APP_AlphaLISAAcceptorBeadConjugationProtocol

Alpha Technology
Antibody Conjugation to AlphaLISA® Acceptor Beads
Detailed Protocol
Recommended Reagents and Materials
*Equivalent sources can be used.
Item
Suggested source
Catalog #
Antibody
N/A. Specific to the target of
interest
N/A
AlphaLISA Acceptor beads
PerkinElmer, Inc.
6772001 (1 mg)
6772002 (5 mg)
6772003 (50 mg)
100 mM Hepes pH 7.4
To prepare
N/A
10% Tween-20
(Surfact-Amps 20)
Thermo Fisher Scientific Inc.
28320
Na cyanoborohydride powder
Sigma-Aldrich Co.
156159
Carboxymethoxylamine (CMO)
Sigma-Aldrich Co.
C13408
100 mM Tris-HCl pH 8.0
To prepare
N/A
800 mM NaOH
To prepare
N/A
Item
Suggested source
Catalog #
Proclin™-300 or Kathon® CG/ICP II
Sigma-Aldrich Co.
48912-U / 48178-U
PBS 1X pH 7.4
Invitrogen (Life Technologies)
10010
1X PBS + 0.05% Proclin-300
To prepare
N/A
Bench centrifuge
Eppendorf AG
5145 R
Sonicator
Fisher
F60
Rotary shaker (Labquake®)
Barnstead Int.
400110
LAMBDA™ 25 UV/VIS
spectrophotometer
PerkinElmer, Inc.
L6020060
Procedure Outline
Antibody assesment and
pre-treatment (if needed)
Assemble Conjugation Rx
(±30 min.)
Overnight Incubation
(18-24h)
CMO blocking step
(60 min.)
Washing steps
(±60 min.)
Note: This procedure can be also used with AlphaScreen Donor and Acceptor beads. Donor beads should be
handled under subdued light.
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1.
ANTIBODY ASSESSMENT AND PRE-TREATMENT
A preliminary check of the antibody to be conjugated is mandatory. The User must check for the
followings:
1.1
1.2
Antibody concentration

To accurately determine the concentration of the antibody, please refer to the method
described in Appendix 1.

The conjugation will perform best when the antibody concentration is at least 1 mg/mL
(when conjugating 1-2 mg of beads) or 0.53 mg/mL (when conjugating 2.5 mg of beads or
higher amounts). Lower concentrations of antibody yield lower coupling efficiency.

If required, antibody solutions can be concentrated using an iCON Concentrator (Thermo
Fisher Scientific Inc.) according to the manufacturer’s instructions
(www.piercenet.com/files/2177as8.pdf) (or equivalent device).
Antibody formulation

The antibodies must not be in any amine-based buffer, including Tris, glycine, bicine,
tricine, etc.

If buffer exchange is necessary, the buffer should be replaced by a neutral to slightly
alkaline buffer, such as PBS or carbonate buffer pH 8.0. Although both buffers can be
utilized, phosphate buffer will be used in the following protocols. See Appendix 2 for buffer
exchange protocol using Zeba™ Desalt Spin Columns.

Ideally, antibody solutions should not contain any protein or peptide-based stabilizers (such
as BSA or gelatin). It has been shown that BSA concentrations up to 0.1% in the antibody
solution do not impact bead coupling. In the presence of higher concentrations of BSA, the
conjugation process might result in lower coupling efficiency in some cases, and should be
avoided if possible.

If removal of protein stabilizers is necessary, we suggest the use of PhyTip affinity columns
(www.phynexus.com/technology/affinity-columns) on a liquid-handling system, such as the
JANUS® Automated Workstation from PerkinElmer Inc.

