by pipetting 200 µL of Octet® Sample Dilution
Buffer into each well of three polypropylene 96-wellplates. Well locations filled with Sample Diluent in the
Hydration Plate should correspond to biosensor locations in Biosensor Tray.
6. Using the Octet® AS Station, hydrate biosensors in the
Hydration Plate at 1000 rpm and 30°C for 1 minute.
7. Incubate hydrated biosensors in the Sample Plate on
the Octet® AS Station at 1000 rpm and 30°C for
30 minutes.
8. During this incubation, prepare the Octet® Fluoresceinanti-CHO Antibody Plate:a. Prepare a 1:100 dilution of Octet® Fluorescein antiCHO antibody in Octet® Sample Dilution Buffer.
b. Pipette 200 µL into each well of a new 96-well
microplate. Filled wells should correspond tobiosensor location and number in the Biosensor Tray.
9. After 30 minutes of sample incubation, replace theSample Plate with the first Wash Plate on the Octet® AS
Station and incubate at 1000 rpm and 30°C for
30 seconds.
10. Replace the Wash Plate with the Octet® Fluoresceinanti-CHO Antibody Plate. Incubate on the Octet® AS
Station at 1000 rpm and 30°C for 30 minutes.

11. During this incubation, prepare the Detection Plate.
Either a 96-well or a 384-well microplate can be used
depending on the Octet® instrument being used. The
fill volumes for 96- and 384-well microplates are 200 µL
and 80 µL, respectively.
a. Prepare an appropriate volume for the plate type of:
A 1:50 dilution of the Octet® Anti-FITC HRP
antibody in Octet® Sample Dilution Buffer
A 1:10 dilution of Metal Enhanced DAB in Octet®
Peroxide Substrate Buffer
b. In a black, flat-bottomed 96- or 384-well microplate,pipette 200 µL or 80 µL respectively of each reagent
specified into the wells of the microplate using theplate layout shown in Figure 8). Each reagent needs
to be filled into all wells of one column (for 8-channel
assays) or two columns (for 16-channel assays) in the
Detection Plate.
12. After 30 minutes of Fluorescein anti-CHO incubation,
replace the Sample Plate with second Wash Plate on
the Octet® AS Station and incubate at 1000 rpm and
30°C for 30 seconds.

--

Octet® AS Sidekick Station:
Hydration plate (200 µL/well)
Octet® Sample Dilution Buﬀer
1 min, 30˚C, 1000 rpm

Octet® AS Station:
Wash Plate (200 µL/well)
Octet® Sample Dilution Buﬀer
30 secs, 30˚C, 1000 rpm

Octet® AS Station:
Wash/hydration plate (200 µL/well)
Octet® Sample Dilution Buﬀer
30 secs, 30˚C, 1000 rpm
Octet® System: 600 sec, RT, 0 rpm

Octet® AS Station:
CHO HCP plate(200 µL/well)
CHO HCP Standards and Samples
30 mins, 30˚C, 1000 rpm

Octet® AS Station:
Octet® Fluorescein anti-CHO plate(200 µL/well)
Fluorescein anti-CHO antibody
30 mins, 30˚C, 1000 rpm

Octet® System:
Detection plate (200 µL/well)Buﬀer (Octet® Sample Dilution Buﬀer)
Enzyme (Octet®Anti-FITC HRP antibody)
2nd Buﬀer (Octet® Peroxide Substrate Buﬀer)
Detection (Octet® DAB/Metal Concentrate)

Figure 6: Flow chart of HCP assay steps on 8- and 16-channel
Octet® systems with the Octet® AS Offline Immobilization Station.

7

1

2

3

4

5

6

7

8

9

10

11

12

A
B
C
D
E
F
G
H

HCP standards
HCP unknown
Figure 7: Sample Plate layout for incubation with biosensors on the
Octet® AS Station.
1

2

3

4

5

6

7

8

9

10

11

12

A

Figure 9: Settings for Advanced Quantitation assays for HCP detection
on 8- and 16-channel Octet systems.

Run the Assay
1.

B
C
D

2.

E
F
G
H

A

1

3.
2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

B
C
D
E

4.

