ge healthcare life sciences · ge healthcare life sciences ... suitable concentration for ph...
TRANSCRIPT
GE Healthcare
Life Sciences
Methodology Guideline 29-0136-74 AA Biacore™ systems
Quantification of influenza hemagglutinin using Biacore T200
2
29-0136-74 AA
Table of Contents
1. Introduction ....................................................................................................................................................... 3
1.1. Steps recommended when modifying the assay for new reagents ............................................................ 3
1.2. General steps in the optimized assay .......................................................................................................................... 4
1.3. Definitions .................................................................................................................................................................................. 4
A list of abbreviations is provided in Table 1. ................................................................................................................... 4
2. Materials ............................................................................................................................................................ 5
2.1. Chemicals/biological material ......................................................................................................................................... 5
2.2. Instrumentation/ disposables ......................................................................................................................................... 5
2.3. Buffers ......................................................................................................................................................................................... 6
3. Assay modification for new reagents ........................................................................................................... 7
3.1. pH scouting ............................................................................................................................................................................... 7
3.1.1. Reagents for pH scouting/immobilization.................................................................................................... 7
3.1.2. Immobilization pH scouting procedure ......................................................................................................... 8
3.1.3. Evaluation ..................................................................................................................................................................... 8
3.2. Immobilization ......................................................................................................................................................................... 9
3.2.2. Reagents for immobilization ............................................................................................................................... 9
3.2.2. Immobilization procedure .................................................................................................................................... 9
3.2.3. Evaluation ................................................................................................................................................................... 10
3.3. Establishing a suitable serum dilution factor ........................................................................................................ 10
3.3.1. Dilution of serum ..................................................................................................................................................... 11
3.4. Surface performance test ............................................................................................................................................... 12
3.4.1. Surface performance procedure .................................................................................................................... 12
3.4.2. Evaluation ................................................................................................................................................................... 12
3.5. Calibration curve set up ................................................................................................................................................... 13
3.5.1. Preparation of serum ............................................................................................................................................ 13
3.5.2. Preparation of calibration curve (standards) ............................................................................................ 13
3.5.3. Preparation of start-up cycles.......................................................................................................................... 14
3.5.4. Evaluation ................................................................................................................................................................... 14
3.5.5. Important considerations ................................................................................................................................... 15
4. Running an optimized concentration analysis ......................................................................................... 15
4.1. Immobilization ....................................................................................................................................................................... 15
4.2. Preparation of serum ......................................................................................................................................................... 16
4.3. Preparation of standards for calibration curve .................................................................................................... 16
4.4. Preparation of samples .................................................................................................................................................... 16
4.5. Preparation of start-up cycles....................................................................................................................................... 16
4.6. Running Concentration analysis using the template ........................................................................................ 16
5. QC and evaluation of results ........................................................................................................................ 16
5.1. Quality check of the run ................................................................................................................................................... 17
5.2. Evaluation ................................................................................................................................................................................ 19
6. References ....................................................................................................................................................... 22
7. Appendix ........................................................................................................................................................... 23
7.1. Serum dilution test using the Method Builder ....................................................................................................... 23
7.2. Robustness of sample buffers ....................................................................................................................................... 24
7.2.1. Test of sample buffer robustness ................................................................................................................... 24
3
29-0136-74 AA
1. Introduction This methodology guideline describes how to develop an assay in Biacore T200 for quantification of hemagglutinin (HA) in
influenza vaccine samples. The assay development needs to be performed every time new influenza reagents are used. The
assay described is an inhibition assay and it is based on the published method “A novel assay for influenza virus
quantification using surface plasmon resonance” (see section 6.). The principle for inhibition assays can be seen in Figure 1.
In addition, a general procedure for concentration analysis and evaluation is also included in this methodology guideline.
Figure 1. Inhibition assay principle: HA is first immobilized on the dextran matrix (red circles). Virus is then mixed with a fixed concentration of serum and
injected over the surface. Free anti-HA antibodies in the serum (not bound to virus at equilibrium) bind to the surface HA, giving a response. A) High
concentration of virus in the sample gives low antibody binding (1), while low virus concentration results in high binding level (2). B) Calibration curve based
on injection of a known concentration series of virus standard. Concentration of vaccine samples are measured against the standard curve.
1.1. Steps recommended when modifying the assay for new reagents
1. pH scouting
- Optimize pH of the immobilization buffer for the HA protein preparation to be immobilized.
2. Immobilization time
- Establish a suitable immobilization time using the buffer from the scouting.
3. Determine suitable serum dilution
- Recommended for new serum and/or when a new immobilization has been performed.
4. Calibration curve
- Determine range of detection and run repeated calibration curves to test surface performance.
5. Optional: Robustness of sample buffers
- Check for matrix effects from the samples.
Sensor Chip CM5
1 2
B A
4
29-0136-74 AA
1.2. General steps in the optimized assay
- Immobilization
- Preparation of serum, samples and influenza reference standard
- Unattended run of analysis
- Evaluation
1.3. Definitions
A list of abbreviations is provided in Table 1.
