Biologic duoflow

The murine monoclonal IgG1 antibody targeting human CA125 was produced from hybridoma B43.13 [19 ] that was kindly provided by Quest Pharma Tech Inc., Edmonton, Canada. The hybridoma cell culture supernatant was used to purify the MAb-B43.13 by protein G affinity (P-7700, Sigma-Aldrich, St. Louis, MO, USA) on a BioLogic DuoFlow™ chromatography system (760-0135, Bio-Rad Laboratories, Inc., Hercules, CA, USA). MAb-B43.13-derived anti-CA125 scFv was produced with modifications to previously described constructs [20 (link)],[21 (link)]. Briefly, the DNA sequence encoding scFv-B43.13 was cloned into a pET22b + vector with a modified inter-chain linker, and the protein was expressed in Escherichia coli Rosetta 2 DE3 (Novagen, 71400-3, Merck KGaA, Darmstadt, Germany). The C-terminal hexa-histidine-tagged scFv was purified from the soluble fraction of recombinant cell lysates by immobilized metal affinity chromatography using a TALON® Superflow resin (635507, Clontech Laboratories, Inc., Mountain View, CA, USA).

A Superose 6 HR 10/30 column coupled to an FPLC instrument (Biologic Duo Flow, Bio-Rad) was equilibrated with 20 mM Tris–HCl pH 8.0, 150 mM NaCl. Samples were incubated with 10–30 µM H₂O₂ for 30–60 min at room temperature before loading them onto the column to form the intermolecular disulfide bonds of VvPrx3 (C73S). Samples were subjected to chromatography, which was performed at a flow rate of 0.2 ml min⁻¹. Several standard preparations were run to calibrate the column: ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa) (Supplementary Fig. S8). The absorbance at 280 nm was used to monitor the presence of the proteins.

Peptides ^AQAp5_(d), ^aqap5 and ^AQAp5 (Table 1) were purchased from Anaspec (Fremont, CA) as crude preparations and were purified by reverse-phase high performance liquid chromatography (RP-HPLC; Biologic DuoFlow; BioRad, Hercules, CA) using a C3 matrix (ZorbaxTM 300SB; Agilent, Santa Clara, CA). A linear gradient of 1–51% acetonitrile in water with 0.05% v/v trifluoroacetic acid served as the mobile phase. A flow rate of 4 mL/min was used to elute the peptides. Fractions were collected, pooled, and integrity of the peptides was verified by mass spectrometry [9 (link), 10 (link)]. Analytical HPLC was performed, essentially as described above, but using a Zorbax SB-C3 1500 mm matrix with a flow rate of 1 mL/min.Table 1

Primary structure of peptides

Peptide	Primary structure
AQAp5(d)	AQAys kaqka qakqa kqaqk aqkaq akqak q
aqap5	aqaYS KAQKA QAKQA KQAQK AQKAQ AKQAK Q
AQAp5	AQAYS KAQKA QAKQA KQAQK AQKAQ AKQAK Q

d-Amino acids are shown in lower case

RgpA protein was purified from OMVs of P. gingivalis ATCC33277 following a previously described protocol [28 (link),29 (link)]. Once the OMVs were obtained, they were sonicated (Vibra-cell VCX130-Sonics) and ion-exchange liquid-phase chromatography (FPLC) (BioLogic DuoFlow™ BioRad) was performed. An initial cycle of anion exchange chromatography allowed for the collection of bioactive fractions, which were separated in the second cycle of cation exchange chromatography, followed by filtration using a 3 kDa filter (Pall Nanosep™). Protein purity was confirmed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot [30 (link)].