Keyhole limpet hemocyanin (klh)

Three mg of purified B. melitensis LPS was used for conjugation with KLH (SoluLink, Inc., San Diego, CA). Briefly, periodate-treated B. melitensis 16M LPS was linked to succinimidyl 4-hydrazinonicotinate (SANH)-modified KLH. To estimate conjugation efficiency, KLH-conjugated B. melitensis LPS was analyzed on 10% Bis-Tris SDS PAGE gels (Invitrogen Corp., Carlsbad, U.S.A) under reducing conditions and stained with periodic acid silver that detects LPS.
Monoclonal antibodies were raised against KLH-conjugated B. melitensis LPS using standard methods [43] (link), [44] . The primary screen for hybridoma supernatants was an ELISA using wells coated with B. melitensis LPS. Positive hybridoma supernatants were isotyped and IgG isotypes chosen for further study (Monoclonal Antibody Isotyping Kit, Pierce, Rockford, IL).

The primary antigen KLH (Thermo Fisher Scientific, Rockford, IL, USA) was prepared as followed; KLH was dissolved in ultrapure water, left on ice for 30 minutes and dialyzed (Thermo scientific, Rockford, IL, USA) against PBS followed by a 30 minutes centrifugation (3220 x g) to remove undissolved particles. The final assay concentration for KLH was 30 μg/ml. Protective antigen (PA) from Bacillus anthracis (List Biological Labs, Campbell, CA, USA) was used in the assay at a final concentration of 3 μg/ml. Cytomegalovirus pp65 protein (CMV; Miltenyi Biotec, Lund; Sweden) was used in the assay at 2 μl/ml (concentration unknown). Infliximab (10 mg/ml), rituximab (10 mg/ml), adalimumab (50 mg/ml) and natalizumab (20 mg/ml) were obtained from the ABIRISK consortium (Novartis, Basel, Switzerland). The BPs were aliqouted and stored at -80°C according to the instructions provided. The Abs were used in the assay at 45 μg/ml (0.3μM).

Peptides were synthesized by RS Synthesis (Louisville, KY), HPLC purified to >95%, and amino acid sequence verified by mass spectrometry. Peptides were conjugated to maleimide activated Keyhole Limpet Hemocyanin (KLH) per manufacturer’s instructions (ThermoFisher, Waltham MA). Specific peptides used to generate antisera were as follows: Cys32 human PIGBOS(32-42)-NH₂, CAKDQKELKEK- NH₂; Cys32 rat PIGBOS (32–54), CSRDQKELKELVKILQESEEKRS.

Keyhole limpet hemocyanin (KLH) was purchased from Thermo Fisher Scientific (Brebières, France). Rtx (Mabthera^®) was purchased from Roche (Neuilly, France) and Ifx (Remicade^®) from Centocor (Horsham, PA, USA). Peptides were purchased from Pepscan (Lelystad, The Netherlands).

The myelin oligodendrocyte glycoprotein peptide 35–55aa (MOG_35–55, MEVGWYRSPFSRVVHLYRN GK) was synthesized by SBS Genetech (San Francisco, CA). The purity of these peptides was in the range of 95.18%. This is MHC-II presented peptide interacting with CD4+ T cells. Complete Freund's adjuvant (CFA) and pertussis toxin (PTX) were from Sigma-Aldrich. Reagents purchased from BD Bioscience (San Jose, CA) were Mycobacterium tuberculosis H37Ra, fixation/permeabilization kit, and leukocyte-activation cocktail (LAC). Sulfo-SMCC and Keyhole Limpet Hemocyanin (KLH) were from Thermo Scientific (Waltham, MA). The following fluorescent antibodies were used: CD4-PerCP (clone RM4-5, BD); IL-17-PE (clone TC11-18H10.1, Biolegend (San Diego, CA)); Foxp3-APC (clone 3G3, Miltenyi Biotec (San Diego, CA)). Foxp3/transcription factor staining buffer set used for Foxp3 intracellular staining was from eBioscience (San Diego, CA). Mouse CD4+ T cell enrichment kits (EasySep) were purchased from Stem Cell Biotech (Vancouver, Canada). Carboxyfluorescein Succinimidyl Ester (CFSE) used for cell tracking and T cell proliferation assay was from Life Technology (Grand Island, NY).

Two peptides (C3 and N1) were described in the previous study [48 (link)]. Briefly, the peptide C3 contained the carboxyl-terminal (C-terminal) sequence of the capsid protein of PCV2b (GenBank: AAC35331.1) between residues 195 and 233, and the peptide N1 contained the sequence of the ORF3 protein of PCV2b (GenBank: AAC35332.1) between residues 35 and 66. The peptides (C3 and N1) were appended with an NH₂-terminal (N-terminal) cysteine during synthesis, which was required for conjugation with the keyhole limpet hemocyanin (Thermo scientific, Rockford, IL, USA). Other peptides were synthesized by Yao-Hong Biotechnology Inc. (New Taipei, Taiwan), as listed in Table 1. These peptides contained the sequence of the ORF3 protein of PCV between residues 35 and 66, overlapping ten-mer peptides and associated peptides (20-mer, 22-mer, and 32-mer). Peptides P48 (PCV2a, GenBank: CBZ47005.1), P49 (PCV2, GenBank: AIA63974.1), and P56 (PCV1, GenBank: U49186) [28 (link)] were also used in this study. These truncated peptides were primarily used for the mapping of linear epitopes for anti-ORF3 mAbs binding. The purity of all peptides was assessed by high-performance liquid chromatography (LC-10ATVP serial dual plunger pump, Shimadzu, Torrance, CA, USA) and tested for the correct mass by Waters Micromass ZQ™ 2000 LC Mass Spectrometer (Waters, Milford, MA, USA).