F
G
H

5.

I
J
K
L
M
N
O
P

Octet® Sample Dilution Buﬀer
Octet® Anti-FITC HRP
Octet® Peroxide Substrate Buﬀer
Octet® DAB/Metal Concentrate
Empty

Figure 8: Detection Plate layout for 8-channel assays on Octet® R8system (top) and for 16-channel assays on Octet® RH16 system(bottom).

8

After the second wash step on the Octet® AS Station iscomplete, place the Biosensor Tray over the second
Wash Plate (the same plate used on the Octet® AS Station for the last wash) into the Octet® instrument.
Place the Detection Plate into the Octet® Instrument.
For 8-channel Octet® systems, place the Detection
Plate in the single plate station (Plate 1) in the instrument. For 16-channel systems, place the Detection
Plate in the reagent plate station (Plate 2).
Launch Octet® BLI Discovery Software and choose theAdvanced Quantitation option in the Experiment wizard. In the Assay Settings window, click Modify to set up
the assay parameters as shown in Figure 9. Note thatthe sample step is specified to have been carried out
offline.
Click OK once the assay parameters have been
defined.
Define the Sample and Detection Plate layouts:
For 16-channel Octet® systems:
a. Define the Sample Plate layout to correspond to thelayout in Figure 7. The Sample Plate Layout needs tobe defined in the Octet® BLI Discovery Software
even though the Sample step has been performed
offline as this enables the software to assign the correct Sample IDs to the biosensors during analysis.
b. Define the Detection Plate layout to correspond to
the appropriate layout in Figure 9:
Octet® Sample Dilution Buffer = B (Buffer)
Octet® Anti-FITC HRP antibody = E (Enzyme)
Octet® Peroxide Substrate Buffer = 2 (2nd Buffer)
Octet® DAB/Metal Concentrate = D (Detection)

---

---

35

The data shown was generated on the Octet® RH16 systemusing the protocol outlined in this technical note.
CHO‑HCP standards at 200, 75, 25, 8, 2, 1, and 0.5 ng/mL
in Octet® Sample Dilution Buffer were prepared and run
in triplicate. Three unknowns (Samples 1, 2 and 3) were runin 8 replicates to assess assay precision.

3

60

80
100
Time (sec)

4

20

15

10
5

0
0

20

40

120

140

160

180

Figure 10: Real time data from detection steps measured on the Octet®
RH16 system with the CHO HCP assay. Processing of the biosensors
with samples and the binding of the Octet®” Fluorescein anti-CHO
antibody were performed using the Octet® AS Station. The steps shown
include 1) baseline in Sample Diluent, 2) binding of Octet® Anti-FITC
HRP antibody, 3) Octet® Peroxide Substrate Buffer equilibration and 4)
detection of signal using Octet® DAB/Metal Concentrate. The assay
was run according to the procedure outlined in this technical note.
40

1.

Example Data from a Routine Assay

2

25

Analyze Data

35
30
Binding Rate (nm)

In Octet® BLI Discovery Software, load the data folder
to be analyzed.
2. Select the reference well and perform reference
subtraction if needed.
3. Group and Concentration information can be
modified in the table if needed.
4. In the Results tab:a. Select R equilibrium as the binding rate equation.
This equation will fit the binding curve generated
during the experiment and calculate a response atequilibrium as the output signal.
b. Click Calculate Binding Rate. Results will be
displayed automatically in the table.
c. Click Save Report or select File > Save Report
to generate a Microsoft® Excel® report file.

1

30

Binding Rate (nm)For 8-channel Octet® systems:a. Specifying the Sample step to be carried out offline
enables two plates to be opened in the software(even though there is only one plate position in the
instrument). The first plate (Plate 1) has the same layout as the Sample Plate layout used in the Octet® AS
Station incubation.
b. The layout of the second plate (Plate 2) is defined
to correspond to the appropriate Detection Platelayout in Figure 9 where:
Octet® Sample Dilution Buffer = B (Buffer)
Octet® Anti-FITC HRP antibody = E (Enzyme)
Octet® Peroxide Substrate Buffer = 2 (2nd Buffer)
Octet® DAB/Metal Concentrate = D (Detection)
6. Enter Sample and Sensor information in the Plate Definition tab and the Sensor Assignment tab as desired.
7. In the Run Experiment tab, enter a delay time of
600 seconds in order to give the plates at least
10 minutes inside the Octet® instrument to equilibrate
to assay temperature.
8. Enter a location and file name for saving the data.
9. Click GO to run the assay.

