Production of monoclonal antibodies
Monoclonal antibodies were prepared in a manner similar to that reported previously25. Briefly, mice were immunized with either recombinant CK18 protein (Prospec, Israel) or the synthetic peptide (381RRLLEDGEDFNLGDALD397) (BEX,Japan) and splenocytes of hyper-immunized mice were fused with myeloma cells (X 63). Positive clones were selected using ELISA with recombinant CK18 protein or the peptide. Large amounts of mAbs were purified using the HiTrap MabSelect SuRe system (Cytiva, Japan) in binding buffer (1.5 M glycine, 3 M NaCl, 10 mM EDTA (pH8.9)) and elution buffer (100 mM sodium citrate (pH 3.0)).
All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Institutional Animal Care and Use Committee at Sysmex corporation. This study was carried out in compliance with the ARRIVE guidelines.
Recombinant fCK18 protein construction, design and purification
Two fragmented recombinant CK18 proteins were prepared according to an established method25. pBLC-fCK18 (241R-397D) and pBLC-fCK18 (261R-397D) were constructed by synthetic DNA into a pBLC expression vector (inhouse) using restriction enzymes, NdeI and EcoRI (Takara, Japan). Note: we defined the initiator protein, Methionine, as number 1 (1 M). Recombinant fCK18 expression used JM109 Escherichia coli (Wako, Tokyo, Japan); cells were transformed with pBLC-fCK18 (241R-397D) or pBLC-fCK18 (261R-397D), cultured at 37 °C in Luria Bertani (LB) medium until optical density at 600 nm reached 0.6, then further cultured at 37 °C for 12 h after induction with 500 mM isopropyl-b-d-thiogalactoside (Wako, Japan). Cells were recovered by centrifugation for 10 min at 10,000×g and kept at − 20 °C until subsequent analysis. The cells were sonicated in a lysis buffer (5 mM Tris–HCl (pH 8.0), inhibitor cocktail, and 0.2 mg/mL lysozyme) and soluble proteins were isolated by centrifugation at 10,000×g for 15 min. fCK18 (241R-397D) or fCK18 (261R-397D) were purified by affinity binding to a K18-624 mAb conjugated HiTrap NHS-activated HP column in buffer (Dulbecco’s phosphate-buffered saline) and eluted in buffer (0.1 M Glycine–HCl (pH 7.5)).The expression of fCK18 (241R-397D) or fCK18 (261R-397D) was assessed using SDS-PAGE and immunoblot analysis, and then fCK18 (241R-397D) or fCK18 (261R-397D) was desalted using a PD-10 column (Cytiva).
SDS-PAGE, western blotting (WB), and immunoprecipitation (IP)
For SDS-PAGE, 0.2–16 ng of recombinant fCK18 was applied to a 4–20% Ready GEL (Bio-Rad, Japan) and stained by an immunofluorescence reagent (Oriole, Bio-Rad, Japan). Immunoblot analysis was carried out using 20 ng of recombinant CK18 and fCK18, applied on 4–20% Ready GEL (Bio-Rad), and transferred from the gel to a PVDF membrane. PVDF membrane was blocked with PVDF Blocking Reagent (TOYOBO, Japan) in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) for 1 h at room temperature and incubated with K18-328 or K18-624 mAbs followed by a secondary HRP-conjugated antibody (MBL, Japan). Immunoreactive bands were visualized using HRP substrate reagents (Nacalai, Japan). For immunoprecipitation we covalently coupled K18-624 mAb or a commercially available antibody to magnetic beads (Magnosphere MS300/Carboxyl, JSR, Japan) using carboxyl and ethylene dichloride. The commercially available M30 antibody (VLVbio, Sweden) was diluted in 0.1 M MES buffer (pH 5.0) with beads and incubated for 1 h at room temperature; the beads were subsequently washed and stored in blocking buffer at 4 °C. Immune complexes were loaded equally on a 4–20% Ready GEL (Bio-Rad) and transferred from the gel to a PVDF membrane. PVDF membrane was blocked using a PVDF Blocking Reagent (TOYOBO, Japan) in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) for 1 h at room temperature and incubated with alkaline phosphatase-conjugated K18-328 Fab. Immunoreactive bands were visualized using colorimetric alkaline phosphatase substrate reagents (Roche, Japan).
CLEIA system
fCK18 in human serum was measured using the HISCL-5000 CLEIA system (Sysmex Corporation, Japan) that was developed using a two-step immunoassay system method and verified for human sera. Using this procedure, CK18-624 mAb conjugated magnetic beads were mixed with human serum, washed, and then allowed to react with alkaline phosphatase-conjugated K18-328 Fab. After the wash step, a chemiluminescent substrate (CDP-Star) was added and luminescence was emitted upon binding to the ALP. The amount of chemiluminescence was measured and its concentration was determined using recombinant fCK18 as a standard. The three different values, low, middle and high, were selected for CV. Low, middle, and high values were selected in the range of 0.465–0.930 ng/mL, 0.930–4.650 ng/mL, and 4.650–9.300 ng/mL from the fCK18 standard, respectively. LoD was defined as the point does not match between blank mean + 3 standard deviation (SD) and low concentration samples (such as a dilution of the lowest Std.) of mean − 3SD. LoQ was defined below 5% of the CV26.
All methods were carried out in accordance with relevant guidelines and regulations.
Human samples
Serum from healthy individuals and NASH patients was purchased from Discovery Life Sciences (CA, USA) with approval and informed consent. Serum was stored at − 80 °C. fCK18 concentration was measured in 100 healthy individuals and 11 NASH patients.
All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Ethics Committee at Sysmex corporation.
Statistical analysis
Continuous variables are presented as mean ± SD, and categorical variables are shown as number of patients. Data were analyzed using Manny–Whitney U test in two groups. All statistical analyses were performed using Prism (GraphPad Software, Inc. La Jolla, CA). Differences were considered to be significant at P < 0.05.
