Patient samples
Peripheral blood was collected from traumatic and pathologic SCI patients in Jessa
Hospital (Hasselt, Belgium), University Hospitals Leuven (Leuven, Belgium), Adelante
Rehabilitation Centre (Hoensbroek, The Netherlands), Hospital East-Limburg (Genk,
Belgium), Antwerp University Hospital (Edegem, Belgium), Radboud University Medical
Centre (Nijmegen, The Netherlands) and Academic Hospital Maastricht and General Hospital
Turnhout (Turnhout, Belgium). Samples of traumatic SCI patients were taken at hospitalization
(T0) or 3 weeks after injury (T1). Samples of pathologic SCI patients (e.g. stenosis,
spinal disc herniation, compression caused by bleeding) were collected preoperatively
(T0) and 3 weeks after surgery (T1). Patients with pre-existing autoimmune disorders
were excluded from the study. Written informed consent was acquired from all participants
after approval by the Medical ethics committee Hospital East Limburg (B371201317091),
Adelante (54-14/CK/JM) and Academic Hospital Maastricht (METC13-4-079). Blood samples
were centrifuged for 10 min at 400g. Plasma was collected and centrifuged for 10 min at 1500g. After processing, plasma samples were aliquoted and stored at ?80 °C. Samples were
processed and stored in collaboration with the University Biobank Limburg (UBiLim)
and Biobank University Hospitals Leuven. A total of 51 SCI patients and 49 age- and
gender-matched healthy controls were involved in the study. Plasma samples from healthy
controls were collected via UBiLim.
Construction of a hSC cDNA phage display library
Commercially obtained poly A+ RNA (size range of 0.2–10 kb, Clontech, Saint-Germain-en-Laye,
France) from spinal cord tissue of 18 Caucasians (ages 25–63 years) was converted
to double-stranded cDNA with EcoRI and XhoI adapters by using the Superscript Choice
System for cDNA synthesis kit (Life Technologies, Gent, Belgium) as described previously
23]. Purified cDNA inserts were directionally ligated into our pVI phage display vectors,
pSPVIA, pSPVIB and pSPVIC, each representing 1 of 3 different reading frames 16]. Ligation mixtures were used to transform Escherichia coli (E.coli) TG1 cells (Lucigen, Middleton, USA) by electroporation to obtain human spinal cord
(hSC)-pSPVIA, hSC-pSPVIB and hSC-pSPVIC libraries.
Serological antigen selection procedure
The serological antigen selection (SAS) procedure was performed as described previously
16], 19]. In brief, an immunotube (Nunc, Roskilde, Denmark) was coated overnight at 4 °C with
10 ?g/ml rabbit anti-human immunoglobulin G (IgG, Dako, Glostrup, Denmark) in coating
buffer (0.1 M sodium hydrogen carbonate, pH 9.6). After washing with 0.1 % (v/v) phosphate-buffered saline-Tween20 (PBS-T, 50 mM Tris, 150 mM sodium chloride, pH
7.5) and PBS, the immunotube was blocked with 2 % (w/v) skimmed milk powder in PBS (MPBS) for 2 h at room temperature (RT). Plasma samples
of 10 traumatic SCI patients were pooled and pre-adsorbed against E. coli and phage components, as described previously 16]. For the first selection round, equal numbers of phage particles from each hSC cDNA
phage display library (hSC-pSPVI-A, hSC-pSPVI-B and hSC-pSPVI-C) were pre-incubated
with the pre-adsorbed SCI plasma pool in 2 % MPBS for 1.5 h at RT on a rotating platform
24]. After washing the immunotube, the pre-incubated phage-plasma mix was transferred
to the coated tube on a rotating platform, followed by standing conditions at RT.
Non-bound phage were removed by extensive washing of the immunotube with 0.1 % PBS-T
and PBS. Bound phage were eluted by adding 100 mM triethylamide (Sigma-Aldrich, Bornem,
Belgium) to the immunotube for 10 min on a rotating platform and neutralized with
1 M Tris-HCl (pH 7.4). The output of each selection round was amplified by infection
of E. coli TG1 bacteria and plated on ×2 YT agar plates containing ampicillin and glucose (16 g/l
bacto-tryptone, 10 g/l yeast extract, 5 g/l NaCl, 15 g/l bacto-agar, ampicillin at
100 ?g/ml, and glucose at 2 %). Five consecutive selection rounds were performed.
To identify enriched cDNA clones, individual colonies were selected and insert cDNA
fragments were amplified with vector primers binding adjacent to the cDNA insert followed
by restriction enzyme digestion (BstNI (Roche Diagnostics, Vilvoorde, Belgium) and
NspI (NEB, Leiden, The Netherlands)). Enriched cDNA products representing identical
cDNA clones were selected and identified by sequencing of the corresponding cDNA phage
insert. Amino acid sequences of identified clones were compared to public protein
databases of the National Center for Biotechnology Information (NCBI) with BLAST analysis.
Phage ELISA
Antibody reactivity levels of individual plasma samples against selected phage clones
were measured by phage enzyme linked immunosorbent assay (ELISA). Ninety-six-well
flat-bottom plates (Greiner Bio-One, Wemmel, Belgium) were coated overnight at 4 °C
with 5 ?g/ml anti-M13 antibody (GE Health care, Diegem, Belgium) in coating buffer.
Plates were washed twice with PBS and blocked with 5 % MPBS for 2 h at 37 °C, while
shaking. After washing three times with 0.1 % PBS-T and once with PBS, polyethylene
glycol-purified phage displaying the candidate antigen (7?×?10
11
colony-forming units/ml) or empty phage (negative control) were added and incubated
for 1 h at 37 °C under static conditions followed by 30 min at RT, while shaking.
Plates were washed, and plasma samples (1/100 in 5 % MPBS) were incubated for 1 h
at 37 °C under static conditions followed by 30 min at RT, while shaking. Washing
steps were repeated, and horseradish peroxidase human IgG-Fc fragment cross-adsorbed
antibody (1/50,000, Bethyl laboratories, Montgomery, USA) was added for 1 h shaking
at RT. 3,3?,5,5?-Tetramethyl-benzidine dihydrochloride (TMB, Thermo Scientific, Erembodegem,
Belgium) solution was added after washing the plates, and the reactions were incubated
in the dark for 11 min. The colour reaction was stopped by adding 1.8 N H
2
SO
4
. Optical density (OD) signals were measured at 450 nm in a Tecan plate reader (Tecan,
Männedorf, Switzerland). Samples were considered positive when the general reactivity
(OD (specific phage)/OD (empty phage)) was higher than 1.5 and the OD-value of a specific
signal was above 0.1. Samples were tested in duplicate in a single ELISA experiment,
and experiments were performed independently for at least two times. Polyreactive
samples (reactive to the empty phage) were excluded from the analysis.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 6 XML. Fisher’s exact test
was applied for the analysis of associations between the presence of antibody reactivity
directed to particular antigenic targets or panels of targets and SCI patients. A
p value of 0.05 was considered statistically significant.