Table 1. List of abbreviations occurring in this methodology guideline.
Abbreviation Complete name
HBS-EP+ HEPES buffered saline with EDTA and 0.05 % Surfactant P20
EDTA Diaminoethanetetraacetic acid
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
NHS N-hydroxysuccinimide
RU Response units
HA Hemagglutinin
Ag Antigen
CV Coefficient of Variation
5
29-0136-74 AA
2. Materials
2.1. Chemicals/biological material
Required chemicals and examples of biological materials are listed in Table 2.
Table 2. Chemicals and biological material (examples) required for quantification of hemagglutinin (HA) in influenza vaccine samples using Biacore systems.
Suppliers and order codes (P/N) are listed as well.
Chemicals Supplier P/N
Amine Coupling Kit (EDC, NHS, ethanolamine) GE Healthcare BR-1000-50
HBS-EP+ buffer, pH 7.4, 10x concentrate GE Healthcare BR-1006-69
Surfactant P20 (10 % solution) GE Healthcare BR-1000-54
HCl, 37% Merck 1.00317.2500
10 mM Sodium acetate, pH 5.5 GE Healthcare BR-1003-52
Maleic acid Fluka 63190-250G
NaH2PO4 Merck 1.06346.1000
Na2HPO4 Merck 1.06586.0500
NaOH Merck 1.06469.1000
Hemagglutinin protein (examples) Supplier
A/H3N2/Wyoming/3/2003 Protein Sciences*
B/Jilin/20/2003 Genway Biotech
A/H1N1/New Caledonia/20/99 Recombinant ProSpec
* In our experience HA from Protein Sciences has been of good quality.
2.2. Instrumentation/ disposables
Required instrumentation and disposables are listed in Table 3.
Table 3. Instrumentation/disposables required for quantification of hemagglutinin (HA) in influenza vaccine samples using Biacore systems. Suppliers and
order codes (P/N) are listed as well.
Instrumentation/disposables Supplier P/N
Biacore T200 GE Healthcare 28-9750-01
Series S Sensor Chip CM5 GE Healthcare BR-1005-30
Rubber caps (for 7 mm plastic vials), type 3 GE Healthcare BR-1005-02
Plastic vials 7 mm (0.7 ml) GE Healthcare BR-1002-12
Rubber caps (for 16mm glass vials), type 2 GE Healthcare BR-1004-11
Glass vials 16 mm (4.0 ml) GE Healthcare BR-1002-09
Microplate, 96 well GE Healthcare BR-1005-03
Microplate foil, 96 well (without glue over well) GE Healthcare 28975816
Plastic vials 11 mm ( 1.5 ml) GE Healthcare BR-1002-87
6
29-0136-74 AA
2.3. Buffers
Prepare running buffer, immobilization buffers and regeneration solution as described below.
Running buffer, pH 7.4: 1 L, HBS-EP+ (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20)
HBS-EP+ 10x 100 ml
Milli-Q water 900 ml Dilute 100 ml HBS-EP+ 10x in 900 ml Milli-Q water.
Immobilization buffer, pH 5.5: 100 ml, 10 mM Sodium acetate, 0.05% Surfactant P2
10 % Surfactant P20 500 µl
10 mM Sodium acetate, pH 5.5 up to 100 ml Add 500 µl P20. Add Sodium acetate up to 100 ml in a volumetric flask
Immobilization buffer, pH 6.0: 100 ml, 10 mM Maleate buffer, 0.05% Surfactant P20
Maleic acid 0.116 g
Milli-Q water up to 100 ml
10 % Surfactant P20 500 µl Dissolve in ~80 ml Milli-Q water. Adjust pH with 1 M NaOH to pH 6.0. Add 500 µl of surfactant P20. Add up to 100 ml with
Milli-Q water in a volumetric flask. Check pH after final volume adjustment.
Immobilization buffer, pH 6.5: 100 ml, 10 mM Maleate buffer, 0.05% Surfactant P20
Maleic acid 0.116 g
Milli-Q water up to 100 ml
10 % Surfactant P20 500 µl Dissolve in ~80 ml Milli-Q water. Adjust pH with 1 M NaOH to pH 6.5. Add 500 µl of surfactant P20. Add up to 100 ml with
Milli-Q water in a volumetric flask. Check pH after final volume adjustment.
Immobilization buffer pH 7.0: 100 ml, 10 mM Na Phosphate, 0.05% Surfactant P20
NaH2PO4 0.06 g
Na2HPO4 0.16 g
Milli-Q water up to 100 ml
10% Surfactant P20 500 µl Dissolve the salts in ~80 ml Milli-Q water. Use of a magnetic stirrer is recommended. Check that the pH is 7.0. If pH <7.0, adjust with 1 M NaOH to pH 7.0. Add 500 µl of surfactant P20. Fill up to 100 ml with Milli-Q water. Check pH after final volume adjustment. Alternatively: Dissolve 0.138 g NaH2PO4 . H2O (Merck) in <100 ml Milli-Q water. Adjust pH with NaOH, add 500 µl surfactant
P20 and fill up to 100 ml with Milli-Q water.