25
20
15
10
5
0

0

10

20

30
Time (sec)40

50

Expected conc.
(ng/mL)Calc. conc.
(ng/mL)

Recovery

%CV

200.0

201.0

101%

2.1%

75.0

73.9

99%

0.7%

25.0

25.5

102%

2.3%

8.0

8.08

101%

2.8%

2.0

2.01

101%

1.8%

1.0

1.00

100%

1.7%

0.5

0.50

100%

3.3%

60

Figure 11: The data shows a dose response for the calibration standards
in triplicate. The calculated concentrations and %CV values resulting
from the analysis of the data are shown in the accompanying table.
940

SampleCalc. con.
(ng/mL)%CV

Sample 1

137.5

4.1%

Sample 2

10.6

4.0%

Sample 3

0.43

5.1%

35

Binding Rate (nm)

30
25
20
15
10
5
0

0

10

20

30
Time (sec)

40

50

60

Figure 12: Graph showing data for three unknown samples, eachhaving eight replicates. The calculated concentrations and %CV
are shown in the accompanying table. The concentrations were
calculated using the calibration data shown in Figure 11.

Assay Troubleshooting Guide
Issue

Possible reasonsSuggested solutions

No signal from HCP standards

Peroxide Substrate Buffer andSample Diluent switched.

Re-run assay taking care to dilute the Metal Enhanced DAB
concentrate in Octet® Peroxide Substrate Buffer and samples
and standards in Octet® Sample Dilution Buffer.

Peroxide Substrate Buffer or Metal
Enhanced DAB is no longer active.

Check for activity by combining Octet® Peroxide Substrate
Buffer and Metal Enhanced DAB with 1 μL of HRP anti-FITC
antibody. A dark precipitate should appear if all are active.

Reagents no longer active.

Confirm that the Metal Enhanced DAB has been stored at-20°C, the Octet® Peroxide Substrate Buffer at either -20°C or
4°C (after opening), the biosensors at room temperature andthe Octet® CHO standards, Fluorescein anti-CHO and HRP
anti-FITC at 4°C.

HCP concentration is below the LOD.

Perform a dilution series of the sample in order to determine
what dilution is required to bring the sample signal within the
dynamic range of the assay.

Azide present in samples.

Dialyze sample to remove azide.

Sample signal is too high

HCP concentration is above theconcentration of the highest standard.

Perform a dilution series of the sample in order to determine
what dilution is required to bring the sample signal within the
dynamic range of the assay.

High variability between runs

Inconsistency in incubation times.

For best results, assay step times should be consistent acrossstandards and samples. For incubation steps involving manualintervention, use a lab timer to ensure all samples and assays
are treated similarly.

Octet® Fluorescein anti-CHO, anti-FITC
HRP and Metal Enhanced DAB notprotected from light.

Protect Octet® Fluorescein anti-CHO, anti-FITC HRP and
Metal Enhanced DAB from light by storing in a dark, dry placewhen not in use. Additionally, dilutions of these materials
should be stored in a dark place until they are pipetted into
micro plates and placed either into the instrument or onto theOctet® AS Station.

No sample signal

10

11

Germany
Sartorius Lab Instruments GmbH & Co. KG
Otto-Brenner-Strasse 20
37079 Goettingen
Phone +49 551 308 0

 F
 or further contacts, visit

www.sartorius.com/octet-support

Specifications subject to change without notice.
Copyright Sartorius Lab Instruments GmbH & Co. KG.
For Research Use Only.
TN-4041 Rev D

USA
Sartorius Corporation
565 Johnson Avenue
Bohemia, NY 11716
Phone +1 888 OCTET 75
Or +1 650 322 1360