Covalent coating of IgG subclass proteins onto Luminex® beads
We covalently coated purified human IgG1 protein (cat#: 400120), IgG2
protein (cat#: 400122), IgG3 protein (cat#: AG506), and IgG4 protein
(cat#: 400126) (MilliporeSigma, St. Louis, MO) onto different Luminex®
MicroplexTM microspheres (or beads) (cat#:
LC10050-01; Luminex Corp., Austin, TX) using the xMAP® Antibody Coupling
Kit (cat#: 40-50016, Luminex Corp., Austin, TX). Briefly, the covalent
coupling is achieved through carbodiimide reactions between the primary
amino groups on the IgG subclass proteins and the carboxyl functional
groups on the beads surface. We removed competing substances from the
stock solution of purified IgG subclass proteins using desalting columns
(cat#: 89877, ThermoFisher Scientific, Waltham, MA), and resuspended
the proteins in 1X PBS (pH 7.4) prior to the coupling reactions. We
followed the manufacturer’s recommendations to covalently coat IgG1
proteins on bead region 90, IgG2 on bead region 92, IgG3 on bead region
93, and IgG4 on bead region 95. Overall, we produced 12 types of
specific beads, where four different proteins (IgG1, IgG2, IgG3, and
IgG4) were coated on the beads at three different concentration (0.5μg,
1μg, and 2μg per 1x106 beads).