Oligoclonal IgG antibodies in multiple sclerosis target patient-specific peptides

Michael Graner; Tiffany Pointon; Sean Manton; Miyoko Green; Kathryn Dennison; Mollie Davis; Gino Braiotta; Julia Craft; Taylor Edwards; Bailey Polonsky; Anthony Fringuello; Timothy Vollmer; Xiaoli Yu

doi:10.1371/journal.pone.0228883

Abstract

IgG oligoclonal bands (OCBs) are present in the cerebrospinal fluid (CSF) of more than 95% of patients with multiple sclerosis (MS), and are considered to be the immunological hallmark of disease. However, the target specificities of the IgG in MS OCBs have remained undiscovered. Nevertheless, evidence that OCBs are associated with increased levels of disease activity and disability support their probable pathological role in MS. We investigated the antigen specificity of individual MS CSF IgG from 20 OCB-positive patients and identified 40 unique peptides by panning phage-displayed random peptide libraries. Utilizing our unique techniques of phage-mediated real-time Immuno-PCR and phage-probed isoelectric focusing immunoblots, we demonstrated that these peptides were targeted by intrathecal oligoclonal IgG antibodies of IgG1 and IgG3 subclasses. In addition, we showed that these peptides represent epitopes sharing sequence homologies with proteins of viral origin, and proteins involved in cell stress, apoptosis, and inflammatory processes. Although homologous peptides were found within individual patients, no shared peptide sequences were found among any of the 42 MS and 13 inflammatory CSF control specimens. The distinct sets of oligoclonal IgG-reactive peptides identified by individual MS CSF suggest that the elevated intrathecal antibodies may target patient-specific antigens.

Citation: Graner M, Pointon T, Manton S, Green M, Dennison K, Davis M, et al. (2020) Oligoclonal IgG antibodies in multiple sclerosis target patient-specific peptides. PLoS ONE 15(2): e0228883. https://doi.org/10.1371/journal.pone.0228883

Editor: Brahim Nait-Oumesmar, UPMC, FRANCE

Received: August 28, 2019; Accepted: January 24, 2020; Published: February 21, 2020

Copyright: © 2020 Graner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: XY received Research Award (RG 3934A1/1) from National Multiple Sclerosis Society, https://www.nationalmssociety.org/. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS) and is characterized by inflammatory demyelination and neuronal damage. Cerebrospinal fluid (CSF) oligoclonal bands (OCBs), a characteristic feature of MS, are associated with increased levels of disease activity and disability [1–3], the conversion from a clinically isolated syndrome to early relapsing-remitting MS [4], greater brain atrophy [5], and increased cortical lesion load/intrathecal inflammation [6]. Furthermore, recent studies have shown that antibodies produced by clonally expanded plasma cells in MS CSF cause demyelination [7], and myelin-specific MS antibodies cause complement-dependent oligodendrocyte loss and demyelination [8]. This evidence supports the notion that intrathecal IgG in MS plays a critical role in disease pathogenesis, consistent with the view that CSF IgG alone remains the best marker of disease activity in individual MS patients [1].

OCBs have been assumed to target antigens relevant to MS pathogenesis, with leading antigen candidates being myelin proteins and/or viruses. However, despite intensive research over the last several decades, the target specificities of the IgG within OCB in MS have remained a mystery. A recent report by Brändle et al. [9] showed that OCBs in MS target ubiquitous intracellular antigens released in cellular debris. We hypothesized that phage-displayed random peptide libraries can be used to identify antigenic peptides specific to intrathecal IgG of MS.

We have previously shown that phage peptides reactive to OCBs are persistent in MS patients [10], suggesting that these peptides can be used as unique tools for investigating the specificity of OCBs and to investigate disease pathogenesis. To further elucidate OCBs specificity in MS, we used 20 OCB-positive MS CSF IgG to screen phage-displayed random peptide libraries and identified 40 high-affinity peptides which were reactive to intrathecal oligoclonal IgG in most MS patients. We also show that these peptide antigens are unique in each patient. Our data suggest that the oligoclonal bands in MS may target patient-specific antigens.

Materials and methods

Patients

With approval of the University of Colorado Institutional Review Board (COMIRB # 00–688), CSF and sera from MS patients and controls were collected at University of Colorado Hospital after obtaining written consent CSFs were immediately centrifuged at 500 x g for 10 minutes, and the supernatant was collected. Both CSF and sera were stored at −80°C until use. The CSF of all MS patients contained oligoclonal bands (OCBs, determined by ARUP Laboratories, SLC, UT). CSF IgG concentration, percent of IgG in CSF, and number of OCBs from each patient are listed in Table 1.

Download:

Table 1. Clinical characteristics of MS patients.

https://doi.org/10.1371/journal.pone.0228883.t001

Identification of high affinity phage peptides with MS CSF IgG

Ph.D.-7^™ and Ph.D.-12^™ Phage Display Peptide Library (New England BioLabs, Beverly, MA) kits were used for affinity selection of specific peptides by all MS CSF. The Ph.D.-12 library is a combinatorial library of random 12-mer peptides fused to a minor coat protein (pIII) of M13 phage. The displayed peptide is expressed at the N-terminus of pIII. The library consists of approximately 10⁹ electroporated (i.e., unique) sequences. Similarly, the Ph.D.-7 library is a combinatorial library consisted of 10⁹ unique random heptapeptides. The panning procedure as well as characterization of positive phage peptides were as described [10]. A streamlined protocol was used to determine phage peptide specificity after affinity selection [10]. Briefly, individual phage plaques were amplified in U96 DeepWell plates and used to determine reactivity to panning MS CSF IgG by 96-well ELISA [11]. Positive clones were confirmed by duplicate phage ELISA with a pre-immune human IgG control. DNA from positive phage clones were purified and sequenced.

Dose–response phage-mediated real-time Immuno-PCR

Phage-mediated real-time Immuno-PCR (IPCR) was performed as described [12]. Reacti-Bind^™ wells of protein A-coated clear strip plates (Thermo Scientific, Rockford, IL) were coated with 50 μl of CSF or serum (1 μg/ml IgG) and with pre-immune human IgG (Alpha Diagnostic) in TBS (50 mM Tris–HCl, 150 mM NaCl) at room temperature for two hours, washed with TBS containing 0.05% Tween 20 (TBST), and blocked with 3% nonfat dry milk/0.05% TBST at room temperature for one hour. Serial 10-fold phage dilutions in duplicate were added to MS CSF/serum IgG-coated wells and incubated at room temperature for two hours. After washing, bound phage were lysed in 50 μl of double-deionized water by heating the plates at 95 °C for 15 minutes. Single-stranded phage DNA was released and used as template for real-time PCR in an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). For standard SYBR^® Green PCR, each reaction (20 μl) consisted of 1× power SYBR^® Green master mix (Applied Biosystems), 750 nM of each M13 phage primer and 4 μl of phage template. Thermal cycle conditions were 95 °C for 10 minutes, followed by 40 cycles at 95 °C for 15 seconds and 60 °C for 45 seconds. Fast real-time PCR was conducted using 1× Fast SYBR^® Green master mix, with thermal cycling at 95 °C for 20 seconds, followed by 40 cycles at 95 °C for three seconds and 60 °C for 30 seconds. A control reaction without template was included in each run.

Isoelectric focusing (IEF) immunoblotting

CSF (200–500 μl) was concentrated on an Amicon Ultra 0.5-ml 30 K cellulose centrifugal filter unit at 14,000×g for 30 minutes at room temperature before IEF (SPIFE^® IgG IEF kit, Helena Laboratories, Beaumont, TX) using SPIFE 3000 electrophoresis analyzer. Wicks were soaked in an anode (0.3 M acetic acid) or cathode (1 M NaOH) solution and applied to the edge of a SPIFE^® IgG IEF gel. Five microliters of concentrated MS CSF/sera (3–5 μg IgG for phage probe and 100 ng IgG for alkaline phosphatase-conjugated anti-human IgG probe) were loaded into wells of an SPIFE IEF gel. After electrophoresis at 700 V for one hour at 15 °C, samples were transferred to PVDF membranes for 45 minutes, followed by blocking in Helena blocking agent (1 g bovine milk protein/50 ml 1× TBS) for one hour at room temperature. Membranes were incubated with the respective phage peptide at concentrations ranging from 5.0×10¹⁰ to 1.5×10¹¹ pfu/ml in 1:10 Helena blocking agent/TBST (blocking buffer) at room temperature for two hours. After washing with 0.05% Tween-TBS, membranes were incubated with mouse anti-M13 mAb at a 1:500 dilution in blocking buffer, followed by incubation with 1:500 dilution of AP conjugated anti-mouse IgG at room temperature for one hour. Membranes were developed with NBT/BCIP substrate. For control blots, membranes were incubated for one hour with 1:1000 dilutions of AP-anti-human IgG (H+L) in blocking buffer, followed by NBT/BCIP detection.

Western blots determining phage peptide reactivity to CSF/serum IgG antibodies of IgG1/IgG3 subclasses

Phage peptides (10¹¹ pfu/per well) in TBS were denatured and reduced by incubation with 1x protein sample buffer containing β-mercaptoethanol (Pierce Biotechnology, Rockford, IL) at 95 °C for 10 minutes, and separated in BioRad 4–15% Tris/Glycine gel for 50 minutes at a constant 200 V. The gels were electro-blotted onto PVDF membranes (Bio-Rad, Hercules, CA) for 60 minutes at a constant 15 V using Trans-Blot^® Semi-Dry Cell (Bio-Rad). After blocking for one hour with 1× casein/TBS (Vector Labs, Burlingame, CA) containing 0.1% Tween 20, the blots were incubated with corresponding MS CSF and serum (primary antibodies, at 1 μg/ml) at 4°C overnight. The bound CSF and serum IgG antibodies were then detected with HRP-mouse anti-human IgG1 and IgG3 antibodies (1:5000) respectively. Isotype-specific mouse monoclonal anti-human IgG1 (I2513, clone 8c/6-39) and anti-human IgG3 (I7260, clone HP-6050) antibodies were used (Sigma). This was followed by secondary antibody anti-mouse IgG (H+L) incubation and detection with SuperSignal^® West Femto Maximum Sensitivity chemiluminescent substrate (Pierce Biotechnology).

For detection of phage pIII protein, duplicate membranes were incubated with a 1: 25,000 dilution of mouse anti-M13 pIII mAb (New England BioLabs, Ipswich, MA), followed by a 1: 25,000 dilution of HRP-conjugated goat anti-mouse IgG (Vector Labs) as secondary Ab and with SuperSignal^® West Pico substrate for chemiluminescent detection (Pierce Biotechnology).

Quantifying band intensity of western blots

The FluorChem Q^™ system was used to detect the signal produced by addition of chemiluminescent substrate to probed blots. Digital images of the blots were collected by the FluorChem Q at several different lengths of time of exposure to optimize for image clarity and quality. The images were then analyzed quantitatively by AlphaView software for FluorChem^™ systems.

DNA sequencing and database searches

Single-stranded phage DNA was purified and sequenced to deduce the amino acid sequence of the peptides. Consensus peptides were identified by sequence alignment using ClustalW (http://www.ebi.ac.uk/clustalw/). To identify candidate proteins, the most abundant peptides panned by CSF from each patient were searched in BLAST (http://www.ncbi.nlm.nih.gov/) using the Swiss Prot protein sequence database.

Results

Specific phage peptides were identified by CSF from 14/20 OCB-positive MS patients, but no common peptide sequences were found

We studied a total of 20 MS patients to investigate peptide antigen specificity of the intrathecal IgG. These patients were all positive for oligoclonal bands. Table 1 lists key clinical immunological features of the patients, including CSF IgG concentration, percent of IgG in the CSF, and the number of oligoclonal bands. We applied Phage-Displayed Random Peptide Libraries technologies (Ph.D.-7^™ and Ph.D.-12^™, New England Biolab) for a minimum of three rounds of panning with MS CSF for this study. After three to five rounds of affinity selection with each of the 20 MS CSF, phage peptides were analyzed for specificity using our streamlined high throughput protocol as previously described [10]. 14 MS CSF selected positive phage peptides, while the CSF from six MS patients failed to identify any positive phage clones with a repeated panning approach, as well as using additional ultra-fast selection of peptide method [13]. All positive phage clones were amplified, and the phage DNA was purified and sequenced to deduce peptide sequences [11]. A total of 40 unique peptides were identified by each of the 14 MS CSF IgG, ranging from one to six peptides per patient. Although homologous peptides were identified within each MS CSF, no shared peptide sequences were found among MS patients (Table 2).

Download:

Table 2. Unique peptides were identified from each MS CSF IgG.

https://doi.org/10.1371/journal.pone.0228883.t002

Phage peptides target intrathecally synthesized oligoclonal IgG and were recognized by IgG1 and IgG3 subclasses

To determine whether CSF-selected phage peptides were specific for intrathecally synthesized IgG in MS patients, we tested equal amounts of CSF and paired serum IgG (50 ng IgG per well) for peptide binding specificity. We utilized our highly sensitive dose-dependent phage mediated immuno-PCR (phage-IPCR) method [12]. MS serum and CSF were coated onto wells of protein A plates, followed by addition of serial 10-fold dilutions of corresponding phage peptides to each well, and phage binding specificity was assessed by real-time PCR [10]. Phage peptides selected by CSF IgG of all 14 MS patients bound more to CSF IgG than to serum IgG of the same patient in a dose-dependent manner, and there is a significant difference of phage binding between CSF and serum (p = 0.0002). Fig 1A and 1B show a representative data of greater binding of phage peptides to CSF IgG than to paired serum IgG in two MS patients (1A: MS 02–19; 1B MS 03–7). Fig 1C is the summary data demonstrating intrathecal IgG binding of the peptides to all 14 MS CSF tested. These results demonstrate that the phage peptides were preferentially targeted by intrathecally-synthesized IgG in MS CSF.

Download:

Fig 1. MS phage peptides target intrathecally synthesized IgG in dose-dependent manner by phage-IPCR.

Representative paired MS serum and CSF, as well as pre-immune human IgG control (50 μl at IgG concentration of 1 μl/ml), were coated in duplicate wells of protein A-plates before addition of the corresponding phage peptides (at serial 10-fold dilutions starting with 10⁸pfu) each well. Bound phage was determined by real-time PCR. Phage peptides bound 5-10-fold higher to MS CSF IgG than to paired serum IgG in a dose-dependent manner. Pre-immune human IgG served as negative control. Error bars represent standard deviation. A, patient 1 (02–19); B, patient 5 (03–7); Data represent at least three independent experiments. C. There is a significant higher binding of phage peptides to CSF than paired serum (p = 0,0002, paired Student’s T-Test). Phage peptides (10⁵−10⁸) were assessed for binding by IPCR as described above.

https://doi.org/10.1371/journal.pone.0228883.g001

To further demonstrate that the intrathecal IgG-specific phage peptides were recognized by oligoclonal IgG bands, we examined phage binding specificity to MS CSF and paired serum by isoelectric focusing (IEF) immunoblotting using phage peptides as probes. Paired CSF and serum were separated on agarose IEF gels, transferred to PVDF membranes, and probed with corresponding phage peptides [10]. In all seven MS patients, phage peptides were recognized by multiple high-intensity IgG bands in the CSF, while fewer, less intense, or no bands were detected in the paired serum (Fig 2). To confirm that the IgG bands recognized by phage peptides represent oligoclonal bands in CSF, duplicate serum and CSF IEF blots were probed with anti-human IgG and are shown in Fig 2 next to the phage blots for comparison. All peptide-reactive bands in the CSF corresponded to bands of oligoclonal IgG detected by anti-human IgG antibody.

Download:

Fig 2. Phage probed isoelectric focused blots demonstrate that phage peptides were recognized by MS oligoclonal IgG bands.

Paired MS CSF and serum (3–5 μg total IgG) from seven MS patients were resolved on agarose IEF gels and transferred to nitrocellulose membranes. The blots were probed with corresponding phage peptides (10¹⁰ pfu/ml) and incubated with mouse anti-pIII antibody followed by AP-anti-mouse antibody. Duplicate blots were probed with anti-human IgG as positive controls to reveal total oligoclonal bands. Peptides selected by MS IgG recognized multiple high-density oligoclonal IgG bands in the CSF, but weaker and reduced number of bands in the paired serum. Arrows indicate extra bands detected in the CSF. Patient ID was listed under each blot.

https://doi.org/10.1371/journal.pone.0228883.g002

Additionally, we carried out Western blots of purified phage to determine the IgG subclass specificity of these peptides. Phage (10¹⁰ pfu/well) were separated on a 4–15% SDS-PAGE gel, blotted, and probed with corresponding CSF and paired serum as primary antibodies, followed by incubation with mouse anti-human IgG1 and IgG3 secondary antibodies. Shown here in Fig 3 are representative blots from two patients MS #7 (MS 04–3), and MS#13 (05–4). Phage peptides were recognized by both IgG1 and IgG3 subclasses with equal band intensity in both CSF and serum (Fig 3A). Band intensity analysis showed that the ratios of IgG1 band between CSF and serum were comparable as ratios of IgG3 band between CSF and serum (Fig 3B), suggesting that phage peptides are recognized by IgG1 and IgG3 antibodies in both CSF and paired serum of MS patients.

Download:

Fig 3. Western blots show that the intrathecal IgG-reactive phage peptides were recognized by both IgG1 and IgG3 subclasses MS CSF and serum.

A. SDS-PAGE Western blots of phage peptides show that they were reactive to both IgG1 and IgG3 subclasses in paired CSF and serum of MS patients. Phage peptides (10¹⁰/well) were separated on a 4–15% SDS-PAGE gel, blotted, and probed with corresponding total CSF and serum (primary antibody) of MS patients from which the original phage peptides were identified. The bound CSF and serum antibodies were then probed with mouse anti-human IgG1 and IgG3 antibodies, followed by anti-mouse-HRP antibodies and detected with SuperSignal^® West Femto chemiluminescent substrate. Representative phage shown here are phage peptide from MS #7 (MS 04–3 B1) and phage peptide from MS #13 (MS07-12 B11). B. Band intensity analysis of phage Western blots with IgG1/IgG3 probes showed that the ratio of IgG1 band between CSF and serum was similar as ratio of IgG3 band between CSF and serum. C. Peptides used for the western blots. *same as peptide selected by #13 (MS05-4) [10].

https://doi.org/10.1371/journal.pone.0228883.g003

Screening large number of MS CSF using phage-IPCR did not reveal any shared binding specificity of the intrathecal phage peptides

To determine whether these selected phage peptides shared common antigen bindings to MS intrathecal IgG, we screened 42 MS CSF and 13 inflammatory controls (acute viral meningitis, Behcet’s disease, paraneoplastic syndrome, viral meningitis, Cryptococcal meningitis, subacute sclerosing panencephalitis, acute disseminated encephalomyelitis, neurosyphilis, sarcoid, VZV myelopathy and radiculomyelitis) with representative phage peptides. Patient CSF characteristic including IgG concentration, diagnosis, and OCBs are included in Supplemental data (S1 and S2 Tables). With highly sensitive and specific phage mediated real-time IPCR, we failed to identify any binding reactivity of the peptides as common antigens to MS CSF nor IC CSF (S1 Fig), suggesting that these peptides are patient-specific.

Protein database searches with selected MS peptides

Representative peptides were used to search for homologous sequences in the protein database (https://blast.ncbi.nlm.nih.gov/Blast) to identify corresponding protein candidates. Because of the short length of the peptide sequences, numerous candidate proteins were revealed including proteins of viral, bacterial and other origins. Once again, no common shared protein/peptides were identified. Table 3 shows a list of candidate proteins from human and bacteria. The most abundant candidates are proteins involved in cellular stress, transcription factors, membrane proteins, neuronal proteins, enzymes, and immunoglobulin fragments with peptide sequence identities ranged 62%–100%).

Download:

Table 3. Protein database search results with selected MS peptides.

https://doi.org/10.1371/journal.pone.0228883.t003

Discussion

We previously showed that there is a temporal stability of peptide antigens for the intrathecal IgG antibodies in the CSF of patients with MS, suggesting the importance of applying a phage peptide approach to identify targets of the intrathecal IgG [10]. In this study, we have expanded the sample size for screening which includes CSF from 20 OCB-positive MS patients for panning phage-displayed random peptide libraries. A total of 40 unique, specific peptides were identified by 14 MS CSF, and no peptide sequences were shared among the peptides identified. We demonstrated that these phage peptides were targeted by intrathecal oligoclonal IgG antibodies/oligoclonal bands. Furthermore, these peptides reacted to both IgG1 and IgG3 subclasses. Phage mediated immuno-PCR screening of 42 MS and 13 inflammatory control CSF revealed that no MS specific peptide antigens were found. We postulate that the oligoclonal IgG antibodies in MS may target patient specific antigens, and that the significance of OCB may not due to the antigens specific to antibodies in MS.

Oligoclonal bands can be detected in the CSF of most (>95%) patients with MS, persist throughout the course of the disease, and are a diagnostic hallmark of the disease [14]. The critical role of intrathecal IgG/oligoclonal bands in MS disease pathogenesis is supported by mounting evidence. For example, actively demyelinating lesions are commonly associated with prominent deposition of immunoglobulins and complement activation products [15–18], OCBs are shown to be associated with increased levels of disease activity and disability [1,3,6], a greater risk of second attack [19], the conversion from a clinically isolated syndrome (CIS) to early RRMS [4,20], and greater brain atrophy [5,6]. Moreover, the presence of OCB in CSF in the specific clinical context is still the most reliable parameter to confirm the likely diagnosis of MS [21,22], supporting the critical pathological role of intrathecal IgG antibodies in MS.

Intrathecal production of antibodies against viruses (measles, rubella, and varicella zoster virus), bacteria and CNS components in MS have up until now shown inconsistent or negative results [23–25]. Further, recombinant antibodies generated from clonally expanded B cells/plasma cells in the CSF and from laser capture microdissection of B cells in MS lesions failed to identify MS specific antigens [26,27]. A recent report showed that OCB in MS target ubiquitous intracellular antigens released in cellular debris [9].

We used a combination of two phage peptide libraries (Ph.D. 12 and Ph.D. 7) and extensive panning strategies [11–13] with increased rounds of panning (up to six rounds) and stringency, and identified 40 specific phage peptides from 70 percent of the MS CSF (14 out of 20) used. Significantly, these phage peptides were recognized by intrathecal IgG/oligoclonal bands as demonstrated by our techniques of phage-probed IEF and phage-mediated immune-PCR, suggesting that phage peptides can be a unique tool for investigating antigen specificity of MS oligoclonal bands which may give critical clues as to the disease causation. Despite the high specificity of the intrathecal IgG specific phage peptides, the highly sensitive and specific phage-mediated immuno-PCR technique used here failed to identify common peptide reactivity shared by all MS CSF screened, implying that MS intrathecal IgG antibodies may target patient specific antigens. Our data are consistent with previous studies and support the notion that the disease targets for OCBs in MS have yet to be reproducibly demonstrated [28,29]. Failing to identify MS specific targets for OCBs does not diminish its crucial roles in disease pathogenesis, as the presence of large amount of IgG antibodies are key features of MS lesions [30,31], and have been shown to be associated with increased disease activity and brain atrophy [3,6]. