Bead-antibody coupling

Luminex beads are carboxyl-terminated polystyrene microspheres available with or without a superparamagnetic core. The magnetic beads are 6.5 μm in diameter while the non- magnetic beads are 5.6 μm in diameter.10,11 _{Early work described in Chapter 2 used non-} magnetic beads, while all subsequent experiments used magnetic beads. The beads are impregnated with two different fluorescent dyes. By varying the concentration ratio of both dyes, Luminex produces 100 unique bead types (called “regions”) suitable for up to 100-plex assays.12 A custom optical filter cube (632 nm/22 nm excitation, 685 nm dichroic, 677 nm/50

nm emission) purchased from Semrock (Lake Forest, IL) was used for distinguishing between beads containing different concentrations of Luminex’s infrared-emitting dye.

Through use of the carboxyl groups covering the beads’ surfaces, antibodies and other ligands of interest can be covalently bound to the beads. Antibody linkage to the beads was done via the carbodiimide chemistry of EDAC and sulfo-NHS. EDAC is a heterobifunctional cross- linker, one end binding to the carboxyl groups on the beads, and the other binding to surface amine groups on amino acid residues in immunoglobulin-G (IgG) antibodies. Sulfo-NHS is used in conjunction with EDAC to yield a less labile chemical intermediate, which allows for a much longer bead-antibody coupling period and thus greater coupling efficiency.13

2.2.2.1 Coupling procedure for non-magnetic beads

The procedure for coupling antibodies to beads was provided by the vendor14 and is summarized here. It utilizes four buffers that are prepared prior to each bead coupling. The activation buffer is 100 mM sodium phosphate monobasic at a pH of 6.2. The coupling buffer is 50 mM MES at a pH of 5.0. Sodium hydroxide and hydrochloric acid were used to adjust buffer pH. The wash buffer is PBS + 0.05% Tween-20. The storage/blocking buffer is PBS + 0.1% (BSA) + 0.02% Tween-20 with 0.05% sodium azide as a preservative. The stock beads are at a concentration of 12.5 million beads/mL. All buffers and beads were brought to room

temperature prior to use. Region 1 beads were used with the control antibody and region 50 beads were used with the VEGF capture antibody.

Stock beads were vortexed at 3000 rpm for 30 s and sonicated for 15 s. 5 million beads (400 µL bead stock) were transferred to a 500-µL LoBind microcentrifuge tube (Eppendorf, Hauppauge, New York) and pelleted by centrifuging them at 8,000 rcf for 3 min. The supernatant was removed and replaced with 100 µL of wash buffer. The beads were

resuspended by vortexing them for 30 s at 3000 rpm, then pelleted. The supernatant was removed and replaced with 80 µL of activation buffer. 10 μL of 50 mg/mL sulfo-NHS and 10 μL of 50 mg/mL EDAC (both prepared in activation buffer) were added and the beads were vortexed at 3000 rpm for several seconds. The beads were activated by placing them on a plate shaker (MS1 S7, Fisher Scientific) at 1000 rpm for 20 min at room temperature. The activated beads were pelleted and the supernatant was replaced with 250 µL of coupling buffer. The beads were resuspended and then pelleted. The supernatant was removed and replaced with 250 µL of coupling buffer. 16 µg capture antibody (prepared in PBS) was added and the total volume of the mixture was brought to 500 µL with coupling buffer. The bead tube was covered with foil, gently vortexed, and placed on a 25 rpm rotator (H5500; Labnet International Inc., Edison, NJ) for 2 h at room temperature to couple the beads to the antibodies.

Coupled beads were pelleted and resuspended in 500 µL storage/wash buffer. They were then pelleted, resuspended in 1 mL storage/wash buffer, and this step was repeated once more. The beads were finally resuspended in 250 µL storage/wash buffer and stored at 4 ºC until use. 2.2.2.2 Coupling procedure for magnetic beads

The procedure for coupling antibodies to beads was provided by Bio-Rad.15 The buffers used for coupling antibodies to magnetic beads are the same as those for the non-magnetic beads, except the coupling buffer is PBS at a pH of 7.4. The stock beads are at a concentration of 12.5 million beads/mL. All buffers and beads were brought to room temperature prior to use.

Stock beads were vortexed at 3000 rpm for 30 s and then sonicated for 15 s. 100 μL of stock beads were transferred to a 500-μL LoBind microcentrifuge tube and pelleted on the bottom of the tube with a large rare-earth magnet. The supernatant liquid was removed and replaced with 100 μL of wash buffer. The beads were resuspended in the wash buffer by

vortexing at 3000 rpm for 30 s and then pelleted with the magnet. With the bead tube still on the magnet, the wash buffer was removed and replaced with 80 μL of activation buffer, then the beads were vortexed at 3000 rpm for 30 s. 10 μL of 50 mg/mL sulfo-NHS and 10 μL of 50 mg/mL EDAC (both prepared in activation buffer) were added and the beads were vortexed at 3000 rpm for several seconds. The beads were then placed on a plate shaker (MS1 S7, Fisher Scientific) at 1000 rpm for 20 min at room temperature. 150 μL of coupling buffer was added and the beads were pelleted on the magnet. The supernatant liquid was removed and then 150 μL of coupling buffer was added. The beads were resuspended by vortexing at 3000 rpm and pelleted again. The supernatant was removed and replaced with 100 μL of coupling buffer. 9 µg of capture antibody (prepared in PBS) was added and the total volume of the solution was

brought to 500 μL with coupling buffer. The beads were vortexed and then incubated on a 25 rpm rotator for 2 h at room temperature to couple them to the antibody.

The coupled beads were pelleted from solution and the supernatant was removed. 250 μL of storage/blocking buffer was added and the beads were vortexed at 1500 rpm for 15 s. The beads were pelleted, the supernatant was removed, and 500 μL of storage/blocking buffer was added. The beads were vortexed for 20 s at 1500 rpm and pelleted again. The supernatant was removed and 100 μL storage/blocking buffer was added. The antibody-coupled beads were stored at 4 ºC when not in use.