7
29-0136-74 AA
Regeneration solution: 50 ml, 50 mM HCl, 0.05% Surfactant P20
HCl, 1 M 2.5 ml
Milli-Q water 47.5 ml
10 % Surfactant P20 250 µl Dilute 2.5 ml 1 M HCl in ~40 ml Milli-Q water and add 250 µl of surfactant P20. Add up to 50 ml with Milli-Q water.
Regeneration solution should be prepared freshly every week.
3. Assay modification for new reagents
3.1. pH scouting
For HA protein preparations suitable pH usually is in the range 5.5 - 7.0, depending on preparation. Note that it is necessary
to dilute the protein sufficiently to obtain effective conditions for the immobilization. Typically a protein stock buffer,
containing ~0.15 M salt needs to be diluted at least 5-10 times to obtain a sufficiently low salt level. Too much salt will shield
the charges on the protein and dextran matrix. The highest pH that will give adequate immobilization level should be
chosen, in order to maintain activity as long as possible.
3.1.1. Reagents for pH scouting/immobilization
If the total volume of a recombinant protein is <10 µl, it is advisable to first dilute the protein to ~40 µl in the buffer specified
for the protein by the manufacturer. Suitable concentration for pH scouting is ~5-10 µg/ml.
Monovalent bulk vaccine products may also be used for immobilization. Good results have been obtained with immobilized
cell derived monovalent bulk from split and subunit vaccines. At present, our experience from immobilizing cell derived
whole virus or egg derived vaccine products are limited. For immobilization of whole virus it may be beneficial to dilute the
virus in immobilization buffer containing 1 % zwittergent (omit P20), to solubilize the virus proteins and incubate 30 min prior
to immobilization. It is however not recommended to use zwittergent for samples and/or standards or in the running buffer.
Egg derived vaccine products have been shown to give higher background binding from sheep sera, possibly connected to
the HA preparation used for immunization of sheep.
Dilute the protein to 10 ug/ml in respective immobilization buffer shown in Table 5. Use 7 mm plastic vials. The pH scouting
wizard Rack editor dialogue will state minimum required volumes. Add 200 µl of regeneration solution (50 mM NaOH) to one
vial. Add rubber caps.
Note: Recombinant HA proteins provided by manufacturers have been shown to vary in quality and concentration. In
addition, quality and concentration differ between lots.
8
29-0136-74 AA
Table 5. List of immobilization buffers (names and pH values) to be used for pH scouting.
Buffer name pH
1 10 mM NaP, 0.05 % P20 7.0
2 10 mM Maleate, 0.05 % P20 6.5
3 10 mM Maleate , 0.05 % P20 6.0
4 10 mM Acetate, 0.05 % P20 5.5
3.1.2. Immobilization pH scouting procedure
1. Choose “Open/New wizard procedure” in the file menu. Choose “Immobilization pH scouting”. Click “New”.
2. Choose an unimmobilized flow path and buffers, see Table 3. Click “Next”.
3. Fill in “ligand solution” (HA protein name). Use contact time: “180 s” and flow: “2 µl/min”. Leave Regeneration solution as
default (50 mM NaOH). Click “Next”.
4. Use the default values (check Prime before run, 25°C for analysis and sample compartment temperature). Click “Next”.
5. Use e.g. Reagent Rack 2. Choose “Eject rack” and insert the capped vials. Positions may be changed if desired by
drag/drop. Click “Next”.
6. Check that the HBS-EP+ buffer and water levels will be sufficient for the run. Click “Next”.
7. Choose “Don’t save” the method (the method will be saved with the result anyway).
8. Save the file under appropriate name and click “Start”.
3.1.3. Evaluation
Open the result in the Evaluation software. Check the slope of the injections. It is desirable to choose a pH as close to neutral
as possible to retain protein activity as long as possible on the chip, once immobilized. However, the slope needs to be high
enough that 4000-15000 RU (see section 3.2.) can be expected to be immobilized during up to ~20 min immobilization time
(in some cases up to 30 min). Thus “extrapolate” the slope visually to e.g. 10-20 min injection time and estimate
immobilization level on the y-axis. An example of a pH scouting result can be seen in Figure 2.
9
29-0136-74 AA
Figure 2. Example of a pH scouting result. In this example the 10 mM Maleate buffer, pH 6.0 (green line), was chosen as most suitable for the experiment.
The buffer was estimated after extrapolation to result in an immobilization level of > 4000 RU for a 20 minutes injection, while at the same time being more
“neutral” than 10 mM Acetate buffer, pH 5.5. An injection time of 20 min was therefore chosen for the immobilization. In the figure an extrapolation up to
10 minutes after injection are shown.
