3 Methods
In the following, a method for antibody coupling is described, for
which magnetic beads and plates are utilized. For coupling batches
not exceeding the number of positions found in common benchtop
microcentrifuges, we suggest using 1.5-mL tubes. For magnetic
beads, magnetic tube holders are available (Magna GrIP™ Rack
(8 well)) and used to attract and temporarily retain the particles.
Coupling of more than 24 bead IDs in parallel is preferably performed in microtiter plates and use plate magnets to facilitate bead
sedimentation and fixation. Depending on the future usage of the
beads, they can be transferred to tubes or kept in plates.
3.1 Coupling
of Antibodies on Beads
1. Prepare antibodies at the desired concentration (e.g., 2μg or a
solution with antibody concentration of 20μg/mL per 5 Â 10
5
beads) in coupling buffer (see Note 5).
2. Distribute the desired proportion of beads (e.g., 40μL ¼ 5 Â 10
5
beads) into the wells of 96-well plate.
3. Wash the beads with 3Â 100μL activation buffer (see Note 6).
4. Prepare fresh solutions of NHS and EDC, both at 50 mg/mL
in activation buffer. Calculate the use of 0.5 mg of each substance per bead ID and coupling, and prepare a mixture by
combining 10μL of NHS solution, 10μL o EDC solution, and
30μL of activation buffer per well (see Note 7).
5. Incubate for 20 min at room temperature in the dark, under
permanent and gentle mixing on a shaking table (650 rpm),
and wash thereafter with 3 Â 100μL coupling buffer.
6. Continue without interruption by adding the antibody solution to the activated beads and incubate for 2 h under continuous, gentle shaking (650 rpm).
7. The beads are washed 3Â with 100μL of wash buffer.
8. The liquid is removed, and 100μL of storage buffer is added
prior to the bead storage at +4
C in the dark overnight.
3.2 Bead Array
Preparation
and Coupling
Efficiency Test
Theoretically, suspending the starting amount of 5 Â 10
5 beads/
ID in 100μL of storage buffer after the coupling procedure yields a
bead concentration of 2500 beads/ID/5μL. We recommend preparing bead array mixtures combining equal volumes of selected
bead identities so that the theoretical count of each identity is
500 beads/ID/5μL. Thus, a bead array mixture should be
prepared so that each bead ID is diluted 1:50 in storage buffer
into a final volume enough for an extra 10% in terms of the number
of assay wells. The theoretical final concentration will be
100 beads/ID/μL. A bead count can be performed on the final
stock solution to ensure a sufficient number of beads before analysis
146
Lucı ´a Lourido et al.
In the following, a method for antibody coupling is described, for
which magnetic beads and plates are utilized. For coupling batches
not exceeding the number of positions found in common benchtop
microcentrifuges, we suggest using 1.5-mL tubes. For magnetic
beads, magnetic tube holders are available (Magna GrIP™ Rack
(8 well)) and used to attract and temporarily retain the particles.
Coupling of more than 24 bead IDs in parallel is preferably performed in microtiter plates and use plate magnets to facilitate bead
sedimentation and fixation. Depending on the future usage of the
beads, they can be transferred to tubes or kept in plates.
3.1 Coupling
of Antibodies on Beads
1. Prepare antibodies at the desired concentration (e.g., 2μg or a
solution with antibody concentration of 20μg/mL per 5 Â 10
5
beads) in coupling buffer (see Note 5).
2. Distribute the desired proportion of beads (e.g., 40μL ¼ 5 Â 10
5
beads) into the wells of 96-well plate.
3. Wash the beads with 3Â 100μL activation buffer (see Note 6).
4. Prepare fresh solutions of NHS and EDC, both at 50 mg/mL
in activation buffer. Calculate the use of 0.5 mg of each substance per bead ID and coupling, and prepare a mixture by
combining 10μL of NHS solution, 10μL o EDC solution, and
30μL of activation buffer per well (see Note 7).
5. Incubate for 20 min at room temperature in the dark, under
permanent and gentle mixing on a shaking table (650 rpm),
and wash thereafter with 3 Â 100μL coupling buffer.
6. Continue without interruption by adding the antibody solution to the activated beads and incubate for 2 h under continuous, gentle shaking (650 rpm).
7. The beads are washed 3Â with 100μL of wash buffer.
8. The liquid is removed, and 100μL of storage buffer is added
prior to the bead storage at +4
C in the dark overnight.
3.2 Bead Array
Preparation
and Coupling
Efficiency Test
Theoretically, suspending the starting amount of 5 Â 10
5 beads/
ID in 100μL of storage buffer after the coupling procedure yields a
bead concentration of 2500 beads/ID/5μL. We recommend preparing bead array mixtures combining equal volumes of selected
bead identities so that the theoretical count of each identity is
500 beads/ID/5μL. Thus, a bead array mixture should be
prepared so that each bead ID is diluted 1:50 in storage buffer
into a final volume enough for an extra 10% in terms of the number
of assay wells. The theoretical final concentration will be
100 beads/ID/μL. A bead count can be performed on the final
stock solution to ensure a sufficient number of beads before analysis
146
Lucı ´a Lourido et al.