Human subjects
CSF and patient sera were collected at the Children’s Hospital of Pennsylvania (CHOP), Philadelphia, PA, with full informed consent and protocols approved by the CHOP Institutional Review Board.
Expression of the ATD fusion protein in 293T cells
We made a fusion gene that expresses the entire 561 amino acids of the N-terminal extracellular domain of human GluN1 (NR1) (UniProtKB - Q05586), including the amino terminal domain (ATD), followed by the Myc tag, a 6XHIS tag, a TEV protease cleavage site, and the transmembrane domain of the human platelet-derived growth factor receptor (PDGFR) (Additional file 1, Genbank Accession #) [13, 15]. The gene was synthesized and inserted into the retroviral vector, pBabe puro by Genscript (Piscataway, NJ) [16]. Amphotropic retroviruses were produced in 293T cells following standard protocols, except that X-tremeGENE 9 DNA Transfection Reagent was used (354,087, Roche, Germany), and the cells were cultured in Advanced DMEM, 1% IFS, penicillin/streptomycin (Invitrogen, Carlsbad, CA) [17].
The retroviral supernatant was used to transduce 293T cells (2.5 X106 in a 10 cm dish), with 4 μg/ml polybrene (TR1003G, Thermo Fisher Scientific, Waltham, MA), for 6 h. 48 h later, cells were selected with 1 μg/ml puromycin (P9620, Sigma-Aldrich, St. Louis, MO). One week later, expressing cells were isolated by FACS staining with the murine anti-NR1 APC mAb (orb149996, Biorbyt, San Francisco, CA) on the BD FACSCanto II (Becton Dickson, Franklin Lakes, NJ). Four rounds of FACS over 4 weeks were performed to isolate a stable, homogeneous population of cells (293T-ATD).
Flow cytometry, FACS, and immunofluorescence studies
To assess antibody binding to 293T-ATD cells by flow cytometry, cells were harvested using 0.05% trypsin, washed, and resuspended at 1 × 106 cells/ml in PBS-1% BSA (A7030, Sigma-Aldrich). Primary antibodies included the Bioorbyt APC-labeled NR1 mAb at 10 μg/mL, an Alexa Fluor 488 labeled Myc tag mAb at 2 μg/mL (16–308, Millipore, Billerica, MA) three human IgG mAbs (5 μg/mL) isolated from a patient with ANRE (5F5, 2G6, 1D1, manuscript in press) and the 6A isotype control mAb [17]. As a secondary antibody for the human mAbs, we used a FITC-conjugated F(ab’)2 goat anti-human IgG (109–096-008, Jackson ImmunoResearch, West Grove, PA). Cells were assayed with a BD FACSCanto II (Becton Dickson, Franklin Lakes, NJ). Data were analyzed using FlowJo 8.8.7. Software (Tree Star, Ashland, OR).
For immunofluorescence studies, 293T-ATD cells were plated at 5 × 104 cells/well on round Corning™ BioCoat™ 12 mm #1 German Glass Coverslips (354,087, Corning, NY) in 24 well plates. 24 h later, the cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, washed with PBS 0.05% Tween-20 (PBST), blocked with 10% Goat serum (Invitrogen) + 1% BSA in PBS (PBS + G + B) for 1 h at 37 °C, and then washed with PBST. Cells were incubated for one hour at room temp in PBS + G + B with one or more of the following added: a murine anti-NR1 APC mAb (orb149996, Biorbyt, San Francisco, CA), an Alexa Fluor 488 conjugated anti-Myc-tag-specific mAb (16–308, Millipore) (1:250 dilution), ANRE patient or normal human CSF (1:20), ANRE patient or normal human sera (1:100), human mAbs, 5F5, 2G6, 1D1 or isotype human control mAb 8E1 (5 μg/ml). After one hour, cells were washed twice with PBST and incubated with the Alexa 568 goat anti-human IgG, 1:1000 (A21090, Thermo Fisher), secondary antibody for the human CSF or mAbs in PBS + G + B for one hour, and then the cells were washed once with PBS and once with dH2O. Coverslips were mounted with ProLong® Gold Antifade reagent with DAPI (P36935, Thermo Fisher) and imaged with a C2+ Nikon confocal microscope with 63×/1.3 NA oil objective; images were analyzed with ImageJ software (https://imagej.nih.gov/ij/). All immunofluorescence studies were performed at least twice.