3.2. Immobilization
Suitable HA concentration for the immobilization may be ~10-15 µg/ml. As mentioned in earlier (see section 3.1.1.), the
quality from the manufacturer may vary for recombinant proteins. Hence, higher concentrations may be needed for
sufficient immobilization, regardless of the stated protein concentration on the vial. (Note the information about salt content
in 3.1). Dilute the protein in the chosen immobilization buffer just prior to immobilization.
3.2.2. Reagents for immobilization
Dilute the NHS and EDC reagents in the Amine Coupling Kit according to Instruction For Use (IFU). Dispense in aliquots (e.g.
100 – 200 µl in 7 mm plastic vials) and store at -20C.
3.2.2. Immobilization procedure
1. Choose the File menu: “Open New wizard/template” and then “Immobilization”. Click “New”.
2. Choose flow path, and enter the “name” of the HA protein (ligand), “contact time” (e.g. 1000 s) and “flow rate” (2-5
µl/min). Click “Next”.
3. In System preparations, use default values except that sample compartment temperature should be set to 10°C. Check
“Normalize” if it is the first immobilization on a new chip. Normalization is then not necessary for the following
immobilizations on the same chip. Click “Next”.
4. Prepare vials with proper volumes of each solution according to the software.
5. Choose “Eject rack” and place the vials according to the set up in the software. “Close” and “Next”.
6. Check that the HBS-EP+ buffer and water levels will be sufficient for the run. Click “Next”.
10
29-0136-74 AA
7. Save the file under an appropriate name and click “Start”.
3.2.3. Evaluation
For an example of immobilization, see Figure 3. The precise level of immobilization is not critical. Ideal immobilization levels
are ~ 6000-10000 RU. Acceptable immobilization levels lie between 4000 to 15000 RU (i.e. new immobilization is not needed
for e.g. 14000 RU result). Below 4000 RU the sensitivity will start to decrease while above 15000 RU an unnecessary amount
of reagent has been immobilized. Too high immobilization levels may cause limitations in HA accessibility for the antibodies
since the surface is very “crowded”. Although this in most cases has little effect on the assays, an excessive amount of
reagent will have been spent needlessly. To modify the immobilization level change the contact time and/or HA protein
concentration.
Figure 3. A typical immobilization of recombinant HA A/H1N1/New Caledonia. Using a contact time of 870 s (14.5 min) gave an immobilization level of 5880
RU.
3.3. Establishing a suitable serum dilution factor
Optimization of serum dilution is needed when a new serum is to be used and/or when a new immobilization has been
performed. Serum is diluted in running buffer to obtain a serum response somewhere in the range 700-2000 RU, using a
contact time of 400 s. Serum diluted to different degrees are injected followed by regeneration after each injection, see
example in Figure 4.
Perform the serum dilution test using Method Builder. The template (Anti-HA serum dilution) can be downloaded from
www.biacore.com/applicationsupporttools Methods. For detailed instructions how to create a method for this purpose,
see section 7.1.
11
29-0136-74 AA
1200
1000
800
600
400
200
0
Response (RU)
Time (s)1200800400
50x
100x
200x400x
Serum dilution
1200
1000
800
600
400
200
0
Response (RU)
Time (s)1200800400
50x
100x
200x400x
Serum dilution
1200
1000
800
600
400
200
0
Response (RU)
Time (s)1200800400
50x
100x
200x400x
Serum dilution
Figure 4. Example of a serum dilution test. The 50x diluted serum was chosen resulting in a response in the range 700-2000 RU.
3.3.1. Dilution of serum
This example is sufficient for 400 s injections at a flow rate of 5 µl/min. Dilute the serum according to Table 6.
1. Mix 16 µl of serum with 384 µl HBS-EP+ to get a 25x dilution.
2. Perform the serial dilution by mixing 200 µl of the dilution from the previous dilution step with 200 µl of HBS-EP+ until a
1600x dilution is reached.
Table 6. Serial dilution of serum in buffer HBS-EP+.
Dilution factor Serum volume Volume HBS-EP+
25x 16 µl undiluted 384 µl
50x 200 µl 25x dilution 200 µl
100x 200 µl 50x dilution 200 µl
200x 200 µl 100x dilution 200 µl
400x 200 µl 200x dilution 200 µl
800x 200 µl 400x dilution 200 µl
1600x 200 µl 800x dilution 200 µl
The results may be checked during the run, and the run can be ended before completion as soon as appropriate serum
binding level has been obtained. In such case, end the run by use of the Run/End run command allowing regeneration to be
performed. Choose a serum dilution that gives a response somewhere in the range ~700-2000 RU. See Figure 4.
12
29-0136-74 AA
3.4. Surface performance test
This may be performed to check the activity of the immobilized surface during repeated serum binding without spending
any standard reagent. Depending on results, final adjustment of the chosen serum dilution factor may be done. Alternatively,
surface performance may also be checked simultaneously when setting up the calibration curve, see section 3.5.
Perform the surface performance test using the Surface performance wizard, see section 3.4.1.
