Protein g affinity column

The genes encoding wild-type HA and NA proteins of H1 NC99 (A/New Caledonia/30/1999 (H1N1)), H1 CA 09 (A/California/4/2009 (H1N1)), H2 SING 57 (A/Singapore/1/57 (H2N2)), H5 IND 05 (A/Indonesia/05/05 (H5N1)), and H9 HK 99 (A/Hong Kong/1074/99 (H9N2)), H1 stabilized stem (SS) H1 NC 99 SS, HIV gp120 control protein, and monoclonal Antibodies CR6261, CR8020, FI6v3, and F10 were synthesized^{21 (link)}. The remaining HA SS probes were constructed by overlapping PCR. Genes encoding these proteins were cloned into a CMVR plasmid backbone for mammalian cell expression^{21 (link),22 (link)}. Δstem mutant probe with two point mutations, Ile45Arg/Thr49Arg (Arg point mutations in HA2; H3 numbering), which prevent binding of bNAb like CR6261 or F10 at the conserved H1 stem epitope were generated using site directed mutagenesis (QuikChange II Site-Directed Mutagenesis Kit; Agilent Technologies, CA, USA). Plasmids encoding these proteins were transfected into 293 F cells (a human embryonic kidney cell line) and supernatants were harvested 72–96 hrs after transfection. HA trimers and stabilized stem proteins were purified as previously described^{21 (link),23 (link)}. IgG Antibodies were purified using a Protein G affinity column (GE Healthcare) as described by the manufacturer.

To inhibit MIF activity, HUVECs were cotreated for 24 h with NS1 and either 100 μM p425 (6,6ʹ-[(3,3-dimethoxy[1,1ʹ-biphenyl]-4,4ʹ-diyl)bis(azo)]bis[4-amino-5-hydroxy-1,3-napthalenedisulphonic acid] tetrasodium salt; Calbiochem, La Jolla, CA, USA) or 50 μM ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid; Calbiochem). In addition, a rabbit anti-MIF polyclonal antibody (10 μg/ml) was used in this study and was purified from recombinant MIF-immunized rabbit serum using a protein G affinity column (GE Healthcare), as previously described [26 (link)]. To inhibit HPA-1, OGT 2115 (Tocris Bioscience, Bristol, UK) was used at the indicated concentration. To inhibit MMP-9, SB-3CT (Abcam, Cambridge, UK) and MMP-9 inhibitor I (Santa Cruz, Dallas, TX, USA) were used at the indicated concentrations. Control mouse and rabbit IgGs were purchased from LeadGene Biomedical (Taiwan).

To generate anti-histone H3 (citrulline R2 + R8 + R17) antibodies, a Wistar rat was subcutaneously immunized in the foot pad with a peptide derived from human histone H3 (citrulline R2 + R8 + R17) (2–20: A(cit)RTKQTA(cit)RKSTGGKAP(cit)RKQ) emulsified in adjuvant (TiterMax Gold; TiterMax). Splenocytes were fused with NSO^bcl2 myeloma cells^{61 (link)} using PEG1500 (Roche, Germany). Hybridoma cells were selected in DMEM/10% FCS containing HAT (Sigma) and 5% BM-Condimed (Roche). The hybridoma supernatants were tested using ELISA, and a monoclonal antibody designed to specifically detect human histone H3 (citrulline R2 + R8 + R17) peptides but not human non-citrullinated histone H3 peptides was obtained (11-11B-4F). The IgG fraction was purified using a Protein G affinity column (GE Healthcare), and the buffer exchange to PBS was performed using PD-10 columns (GE Healthcare). The antibody was biotinylated using EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) according to the manufacturer’s recommendations.

Carboxylic acid-modified CdSe/ZnS QDs with a maximum emission wavelength at 620 nm (QD₆₂₀) was purchased from Wuhan Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), glucosamine hydrochloride (glu-NH₂), bovine serum albumin (BSA), ovalbumin (OVA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Agarose gel was from BIOWEST (Logan, UT, USA). RUS was isolated from the tubers of Ophiopogon japonicus by successive chromatographic steps and the purity was 99.6% by HPLC-ELSD. Diammonium glycyrrhizinate, sarsasapogenin, notoginsenoside R1, oleanolic acid and diosgenin with purities of 99.0% were purchased from the China Pharmaceutical Biological Products Analysis Institute (Beijing, China). The anti-RUS mAbs was derived from a hybridoma cell line which secreted stable monoclonal antibodies against RUS and purified by a Protein G affinity column (GE, Stockholm, Sweden) [30 (link),31 (link)]. All the reagents were analytical grade.

The BP patients fulfilled both inclusion criteria: (i) clinical blistering or erosions on the skin and (ii) circulating autoantibodies against COL17 as detected by BP180-NC16A enzyme-linked immunosorbent assay (ELISA)/CLEIA (MBL, Nagoya, Japan). BP-IgG was purified from plasma obtained by apheresis in a severe BP patient. Total IgG was purified using a protein G affinity column according to the manufacturer’s instructions (GE Healthcare, Amersham, UK). In accordance with the Hokkaido University Hospital bylaws and standard operating procedures approved by the Hokkaido University Hospital Review Board, we obtained patient consent for experimental procedures to be performed at Hokkaido University Hospital. A full review and approval by an ethics committee were not required, according to local guidelines. The studies were conducted in accordance with the Helsinki guidelines.

To produce R4.C6 Fab, the hybridoma was grown in serum free medium in a 7 L bioreactor (Southern Biotech, Birmingham, AL, USA). The cleared supernatant was concentrated and applied to a protein-G affinity column following the manufacturer’s protocol (GE Healthcare Life Sciences). Purified R4.C6 was digested with papain (Pierce Fab Preparation Kit, Thermo Fisher Scientific) with mild reduction. Antigen-binding fragments (Fab) were purified from undigested IgG and Fc fragments by ion exchange chromatography (Southern Biotech, Birmingham, AL, USA). The Fab was 98.8% pure as determined by reverse phase HPLC.