Flowcytometry
1321N1 human astrocytoma cells stably expressing rP2X4Rs14 were maintained in Dulbecco’s-modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were detached by exposing to 0.05% trypsin–EDTA and collected by pipetting. After washing with Hanks’ balanced salt solution containing 2% FBS (HBSS-FBS), cells were incubated with the pre-warmed antibodies (40 μg/ml) for 30 min at 37 °C. After washing, the pellet was resuspended in ice-cold HBSS-FBS and filtered through a 35-μm nylon cell strainer (BD Biosciences). The antibody binding was detected using a channel for FITC of FACSVerse (BD Biosciences).
Expression heavy chain and light chain from 12–10H and light chain from R5 mutant
A DNA sequence encoding heavy chain in Fab region and light chain from 12–10H, and light chain from 5 mutant was inserted into pET22a vector, and transformed into Escherichia coli strain BL21(DE3) codon plus RIL cells (Novagen, Madison, WI, USA), respectively. The transformant cell was grown at 37 °C in 1 L of LB medium containing 50 μg/ml ampicillin. The culture was allowed to grow until mid-log phase (OD600 = 0.6–0.8) and protein expression was induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for 4 h at 37 °C.
Purification of each heavy chain and light chain from 12–10H and light chain from R5 mutant from inclusion body
Antibody heavy chain in Fab region and light chain from 12–10H were purified, respectively, as described in our previous study19. The cultured cells were harvested by centrifugation for 10 min at 8000 rpm. The insoluble fraction was suspended in 20 ml of 20 mM MOPS buffer, pH 7, and then sonicated 3 times for 3 min in an ice-water bath. The mixture was centrifuged for 60 min at 12,000 rpm, 4 °C. The precipitates were resuspended in 10 ml of 20 mM MOPS buffer, pH 7. DNase I (final concentration of 5 μg/ml), RNase A (final concentration of 20 μg/ml) and MgCl2 (final concentration of 10 mM) were added into the mixture to degrade nucleic acids. The mixture was incubated at 40 °C for 1 h followed by centrifugation. The collected pellets was washed with 25 ml of 0.8 M sodium chloride and centrifuged. This washing step was repeated 3 times. The insoluble material was next dissolved in 20 ml of 6 M guanidine solution (0.584 M Tris–HCl buffer, pH 8.6, containing 5.37 mM EDTA and 6 M guanidine hydrochloride) and reduced with 50 μl of mercaptoethanol at 40 °C for 90 min under a nitrogen atmosphere. TAPS sulfonate (225 mg), which reversibly reacts with the thiol groups to give protein strong positive charge, was added, and the reduction solution was incubated at 40 °C for 30 min to increase the solubility of the denatured Fab fragments. The reaction mixture was dialyzed against 50 mM sodium acetate buffer, pH 5.5, containing 8 M urea and passed through an anion exchange (DEAE-Toyopearl) column (1.5 cm × 20 cm) equilibrated with 50 mM sodium acetate buffer at pH 5.5, containing 8 M urea to remove the residues of nucleic acids. The flow-through solution was applied to a cation-exchange (SP-Toyopearl) column (1.5 cm × 30 cm) equilibrated with 50 mM sodium acetate buffer at pH 5.5. The column was eluted with NaCl gradient (0–1 M). The protein fraction was collected and lyophilized.