Dilute the serum in HBS-EP+ according to the results in 3.3 to a total volume of e.g. 3 ml. Transfer the diluted serum to a 16
mm glass vial and add rubber cap.
3.4.1. Surface performance procedure
1. Choose the File menu: “Open New wizard/template” and “Surface performance”. Click “New”.
2. Choose flow path and chip type (CM5). Click “Next”.
3. Choose ~30 cycles. Click “Next”.
4. Add “sample name”, contact time “400 s” and flow rate “5 µl/min”. Add “regeneration name” (50 mM HCl, 0.05% P20),
contact time “30 s”, flow rate “30 µl/min” and stabilization period “30 s”. Click “Next”.
5. In System preparations, use default values except set sample compartment temp to 10°C. Click “Next”.
6. Go to “Menu” and choose “automatic positioning”. Choose to pool the sample in the reagent rack.
7. Click “Eject rack” and place the serum and regeneration buffer in the designed positions. “Insert rack”. Click “Next”.
8. Check that the HBS-EP+ buffer and water levels will be sufficient for the run. Click “Next”.
9. Choose “Don’t save” the method (the method will be saved with the result anyway).
10. Save the surface performance test under appropriate name and click “Start”.
3.4.2. Evaluation
Open the result in the Evaluation software. Open the “Binding stability” plot from Evaluation Explorer at the left of the main
screen. The first 10 cycles will show the largest drop in relative response, e.g. 500-1000 RU (for concentration analysis 5-10
startup cycles are typically used). Thereafter the response level should stabilize. The complete concentration assay will be
adjusted for remaining drift by choosing the “Trend calibration” functionality when evaluating the results. Check that the
response between last startup cycle and the number of cycles you intend to run is not likely to drop below ~50 % of
response of the last startup cycle. See Figure 5 for an example of a surface performance result.
13
29-0136-74 AA
1200
1400
1600
1800
2000
2200
2400
2600
0 5 10 15 20 25 30 35
Binding stability
Rel
ati
ve
res
po
nse
- s
tab
ility
RU
Cycle number
Sample
Figure 5. An example of a surface performance result. The binding stability plot (report point Stability 10 s after injection end versus cycle number) shows
the change in serum response during 30 cycles.
3.5. Calibration curve set up
Establish the concentration range of the calibration curve by injection of a dilution series of the reference virus. By running
repeated calibration curves this may also serve as a test of surface activity. Perform the calibration curve setup using the
Method Builder template “HA quantification assay” with preferred modifications included (such as removing samples and
controls). The template can be downloaded from www.biacore.com/applicationsupporttools Methods.
Note: The use of a reference surface for subtraction of responses is not recommended for concentration analysis using
Biacore systems. Run the assay in one flow cell at the time.
3.5.1. Preparation of serum
Prepare e.g. 5 ml serum by dilution in HBS-EP+ to half the serum dilution factor chosen in 3.3. For example, if a 500x dilution
of the serum was chosen, dilute the serum to x250. The serum will then be mixed 1:1 with standards or samples, thereby
obtaining the chosen final serum dilution factor of x500.
3.5.2. Preparation of calibration curve (standards)
First the standard is serially diluted into HBS-EP+ buffer. Thereafter serum is added to all concentrations. Upon addition of
the serum the standard concentrations will become half to that originally. This is intended since the samples will be treated
in the same way and the concentrations of standards/samples will then be matched.
Note: Do not make the dilution series of the standard directly into the serum!
14
29-0136-74 AA
Example: One ampoule of standard antigen B/Brisbane, is first dissolved in Milli-Q water according to the manufacturer’s
instructions, to obtain a concentration of 74 µg/ml HA. Store in aliquots at -20ºC.
Mix 24 µl antigen (74 µg/ml) with 198 µl HBS-EP+ to get a concentration of 8 µg/ml HA in 222 µl buffer. Dilute serially 110 µl
of the antigen mixture into 110 µl HBS-EP+ to obtain the concentration series 8, 4, 2, 1, 0.5, 0.25 and 0.125 µg/ml. Transfer
100 µl of each concentration into 7 mm plastic vials, add 100 µl of serum to all vials (final volume 200 µl/vial), mix and add
rubber caps. Make sure there are no air bubbles on the bottom of the vials.
3.5.3. Preparation of start-up cycles
Transfer 300 µl of the diluted serum from 3.5.1. to a 7 mm plastic vial. Add 300 µl of HBS-EP+ to obtain the same final serum
dilution factor as for the standards. Add rubber cap.
3.5.4. Evaluation
Open the result in the Evaluation software. Open the “Binding stability” plot from Evaluation Explorer at the left of the main
screen. The start-up cycles will show the largest drop in relative response (can be as large as 500-1000 RU). Thereafter the
response level should stabilize. Check that the response between last startup cycle and the number of cycles you intend to
run is not likely to drop below ~50 % of response of the last startup cycle. The remaining response drift will be adjusted for in
the complete concentration assay by choosing the “Trend calibration” functionality when evaluating the results (see
evaluation example, Figure 6).