Glycerol will significantly impact coupling efficiency. For that reason, extensive dialysis of
the antibody is recommended prior to coupling if the antibody is stored in glycerol.
Alternatively, sequential purification on two Zeba Desalt Spin Columns can be used
(Appendix 2).
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2.
BEAD CONJUGATION REACTION
The ratio of antibody to mg of beads is an important parameter for successful assay development.
Typical coupling weight ratios (amount of beads to amount of antibody) are either 10:1 or 50:1.
When preparing low amounts of beads (1-2 mg), a 10:1 ratio is recommended (i.e. 1 mg of Acceptor
beads to 0.1 mg of antibody), while a ratio of 50:1 is used with bead amounts equal to or higher than
2.5 mg to minimize the antibody consumption (i.e. 5 mg of Acceptor beads to 0.1 mg of antibody).
Please note that the 50:1 protocol was also successfully used with 1 mg of beads, although smaller
vials (0.5 mL Eppendorf tubes) are recommended due to the low volumes involved.
2.1
Protocol for conjugating 1 mg AlphaLISA Acceptor beads (10:1 coupling ratio)
This procedure is appropriate for an antibody solution of ≥ 1 mg/mL.
Bead washing
In a 1.5 mL Eppendorf tube, wash AlphaLISA Acceptor beads (50 L at 20 mg/mL) once: add
50 L PBS, centrifuge at 16,000 × g (or maximum speed) for 15 min and then discard the
supernatant using a pipet tip. (Do not tilt the tube to prevent the pellet from coming off).
Conjugation
Prepare a fresh working NaBH3CN solution at 400 mM in water using NaBH3CN powder (25
mg in 1 mL H2O).
In the Eppendorf tube containing 1 mg of AlphaLISA Acceptor bead pellet (prepared as
described above), add:

0.1 mg of antibody

the appropriate volume of 100 mM Hepes pH 7.4 to obtain a final reaction volume of 200
µL

1.25 L of 10% Tween-20

10 µL of a 400 mM solution of NaBH3CN in water
Incubate for 18-24 hours at 37ºC with mild agitation (6 – 10 rpm) using a rotary shaker.
Blocking

Prepare a fresh 65 mg/mL solution of carboxymethoxylamine (CMO) in 800 mM NaOH.

Add 10 µL of CMO solution to the reaction (to block unreacted sites).

Incubate for 1 hour at 37ºC using a rotary shaker (6 – 10 rpm).
Washing

Centrifuge for 15 minutes at 16,000 × g (or maximum speed) at 4ºC.

Remove the supernatant with a micropipette and resuspend the bead pellet in 200 L of
100 mM Tris-HCl pH 8.0.

Briefly sonicate the bead solution (10 short pulses of 1 second using a probe sonicator to
ensure that the beads are not aggregated; the sonicator power should not exceed 20% of
the maximal power).
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
Centrifuge for 15 minutes at 16,000 × g (or maximum speed) at 4ºC, and then remove the
supernatant.

Repeat the washing step (resuspend the pellet, sonicate and centrifuge) another time.

After the last centrifugation, resuspend the beads at 5 mg/mL in storage buffer (200 L of
PBS + 0.05% Proclin-300 as a preservative).

Vortex, briefly spin down and sonicate the bead solution (10 short pulses of 1 second
using a probe sonicator; the sonicator power should not exceed 20% of the maximal
power).
The sonication steps are recommended but not mandatory. Note that a bath sonicator is
not efficient and should not be used.
Storage
2.2

Store the conjugated Acceptor bead solution at 4ºC in an opaque vial.

Important note: always vortex conjugated AlphaLISA Acceptor beads before use, as
beads tend to settle with time.
Protocol for conjugating 5 mg AlphaLISA Acceptor beads (50:1 coupling ratio)
This procedure is appropriate for a solution of antibody ≥ 0.53 mg/mL.
Washing
In a 1.5 mL Eppendorf tube, wash AlphaLISA Acceptor beads (250 L at 20 mg/mL) once: add
250 L PBS, centrifuge at 16,000 × g or maximum speed for 15 min and then discard the
supernatant using a pipet tip. (Do not tilt the tube to prevent the pellet from coming off).
Conjugation
Prepare a fresh working NaBH3CN solution at 400 mM in water using NaBH3CN powder (25
mg in 1 mL H2O).
In the Eppendorf tube containing 5 mg of AlphaLISA Acceptor bead pellet (prepared as
described above), add:

0.1 mg of antibody

the appropriate volume of 100 mM Hepes pH 7.4 to obtain a final reaction volume of 200
µL

1.25 µL of 10 % Tween-20

10 µL of a 400 mM solution of NaBH3CN in water
Incubate for 18-24 hours at 37ºC using a rotary shaker (6 – 10 rpm).
Blocking

Prepare a fresh 65 mg/mL solution of carboxymethoxylamine (CMO) in 800 mM NaOH.

Add 10 µL of CMO solution to the reaction (to block unreacted sites).
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
Incubate for 1 hour at 37ºC using a rotary shaker (6 – 10 rpm).
Washing

Centrifuge for 15 minutes at 16,000 × g (or maximum speed) at 4ºC.

Remove the supernatant with a micropipette and resuspend the bead pellet in 1 mL of
100 mM Tris-HCl pH 8.0 (200 L per mg of beads).

Briefly sonicate the bead solution (10 short pulses of 1 second using a probe sonicator
to ensure that the beads are not aggregated; the sonicator power should not exceed
20% of the maximal power).

Centrifuge for 15 minutes at 16,000 × g (or maximum speed) at 4ºC, and then remove
the supernatant.

Repeat the washing steps (resuspend the pellet, sonicate and centrifuge) another time.

After the last centrifugation, resuspend the beads at 5 mg/mL in storage buffer (1 mL of
PBS + 0.05% Proclin-300 as a preservative).

Vortex, briefly spin down and sonicate the bead solution (10 short pulses of 1 second
using a probe sonicator; the sonicator power should not exceed 20% of the maximal
power). This step is recommended but not mandatory.
The sonication steps are recommended but not mandatory. Note that a bath sonicator is
not efficient and should not be used.
Storage
2.3

Store the conjugated Acceptor bead solution at 4ºC in an opaque vial.

Important note: always vortex conjugated AlphaLISA Acceptor beads before use, as
beads tend to settle with time.
Protocol for conjugating large amounts of AlphaLISA Acceptor beads
If more than 5 mg of beads are to be conjugated, adapt the 5 mg protocol accordingly. In
addition:

Use 50 mL 3118 Oak Ridge centrifuge tube from Nalgene (Thermo Fisher Scientific,
Cat. # 3118-0050) instead of 1.5 mL Eppendorf tubes. The maximum volume in each
tube should be ≤ 30 mL to allow proper centrifugation of the beads.

Perform all centrifugations steps in a Sorvall RC-5B centrifuge (or equivalent) at 16,000
rpm for 40 min at 4ºC.
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2.
BEAD COUPLING OPTIMIZATION
Although the suggested coupling protocols presented in the previous sections work well for most
conditions, the bead coupling process can be optimized further with the goal to increase the number
of antibodies on beads, by following these recommendations:

Reduce the volume of reaction. Coupling efficiency will usually increase with the bead
concentration. For instance, the bead concentration could be tested up to 100 mg/mL
(compared to 25 mg/mL in the protocol described in Section 5). Adding less volume of a
more concentrated buffer could help to achieve this increased concentration.

Increase the antibody/beads ratio. Coupling efficiency will usually increase with the
antibody concentration. However, the antibody stock solution should be concentrated
enough to allow the addition of a minimal volume of antibody while maintaining the bead
concentration as high as possible. Therefore, the 10:1 ratio, when performed at 25
mg/mL of beads should improve results. By increasing bead concentration to 75 mg/mL,
while maintaining the ratio at 50:1, an antibody concentration of 1.7 mg/mL is required.
By increasing simultaneously the ratio to 25:1, twice that concentration of antibody is
needed, which lies close to the upper practical limit.

