Captureselect iga

Methods for IgA1 Fab profiling have previously been extensively detailed (3 (link), 4 (link)). Briefly, all IgA was captured using CaptureSelect IgA affinity matrix (Thermo Fisher Scientific). Human milk samples were assumed to contain 0.8 μg/μL SIgA and added to excess amount of bead slurry, PBS, and 200 ng of the monoclonals anti-CD20 mIgA1 (7D8-IgA1) and anti-cMET (5D5v2-IgA1). These monoclonals were used as internal standards for quantification, and were a gift from Genmab (Utrecht, NL). Samples were incubated followed by removal of the flow through, containing all non-IgA human milk components. The samples were then washed several times and IgA was digested overnight with the O-glycopeptidase from Akkermansia muciniphila, OgpA (OpeRATOR®, Genovis, Llund, Sweden). Digestion was performed using 40 U SialEXO (a sialidase cocktail to remove sialic acids from the O-glycans) and 40 U of OgpA enzyme, and incubated overnight at 37°C, in an Eppendorf thermal shaker (Eppendorf, The Netherlands). Following overnight digestion with OgpA, Ni-NTA agarose slurry was added to the samples to bind the enzyme and incubated for 30 min. Finally, the flowthrough, containing the IgA1 Fabs, was collected by centrifugation.

Human plasma Visucon-F was obtained from Affinity Biologicals (Ancaster, ON, Canada). Affinity matrix beads for IgG (CaptureSelect FcXL, capacity 25–35 g/L) and IgA (CaptureSelect IgA, capacity 8 g/L) were obtained from ThermoFisher Scientific (Leiden, Netherlands). All used chemicals were from Sigma-Aldrich (Zwijndrecht, Netherlands) except for trifluoroacetic acid (Merck, Darmstadt, Germany) and acetonitrile (Biosolve, Valkenswaard, Netherlands). Purified water was used from a Purelab Ultra system (Veolia Water Technologies Netherlands B.V., Ede, Netherlands). Sequencing-grade trypsin was obtained from Promega (Madison, WI).

Human nasal IgA and recombinant IgG and IgA antibodies were subjected to glycan analysis. Human nasal IgA was purified from nasal wash concentrates using CaptureSelect IgA (Thermo Fisher Scientific), according to the manufacturer’s instructions. Purified antibodies were concentrated using Amicon Ultracell (Merck, Darmstadt, Germany) centrifugation units with a cut-off of 30 kDa. The human nasal wash concentrate samples used here were residual samples collected from a past clinical study [60 (link)]. Antibodies were deglycosylated by PNGase F (Takara Bio, Shiga, Japan) treatment. The released N-glycans were purified and labeled with 2-aminobenzamide using BlotGlyco (Sumitomo Bakelite, Tokyo, Japan), according to the manufacturer's instructions. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed by Sumitomo Bakelite using a LC-MS-IT-TOF apparatus (Shimadzu, Kyoto, Japan). N-glycan structures were predicted using the GlycoMod Tool (http://web.expasy.org/glycomod/) [61 (link)].