Figure 6. Example showing a successful test of the calibration curve range and surface activity. Three start-up cycles were run using serum, displaying an
initial large drop in response after which responses started to stabilize to an acceptable degree. Concentrations between 0.15-10 µg/ml of H3N2 virus
antigen were tested in this example. The calibration was repeated every 3rd samples. Samples consisted of serum only with no virus added. A control
sample (1 µg/ml virus antigen) was also included and repeated every 3rd samples. (To save virus antigen it may be preferred to run fewer calibration curves
and more serum samples).
0
500
1000
1500
2000
2500
3000
3500
0 10 20 30 40 50 60
Binding stability
Rel
ativ
e re
spo
nse
- s
tab
ility
RU
Cycle number
Calibration
Control Sample
Sample
Startup
15
29-0136-74 AA
3.5.5. Important considerations
Preferably a response difference of at least 200 RU between the highest and the lowest calibration points should remain
for the last calibration curve, depending on the need for precision in calculated sample concentrations.
The concentration range of the calibration curve may be adjusted by adjusting the serum dilution. Higher serum dilution
factor (less number of antibodies) shifts the curve to the left, i.e. increases the assay sensitivity while losing ability to
measure higher concentrations (see Figure 7). A lower serum dilution factor (more antibodies) on the other hand shifts
the curve to the right and allows measurement of higher virus concentrations, losing sensitivity for low sample
concentrations.
To save reference antigen 3 calibration curves are sufficient; run calibration first, in the middle and last. Include serum
samples in between (not just buffer) to obtain the approximate number of samples that is intended to be run in the
assay.
Figure 7. Example showing an assay with too high serum dilution factor. The highest virus concentrations bind the majority of all antibodies in the solution.
This is causing the curves to flatten out since very few antibodies remain to bind the sensor surface. Sample concentrations > 2 µg/ml cannot be measured
with high precision. This is solved by using a higher concentration of the serum. If the curve instead flattens out in the beginning, then the serum should be
diluted more. The calibration curve is run 3 times during the assay.
4. Running an optimized concentration analysis The calibration curve is run 3 times during the assay. Further replicates of the calibration curve are thus not necessary but
run if desired. Perform the quantification assay using the Method Builder template “HA quantification assay”. The template
can be downloaded from www.biacore.com/applicationsupporttools Methods.
Note: The use of a reference surface for subtraction of responses is not recommended for concentration analysis using
Biacore systems. Run the assay in one flow cell at the time.
4.1. Immobilization
Perform an immobilization according to the optimized settings from 3.1. and 3.2.
100
200
300
400
500
600
700
800
0,1 1 10
RU
Concentrat ion µg/ml
B/Brisbane
2
16
29-0136-74 AA
4.2. Preparation of serum
About 7 ml of diluted serum is needed for a full 96 well microplate using 50 µl sample/well, 7 standards and the start-up
cycles. Prepare serum in e.g. a 15 ml plastic tube by dilution in HBS-EP+ to half the serum dilution factor chosen in the
previous section. For detailed information see section 3.5.1.
4.3. Preparation of standards for calibration curve
Prepare the standards as described in the previous section under 3.5.2.
4.4. Preparation of samples
Dilute the samples in x2 serial dilutions in HBS-EP+, with the aim of obtaining concentrations covered by the calibration
curve. Add 50 µl of each sample into a 96 well plate. Add 50 µl of the diluted serum to each well (final volume 100 µl/well).
Cover the 96-well plate with a micro plate foil, mix e.g. with a plate shaker.
4.5. Preparation of start-up cycles
Mix 350 µl serum with 350 µl HBS-EP+ (resulting in the same serum dilution as for the standards and samples). Transfer to a
7 mm plastic vial. Add rubber caps.
4.6. Running Concentration analysis using the template
1. Choose the File menu: “Open New/Method”. Choose “Browse” and go to the folder where the “HA quantification assay”
template is. Click “Open”.
2. Go to “Setup Run”.
3. Choose flow path. Click “Next”.
4. Fill in the samples and their dilution. Click “Next”.
5. Check the run cycle list: Are all samples are included and the calibration curves distributed first, in the middle, and last of
the samples? Click “Next”.
6. Put the samples in correct order in your plate, according to the Rack Positions dialogue.
7. “Eject rack” and place all the samples, references, start up and regeneration solution according to the protocol. Click
“Insert rack” and then “Next”.
8. Check that enough running buffer and water is added, empty the waste bottle. Click “Save as” and enter result file
name.
9. Click “start”.
5. QC and evaluation of results This section describes the steps for quality control (QC) and evaluation of a concentration determination run. After the run,
the data is automatically opened in the Biacore T200 evaluation software. Alternatively, during a run the file can be opened
in the evaluation software through the control software (Tools Biacore T200 Evaluation Software).
17
29-0136-74 AA
5.1. Quality check of the run
1. Check the shape and curve of the sensorgrams (see Figure 8).
Figure 8. Evaluation of data in the Biacore T200 evaluation software. Check for disturbances e.g. air injections. A cycle can be excluded by right clicking on
it. Select to view only certain sensorgrams by “Assay step purpose” or “Cycle”. Under “Tools” in the sensorgram window sensorgrams may be colored
differently, baseline adjusted to zero for all sensorgrams, report point placement made visible etc.
2. Double click on “Binding stability” to the left in Evaluation explorer to view the Binding stability plot (see Figure 9).
- Check approximate serum response level (level of last start up cycle and the calibration points with highest
response levels). Level will decrease a little but should ideal start at approximately 600-2000 RU. At least ~250 RU
response difference between highest and lowest calibration points is recommended for the last calibration curve,
depending on the need for precision in calculated sample concentrations.
- Very high levels are caused by high serum concentration, which will decrease sensitivity (but also allow higher
concentration of calibration to be used, see section 3.2.3.). Very low levels are caused by too high serum dilution,
low affinity serum, too low immobilization level or inactive reagent immobilized.
- For some cases high background response levels, approximately 400 – 1000 RU, remain also when it is clear that
high virus concentrations have indeed inhibited binding to the surface. The calibration curve will potentially reach
a lower plateau, like in Figure 7, but on a high “lowest level” level, e.g. >400 RU. This indicates presence of another
antibody/reagent binding to the surface. High background response levels have been seen when egg derived bulk
vaccine or whole virus have been immobilized. High background could possibly be caused by components in the
preparation used for immunisation.
18
29-0136-74 AA
Figure 9. Response levels (at report point Stability) versus cycle number shown in a Binding Stability plot. By right clicking on a data point the sensorgram
can be viewed and/or a cycle excluded.
3. Manual corrections of accidental errors in set up.
- For instance: Samples were placed in the wrong well or given wrong sample name.
- Choose “Tools”/”Keyword table” in the top bar, and make appropriate changes. Apply and save new settings.
4. Double click on “Baseline sample” to the left in Evaluation explorer to view the Baseline: Sample plot (see Figure 11).
Figure 10. Baseline level versus cycle number shown in a Baseline: Sample plot. The baseline may vary several 100 RU. Variations are considered
acceptable as long as the calibration curves/controls show good assay performance throughout the run. A variation of several 1000 RU however indicates
assay problem.
200
400
600
800
1000
1200
1400
1600
0 10 20 30 40 50 60
Binding Stability
Re
lati
ve R
esp
ons
e -
Sta
bil
ity
3
RU
Cycle Num ber
Calibration
Sample
Startup
19
29-0136-74 AA
5.2. Evaluation
1. Click “Concentration analysis”/”Using calibration” in the top bar (see Figure 11).
Figure 11. To evaluate a concentration analysis assay click “Concentration analysis” in the top bar of the Biacore T200 evaluation software window.
2. Click “Use calibration trends”. Make sure the correct Flow cell is used. Click “Finish” (see Figure 12).
Figure 12. Calibration trends for a concentration analysis opened in the Biacore T200 evaluation software. To obtain log scale for concentration double click
on x-axis and enter e.g. 0.1 and 10 as Min and Max respectively. Check the calculated concentrations, a recovery of 95-105 % is expected (not obtained in
this example for the lowest concentration).
20
29-0136-74 AA
3. View the Calibration Trends tab (see Figure 13).
Figure 13. The Calibration Trends tab for a concentration analysis viewed in Biacore T200 evaluation software. This is mainly a quality control check that
the software has performed a visually reasonable fit for each concentration. If a calibration point is missing for some reason, the data may still be valid
depending on how much responses are decreasing. Concentrations may also be calculated against the preceding or average curve, see Figure 12.
4. View calculated concentrations of samples and select your samples:
- Select samples with responses within the steep part of the calibration curve (for example between 300-750RU in
the curve shown in figure 14). Select samples where preferably a few different dilutions of the same sample should
report similar calculated concentration.
21
29-0136-74 AA
Figure 14. Example showing results from process samples. For sample 1 (upper figure) different calculated concentrations were obtained for the dilutions
x20, x40 and x80. The x40 dilution was judged to be most reliable as these responses were obtained from the steep part of the curve. For sample 2 (lower
figure) the two dilutions x20 and x40 report similar calculated concentrations and both gave responses within the steep part of the curve. Therefore, an
average value from the four calculated concentrations (51.9, 50.66, 52.95 and 51.85) was used.
B
A
22
29-0136-74 AA
6. References Estmer Nilsson C, Abbas S, Bennemo M, Larsson A, Hämäläinen M D, Frostell-Karlsson Å. A novel assay for influenza virus
quantification using surface plasmon resonance. Vaccine (2010) 28, 759–766.
23
29-0136-74 AA
7. Appendix
7.1. Serum dilution test using the Method Builder
This part of the guideline shows you how to create a method in Method Builder step by step.
Start by choosing “Cycle types”, to the left.