Try other buffers, such as 100 mM sodium phosphate, pH 8.0.
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Appendix 1: Determination of antibody concentration

Measure the OD of the antibody solution at 450 nm and 280 nm on a spectrophotometer (scan).

Use the 1X PBS, pH 7.4 buffer to prepare the blank and dilutions, if necessary.

The OD value at 280 nm must be within the linear limits of the device used (e.g. 0.200 and 1.800 on the
spectrophotometer). Repeat dilutions as needed.

An A450 reading ≥ 0.1 possibly indicates protein aggregation or the presence of a precipitate in the
antibody solution which can be removed by centrifugation. If necessary, centrifuge at 16,000 x g for 10
minutes at 4°C. Transfer the supernatant and measure OD at 280 nm.

Formula to calculate the antibody concentration:
(OD280 x dilution factor) ÷ 1.34 mg/mL* = Antibody concentration (mg/mL)
(considering that 1 unit OD280 = 0.75 mg/mL protein)
(*The 1.34 mg/mL concentration comes from the following calculation:
-1
Ext. coeff. / molar mass of the Ab = 214,400 M /160,000 g/mol = 1.34 mg/mL)
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Appendix 2: Buffer exchange using Zeba™ Desalt Spin Columns
Selection of column
The choice is based upon the initial volume of the sample. A combination of many columns (alike or different)
can be used to accommodate the sample volume.
Column
Sample volume
Source and Catalog #
Zeba Desalt Spin
Column, 0.5 mL
30 – 130 L
Pierce # 89882 or 89883
(25 or 50 columns)
Zeba Desalt Spin
Column, 2 mL
200 – 700 L
Pierce # 89889 or 89890
(5 or 25 columns)
Zeba Desalt Spin
Column, 5 mL
500 – 2000 L
Pierce # 89891 or 89892
(5 or 25 columns)
Zeba Desalt Spin
Column, 10 mL
700 – 4000 L
Pierce # 89893 or 89894
(5 or 25 columns)
Column preparation

Remove the column’s bottom closure and loosen the cap (do not remove the cap). Place the column in
an appropriate collection tube.

Centrifuge the column (1 min at 1,500 x g at 4°C) to remove the storage solution.

Place a mark on the side of the column where the compacted resin is slanted upward when using fixedangle rotors. Place the column in the centrifuge with the mark facing outward in all subsequent
centrifugation steps. (Note: resin will appear compacted after centrifugation.)

Add buffer (1X PBS pH 7.4) to the column

Column
Volume of 1X
PBS
0.5 mL
300 µL
2 mL
1 mL
5 mL
2.5 mL
10 mL
5 mL
Centrifuge to remove the buffer:
o
0.5 mL columns: 1,500 x g for 1 min at 4°C
o
2 – 10 mL columns: 1,000 x g for 2 min at 4°C
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
Repeat the addition of buffer and centrifugation steps four additional times, discarding buffer from the
collection tube after each cycle.
Buffer exchange



Place the column in a new collection tube, remove the cap and slowly apply the sample to the center of
the compact resin bed. To ensure maximal protein recovery from low-volume samples, apply a stacker of
buffer to the resin bed after the sample has fully absorbed. (The stacker is only applied if the sample
volume is lower than indicated below.)
o
0.5 mL columns: for samples < 70 L, add 15 L buffer
o
2 mL columns: for samples < 350 L, add 40 L buffer
o
5 mL columns: for samples < 750 L, add 100 L buffer
o
10 mL columns: for samples < 1500 L, add 200 L buffer
Centrifuge to collect the sample
o
0.5 mL columns: 1,500 x g for 2 min at 4°C
o
2 – 10 mL columns: 1,000 x g for 2 min at 4°C
Discard column after use.
Determination of antibody concentration

Measure the OD of the product at 450 nm and 280 nm on a spectrophotometer after pre-treatment, as
described in Appendix 1. Use the buffer in which the antibody is diluted to make the blank and the
dilution.
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