Peptide m agarose

To determine the effect of autoantibodies on PAD4 enzymatic activity, IgG and IgA were co-purified from anti-PAD-negative, anti-PAD4 mono-reactive, and anti-PAD3/4 cross-reactive serum and sputum using an equal mixture of Protein A/G agarose (Pierce; cat#20423) and Peptide M agarose (InvivoGen; cat# gel-pdm-2) beads. This mixture purifies predominantly all four IgG subclasses and IgA isotypes, with minimal purification of IgM. The concentration of total Ig was determined by NanoDrop (Thermo), >95% purity confirmed by Coomassie stain, and composition confirmed by immunoblot with goat anti-human IgG antibody and rabbit anti-human IgA antibody (Jackson Laboratories). The effect of purified Ig on PAD4 activity was evaluated using recombinant human PAD4, purified in-house as previously described [18 (link)]. PAD4 (10 nM) was pre-incubated with 1 μM purified Ig for 45 min at 4°C, followed by incubation with 700 μM histone H3 substrate for 3 h at 37°C at increasing calcium chloride concentrations (i.e., 0.2 and 2 mM), as previously described [2 (link)]. Citrullination of histone H3 was evaluated by anti-citrullinated histone H3 immunoblotting (ab5103, Abcam).

The IgM fraction was purified using a home-made IgM resin. Briefly, the IgM antibody (SICGEN ANTIBODIES, AB0405-500) was covalently bonded to glyoxal agarose beads resin (ABT, 6BCL-GM3) according to the manufacturer’s instructions. IgA and IgG from plasma samples were purified through Peptide M/Agarose (Invivogen, 6457-43-01) or Protein G (Thermo Fisher Scientific, 20398), respectively, according to the manufacturers’ instructions. Briefly, 100 μL of plasma was incubated with 200 μL of Peptide M/Agarose, Protein G, or IgM resin for 20 minutes. The resins/beads were washed 5 times with wash buffer (10 mM sodium phosphate, 150 mM sodium chloride; pH 7.2) and eluted in 100 μL fractions with 0.1 M glycine pH 2.76. The pH of the collected fractions was adjusted to 7 with 1 M Tris (pH 8.83). All steps were carried out at 4°C. Western blotting was performed according to standard procedures. Secondary antibodies used were from Abcam, diluted 1:5000: goat anti–human IgA alpha chain HRP (ab97215), goat anti–human IgG Fc HRP (ab97225), and goat anti–human IgM mu chain HRP (ab97205). Protein bands were imaged using ECL on a GE Amersham Imager 680.

Total IgA and IgG was purified from CVL that had been concentrated using Amicon Ultra 15 centrifugal filters and from serum using Peptide M agarose (InvivoGen) and recombinant Protein G (PG) sepharose (GE Healthcare), respectively. Isolation was done by placing 500 μl of a 10% Protein G slurry in PBS in the upper chamber of a 0.45 μm SpinX cellulose acetate microfuge tube (Costar), then centrifuging the tube at 15,000 RCF for 1 minute to remove the PBS. The dry PG in the upper chamber was immediately reconstituted with serum or CVL and placed in a new lower chamber. After 1 hour of mixing, the tube was centrifuged and the IgG-depleted fluid in the lower chamber was placed on ice for later isolation of IgA. The PG-bound IgG in the upper chamber was washed 2 times with PBS. The IgG was then eluted into the lower chamber by adding 100 μl of 0.1 M glycine, pH 2.5, and centrifuging after 1 minute, then repeating this procedure. The 200 μl of IgG in the lower chamber was immediately neutralized using saturated Tris base. The IgA in the initial flow-through was then similarly purified using a 20% slurry of Peptide M. The purified IgG and IgA preparations were dialyzed overnight in 500 ml of PBS at 4°C, then sterile-filtered using sterile 0.22 μm SpinX cellulose acetate microfuge tubes (Costar) and stored at 4°C.