Glycosylation of the Fc domain name is an important driver of antibody effector function. of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such populace level glycosylation screening. We demonstrate the power of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain name. to mis-orient IgG; however, microbes possessing a number of diverse alternative means of antibody-evasion exist (Collin and Killian, 2014). Enzymes such as IdeS and SpeB restrict the Fc domain name by cleaving Abs in their hinge region, Tozadenant and a number of glycosidases with activity against IgG and IgA glycans have been recognized. While one of these glycosidases, EndoS is usually relatively specific to the IgG Fc domain name (M. Collin and Olsen, 2001) and is consequently of interest in therapy of antibody-mediated autoimmune diseases (Collin et al., 2008) Endo S cleaves the IgG glycan after the N-linked acetylchitobiose core, which is variably fucosylated. Thus, its use as an alternative to the pan N-glycosidase PNGaseF is limited by the producing loss of resolution of core-fucosylation, which is known to dramatically modulate IgG Fc binding to FcR3a and FcR3b. Nonetheless, collectively these microbial defense mechanisms represent useful biotechnological tools for IgG glycan analysis. Here, IdeS (von Pawel-Rammingen et al., 2002), a hinge protease, was chosen as a means to separate antibody Fc for glycan profiling. In this work, we present a 96 well plate-based method for microscale purification of antigen-specific antibodies in high throughput, suitable for profiling of large-scale, population-based studies, such as vaccine trials or clinical cohorts. In addition, the method can be used to separately elute the Fc domain name alone without additional actions, via an on-resin digestion with the IdeS enzyme that cleaves the hinge portion of the antibody. With this method we demonstrate isolation of various antigen-specific antibodies from human and non-human primate (NHP) samples in sufficient yield to permit highly quantitative chromatography-based glycan analysis. This method has proved useful across a variety of forms Chuk of antigens, including peptides, and for purification of even epitope-specific antibodies. We have been able to quantify routine antigen-specific Ab enrichment of several hundred fold over serum concentrations in clinically relevant settings, as well as the ability to obtain useful glycan data from relatively small sample volumes (200 L of plasma). 2. Materials and methods 2.1. Sample processing IgG from human or NHP plasma was either separated from other common serum proteins via Melon Gel purification according to the manufacturer’s instructions (Thermo 45214) or simply diluted 10-fold in PBS and then filtered through a 0.22 m syringe filter (Millipore SLGP033RB). Filtered or purified samples were then concentrated to approximately 10 mg/mL total antibody concentration via centrifugal concentration (Amicon UFC801024). Pooled polyclonal human IgG from healthy donors, IVIG, Sigma (#I2511-10 mg), and HIV-infected subjects, HIVIG (NIH AIDS Reagent Program #3957), were used as controls. 2.2. Preparation of affinity resin cartridges HIV gp41, gp120, p24, and influenza HA antigens (Immune Technologies IT-001-005p, IT-001-0027p, IT-001-017p and IT-003-0011p), and SIVmac239 gp120 (IT-001-022p) were Tozadenant diluted to 0.1 mg/mL in 20 mM Tris pH 8.2 to which a 5-fold molar Tozadenant excess of 10 mM Sulfo-NHS-Biotin (Themo 21335) dissolved in dH2O was added. Biotinylation was allowed to proceed for 1 h at RT with end-over-end mixing. To remove extra biotin, the biotinylated antigen was then buffer exchanged 3 times into Phosphate Buffered Saline (PBS) using Amicon spin concentrators with 10 min spins at 3000 (Amicon UFC801024); final volumes were brought up with PBS to establish a biotinylated antigen concentration of 0.5 mg/mL. A synthetic N-terminally biotinylated cyclic SIVsmE543 V2 peptide (Barouch et al., 2012) (JPT Peptide Technologies GmbH); GenBank “type”:”entrez-nucleotide”,”attrs”:”text”:”U72748″,”term_id”:”71025136″U72748) was similarly prepared in PBS. Agilent Bravo Streptavidin Cartridges (Agilent G5496-60010) were loaded into the provided 96-well cartridge racks and 170 L of PBS was added to each tip. Loaded tip racks were placed in receiver plates and spun at 1000 for 2 min. A.