Folding and purification of Fab from 12–10H and from R5 mutant
The folding of Fab was carried out using the dialysis method according to the method of Fujii et al.19 with slight modification. The lyophilized TAPS heavy chain and light chain from 12–10H or the lyophilized TAPS heavy chain from 12–10H and light chain from R5 mutant were separately dissolved in 3 ml of 6 M guanidine solution, respectively. Each fragment solution (82 nmol) were quantified by the absorption at 280 nm and then were mixed in 1:1 molar ratio. Cysteamine (final concentration of 72 mM) was added to the mixed solution of heavy chain and light chain from 12–10H or the solution of heavy chain from 12–10H and light chain from R5 mutant for the reduction. After incubation at 40 °C for 90 min, cystamine (final concentration of 26 mM) was added to the respective reduced solution (redox solution). To the redox solutions, 3 ml of the dialysis buffer (0.1 M Tris–HCl buffer, 1 mM EDTA at pH 8.0 containing 0.3 mM cysteamine, and 0.3 mM cystamine) in the presence of 8 M urea were added respectively (protein concentrations are 30 μM), and the diluted solution was dialyzed twice against the dialysis buffer (0.1 M Tris–HCl buffer, 1 mM EDTA at pH 8.0 containing 0.3 mM cysteamine and 0.3 mM cystamine) in the presence of 8 M urea for 4 h. Then, the dialysis was performed against the dialysis buffer in the presence of 4, 2, 1, and 0 M urea. Finally, the dialysis was performed against 0.1 M buffer containing 1 mM EDTA, at pH 8) for 100 h. The separation of folded Fabs were conducted by ion-exchange HPLC. The refolded Fab from 12–10H and that from R5 mutant were applied to the column of Resource S (1 ml, cytiva) equilibrated with 30 mM MOPS-NaOH at pH 7.0, respectively. NaCl gradient (a 0–1 M) was applied at a flow rate of 1 ml/min and the elution was monitored by absorbance at 280 nm.
Preparations of mutant 12–10 H antibodies by CHO expression system
R5 mutant antibody where Asp60, Thr63, Ser65, Ser67 and Asp70 of the 12–10H light chain were replaced with Arg, simultaneously, and A3 mutant antibody where Tyr33 and Arg52 in the heavy chain and Tyr32 in the light chain of 12–10H were mutated with Ala, simultaneously, were prepared by CHO expression system following the methodology described previously24. In principle, the DNA sequences of the heavy and light chains of the antibody were subcloned into the pcDNA3.4 vector (Thermo Fisher Scientific). The vectors were transiently transfected into ExpiCHO cells (Thermo Fisher Scientific) using ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s standard protocol. The cells were cultured for 8 days at 37 °C and 8% CO2. The cultures were centrifuged at 400 × g for 15 min, and the supernatant was collected. Each supernatant was applied onto an rProtein A Sepharose Fast Flow column (GE Healthcare) equilibrated with PBS at pH 7.4. The fraction bound to the column was washed with the PBS and subsequently eluted with Pierce IgG Elution Buffer (Thermo Fisher Scientific). The eluted fraction was neutralized by addition of 2 M Tris–HCl (pH 8.0) and further purified by size exclusion chromatography using a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) equilibrated with PBS at pH 7.4.
ELISA
ELISA was carried out as described in our previous report14. A 96-well immunoplate was coated with 100 μl of 1 μM rHD in carbonate buffer at pH 9.6 for 1 h. The plate was blocked by phosphate-buffered saline (PBS) containing 5% skim milk (Nakalai Tesque) and 0.005% Tween 20 for 1 h to reduce nonspecific adsorption. Then, the plate was washed with PBS containing 0.005% Tween 20 (PBST) and reacted with 100 μl of the prepared monoclonal antibody dissolved in PBS for 1 h, followed by washing with PBST three times. The bound antibody was reacted with 100 μl of 10,000-fold diluted alkaline phosphatase–conjugated goat anti-mouse IgG (Abcam, Cambridge, UK) for 1 h. After the plate was washed three times with PBST, alkaline phosphatase conjugation substrate dissolved in AP color developing buffer (BIO-RAD, San Francisco, USA), was added to each well and incubated for 15 min. Absorbance at 405 nm was measured with a Multiskan FC plate reader (Thermo Scientific).
Labeling of monoclonal antibodies with succinimidyl ester of Alexa Fluor 488
The 12–10 H antibodies were labeled with Alexa Fluor 488 (Thermo Fisher scientific) at amino group, according to the manufacturer’s instruction. After dialysis of purified antibodies against phosphate-buffered saline (PBS; pH 7.4) at 4 °C, one-tenth amount of 1 M sodium bicarbonate solution was added to the antibody solution. Alexa Fluor 488 dissolved in anhydrous dimethyl sulfoxide (10 mg/ml) was added to the antibody solution at a dye/protein molar ratio of 1 ~ 20. The reaction mixture was stirred overnight at room temperature in the dark followed by the addition of ethanolamine at a final concentration of 100 mM. Unconjugated dye was removed by dialysis against PBS at 4 °C. The dye/protein ratio in the Alexa Fluor 488-labeled antibody was determined by absorbance measurements at 280 and 494 nm. Labeled antibodies were kept at 4 °C in the dark until use.