1. Keep General settings as default.
2. Create Cycle type “Serum”. Insert command “Sample” and Type: Low sample consumption. Use contact time: 400 s and
flow rate: 5 µl/min. Choose flow path for the immobilization. Insert command “Regeneration” and Type: Regeneration
solution (50 mM HCl, 0.05% P20), contact time: 30 s and flow rate: 30 µl/min. Choose the same flow path as for Sample.
3. Choose “Assay step” to the left. Create assay step “Sample” and connect to cycle type “Serum”.
24
29-0136-74 AA
4. Choose “Setup Run” and Detection 1,2,3,4. Click “Next”.
5. Enter Sample solution (dilutions) starting with the highest dilution factor. The highest dilution is run in triplicates as
startup to stabilize binding responses since the response from the first cycle is not reliable.
6. Click “Next” until the Rack Positions dialogue is reached.
7. Click “Eject rack” and place the samples and regeneration buffer in the positions assigned by the software. Click “Insert
rack” and then “Next”.
8. Check that the HBS-EP+ buffer and water levels will be sufficient for the run. Click “Next”.
9. Choose “Don’t save” the method (the method will be saved with the result anyway). Save the serum dilution test under
appropriate name and click “Start”.
7.2. Robustness of sample buffers
Robustness tests are performed to check for matrix effects from the samples. To run the test, mix different concentrations of
additives that are likely to be present in your samples with a fixed concentration of a standard virus. The virus concentration
should preferably come from the steep part of the calibration curve.
7.2.1. Test of sample buffer robustness
Perform the testing according to the general working protocol outlined in section 4. Replace the samples in section 4.4. with
diluted standard containing additives of different concentrations, for example using the schedule below.
1. Dilute stocks of additives in HBS-EP+ (for example):
a. Sucrose 20%
b. Host cell DNA: 20000ng/ml
c. NaCl 2M
d. Cell culture medium 100%
25
29-0136-74 AA
2. Serial dilute the additives in HBS-EP+ buffer in 1.5 ml plastic tubes (for example):
a. Sucrose: 1.3%, 2.5%, 5%, 10%, 20%
b. Host cell DNA: 2500ng/ml, 5000ng/ml, 10000ng/ml, 20000ng/ml
c. NaCl: 0.25M, 0.5M, 1M, 2M
d. Cell culture medium: 13%, 25%, 50%, 100%
3. Make a stock concentration of the specific standard antigen in HBS-EP+, for example 5µg/ml.
4. Transfer 25µl of the additive solutions from step 2 into a 96 well microplate. Add replicates of all samples. Remember to
transfer control samples without additives as well, e.g. 9 samples with HBS-EP+ only.
5. Add an equal amount of stock antigen (25µl) to each of the additives (final volume 50µl). The final concentration of the
antigen will be 2.5µg/ml. The final concentration of the additive solutions will be:
a. Sucrose: 0.63%, 1.3%, 2.5%, 5%, 10%
b. Host cell DNA: 1250ng/ml, 2500ng/ml, 5000ng/ml, 10000ng/ml
c. NaCl: 0.13M, 0.25M, 0.5M, 1M
d. Cell culture medium: 6.3%, 13%, 25%, 50%
2. Add 50µl of diluted serum (as determined in 3.3) to each sample, seal the plate with microplate foil and continue working
after the general protocol. Run the control samples spread out during the run, for example run 3 controls first, 3 controls
in the middle and 3 controls in the end of the run.
7.2.2. Evaluation of robustness
For detailed information about evaluation, see the evaluation guidelines in section 5. Compare the calculated concentrations
of the samples containing additives with the control samples containing HBS-EP+ buffer only. For example, set the average
of the first and last control to 100 (%), and then make a ratio for each of the samples in the same run (concentration of
sample/concentration of first control x 100). An example of a result is shown in Figure 15.
Figure 15. Example showing results from a robustness testing. The graph shows the concentration compared with expected concentration in percent.
Tested additives were different concentrations of bovine serum albumin (BSA 0.13-1 mg/ml), λ-DNA (1.13-10 µg/ml), gDNA (genomic DNA from MDCK cells
5-0.025 µg/ml), cell culture medium (medium 0.01-10%), sodium chloride (NaCl 0.5-0.15 M) and sucrose (5-1%). The results showed that the sucrose level
should be <1% and the NaCl concentration <0.2M. In addition, gDNA from MDCK cells (>2500 ng/ml) increased the response (decrease the calculated
sample concentration). Lambda-DNA was found not to affect the response.
For local office contact information, visit www.gelifesciences.com/contact www.gelifesciences.com/biacore GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden
GE, imagination at work and GE monogram are trademarks of General Electric Company. Biacore is a trademark of GE Healthcare companies. All third party trademarks are the property of their respective owners. © 2012 General Electric Company — All rights reserved. First published January 2012 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Ltd Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK GE Healthcare Bio-Sciences Corp 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327 USA GE Healthcare Europe GmbH Munzinger Strasse 5, D-79111 Freiburg, Germany GE Healthcare Japan Corporation Sanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073 Japan
29-0136-74 AA 01/2012