Mobilization of membrane-bound ATD with TEV protease
The 293T-ATD cells were plated at 2 × 105 cells/well in 12 well plates. 24 h later, they were washed with PBS and then treated with 25 μg rTEV Protease (4469; R&D Systems, Minneapolis, MN) with Xpert Protease inhibitor cocktail solution (P3100–001; GenDEPOT, Barker, TX) in PBS for the indicated time period (10–40 min). The cells were pipetted up and down, transferred to Eppendorf tubes, and centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was collected and immediately dialyzed against cold PBS overnight. The protein concentration was measured using NanoDrop 1000 (Thermo Fisher) and protein was visualized on a Coomassie-stained SDS:PAGE gel.
ATD ELISAs
To analyze the timecourse of ATD mobilization by TEV, we performed a capture assay in which we coated Black 96-well immune plates (12–566-24, Thermo Fisher) with 5 μg/mL anti HIS tag antibody (ab18184, Abcam, Cambridge, MA) overnight, washed the plates 3 times with PBST, blocked with 5% inactivated fetal bovine serum and 3% Goat serum (Invitrogen) in PBST for 1 h at 37 °C, then washed 3 times. ATD samples cleaved at the stated timepoints were added at 5 μg/mL and supernatant from un-cleaved cells was added as negative control, followed by 1 h incubation at 37 °C. The plates were washed three more times, and biotinylated human mAb 5F5 was added at 5 μg/mL (100 μl/well), and then incubated for 1 h at 37 °C. After three additional PBST washes, Streptavidin-poly-HRP conjugate at 1:2000 (Thermo Fisher) was added and incubated for 1 h at 37 °C. After three additional washes, Super Signal ELISA Femto Substrate was used for detection (Thermo Fisher). Relative luminescence values were measured using the Biotek Synergy II Microplate reader (BioTek Instruments, Winooski, VT, USA). Microsoft Excel was used to process the data.
To test binding of human NR1 antibodies to plate-adherent ATD, we added 5 μg/mL Myc antibody (C3956, Sigma-Aldrich) (100 μl/well) to Black 96-well plates (12–566-24, Thermo Fisher) overnight, washed the plates 3 times with PBST, blocked with 5% inactivated fetal bovine serum and 3% Goat serum (Invitrogen) in PBST for 1 h at 37 °C, washed 3 times, added 5 μg/mL ATD, and then incubated for 1 h at 37 °C and washed 3 more times. We added human mAbs, 5F5, 2G6, 1D1, and control isotype 6A at 5 μg/mL (100 μl/well), or 5 μg/mL anti-NR1 mAb (MAB 1586 R1JHL, Millipore), in triplicate samples, and incubated for 1 h at 37 °C. After three additional PBST washes, secondary antibodies were added, either an anti-human IgG HRP conjugate (9040–05 SouthernBiotech, Birmingham, AL) or anti-mouse IgG HRP conjugate (1010–05, Southern Biotech), at 1:2000 and incubated for 1 h at 37 °C, followed by 3 washes. Super Signal ELISA Femto Substrate was used for detection. Data was collected in the Biotek Synergy II Microplate reader and analyzed with Microsoft Excel.
To test binding of TEV-mobilized ATD to plate-adherent human IgG by ELISA, we first biotinylated the ATD using the EZ-Link™ Sulfo-NHS-Biotin kit (21,326, Thermo Fisher). We added 5 μg/mL 5F5 (100 μl/well) to Black 96-well plates (12–566-24, Thermo Fisher), incubated overnight at room temp, washed the plates 3 times with PBST, blocked the wells with 2% non-fat milk in PBST for 1 h at 37 °C, and again washed 3 times. We added triplicate serial dilutions of the biotinylated ATD (diluted in 50 μL PBS/well), and incubated for 1 h at 37 °C. After three PBST washes, Streptavidin-poly-HRP conjugate at 1:2000 (Thermo Fisher) was added and incubated for 1 h at 37 °C. Super Signal ELISA Femto Substrate was used for detection. Data was collected in the Biotek Synergy II Microplate reader and analyzed with Microsoft Excel.