Epoxy Bead Labeling
PEGylated BMS drug A was directly conjugated to epoxy beads (BioScale’s AMMP Type II Labeling Kit) per manufacturer’s procedure.
Streptavidin Bead Labeling
An AMMP Type I Labeling Kit was used for binding of biotinylated PEG of 20 and 40 kDa to paramagnetic streptavidin beads. The method for binding biotinylated PEG to the beads was modified slightly. Briefly, biotinylated PEG was diluted in PBS buffer and reacted with streptavidin beads. The manufacturer’s recommendations were followed except that biotinylated bovine serum albumin (BSA) was not used to saturate open binding sites that were not specifically blocked with a biotinylated reagent.
Protein A Detection With or Without Off-Line Bead Wash
The ViBE platform, for this assay, uses Protein A on a piezoelectric membrane to capture the antibody complex. In the detection step, bound antibody complexes are separated from other matrix components present in the sample by magnetically attracting the beads to the membrane surface, but allowing beads without bound anti-PEG to fall away from the protein A-coated membrane as the magnet is then removed.
Experiments were designed to compare the assay with or without off-line wash, that is, homogenous vs. a non-homogeneous assay. PEG.2 dilutions ranging from 0.250 to 4.0 μg/mL were prepared in PBS with 1% BSA (w/v) and incubated with epoxy beads conjugated with drug A for 1 h. The plate was then placed either on a BioTek washer equipped with a magnet block to allow aspiration of non-binding components, or analyzed on the ViBE without the wash step. In both cases, a Protein A surface was used for detection.
Multiple buffers were tested for suitability, and three buffers (Blocker Casein in PBS, Super Block in PBS, and Super Block in TBS) were further tested for bead blocking and sample dilution. Conjugated type I beads were diluted in each buffer and blocked for 2 h at ambient temperature prior to use. Control samples were prepared by adding PEG.2 positive control to normal human serum pool at 0.625 to 40 μg/mL and diluted to a final concentration of 5% (v/v) in each of the buffers. Detection of the controls was evaluated using both sets of biotin-20 kDa PEG and biotin-40 kDa PEG type I beads (20 and 30 μg PEG/mg beads) without off-line wash. Blocking and serum dilutions were paired for these comparisons.
Assay Sensitivity and Reproducibility Using Optimized Assay Format
The optimized assay format using 10% serum (v/v) is shown in Fig. 1. Control samples were prepared by adding PEG.2 positive control to normal human serum pool at 0.625 to 40 μg/mL and stored at −70°C for 24 h prior to use. The spiked controls were thawed at room temperature and diluted tenfold in Blocker Casein in PBS. To perform the assay, Biotin-PEG 20 kDa labeled beads were first diluted in Blocker Casein in PBS and incubated for 1.5 h at room temperature. Then, 80 μL of each positive control diluted to 10% serum were combined with 40 μL of blocked bead solution in a 96-well polypropylene plate and incubated for 1 h on the ViBE instrument integrated shaker. Once the incubation was complete, the online assay steps were initiated and data were collected by the ViBE software version 0.7.4.14126. The controls were run a total of 12 times over 2 days, one plate per day. Each plate contained six replicates. The mean response and intra- and inter-run %CV were calculated.