Surface plasmon resonance
The kinetic parameters for interactions between the anti-rP2X4 antibody and rHD mutants were measured by SPR using a Biacore X100 instrument (GE Healthcare) with a running buffer of 10 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 0.05 mM EDTA, and 0.005% (v/v) Tween 20. The rHD was immobilized on a CM5 chip by amine coupling chemistry. Serial dilutions of purified 12–10H Fab or 12–10 Fab R5 mutant were injected into the rHD-immobilized and blank channels (for reference subtraction) at a flow rate of 30 μl/min, with an association time of 100 s and a dissociation time of 100 s. Kinetic parameters were calculated by fitting to a 1:1 (Langmuir) binding model using Biacore Evaluation Software (GE Healthcare). To assess the effect of Alexa Fluor 488 labeling on binding affinity of 12–10 H to rHD, the intact 12–10H IgG or Alexa Fluor 488-labeled 12–10H IgG was immobilized on a CM5 sensor chip via amine coupling. Purified rHD was then injected at a series of concentration in the running buffer at a flow rate of 30 μl/min.
Primary cultured microglial cells
Primary cultured microglial cells were prepared according to a previously described method25,26. 1321N1 human astrocytoma cell lines were a gift from Dr. Michael W. Salter, The Hospital for Sick Children, Toronto, Canada. In brief, a mixed glial culture was prepared from neonatal Wistar rats (CLEA Japan) and maintained for 13 days in DMEM with 10% FBS. Immediately before experiments microglia were collected as the floating cells over the mixed glial culture by a gentle shake of the culture flasks. The collected cells were centrifuged and resuspended with FBS-HBSS. The cell suspension was blocked by incubating with Fc Block (anti-rat CD32, BD Biosciences) for 5 min at 4 °C and immunostained with the antibodies (3A or R5 mutant) and CD11b-Alexa Fluor 647 (BD Biosciences) for 30 min at 37 °C. After washing, the cells were analyzed as described above.
Microglia isolated from spinal cord tissue
Methods employed in this study are are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Male Wistar rats (CLEA Japan) were aged 9 weeks at the start of the experiment and were housed in individual cage at a temperature of 22 ± 1 °C with a 12/12 h light/dark cycle (lights on 8 A.M. to 8 P.M.), and fed food and water ad libitum. All animal experiments were conducted according to the national and international guidelines contained in the Act on Welfare and Management of Animals (Ministry of Environment of Japan) and Regulation of Laboratory Animals (Kyushu University) and under the protocols approved by the Institutional Animal Care and Use committee review panels at Kyushu University. To develop a model of neuropathic pain27,28, the left fifth lumbar (L5) spinal nerve was tightly ligated with 5–0 silk and cut just distal to the ligature under isoflurane (2%) anesthesia. The wound and the surrounding skin were sutured with 3–0 silk. Fourteen days later, rats were deeply anesthetized by i.p. injection of pentobarbital and perfused transcardially with PBS, and the fourth and fifth lumbar segments of the spinal cord ipsilateral to the injury was isolated. Spinal tissue pieces were treated with pre-warmed 0.8 ml enzymatic solution [0.2 U/ml collagenase D (Roche) and 4.3 U/ml of dispase (GIBCO)] in HBSS-FBS for 30 min at 37 °C. The tissues were homogenized by passing through a 23G needle attached with a 1 ml syringe and were further incubated for 15 min at 37 °C. After that, the tissues were homogenized by passing twice through a 26G needle, and the enzymatic reaction was stopped by adding EDTA (0.5 M). Myelin debris was removed from the cell suspension using Myelin Removal Beads II and a MACS LS column (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. After centrifugation, the cells were resuspended in HBSS-FBS. The suspension blocked with Fc Block were immunostained and analyzed by flow cytometry as described above.
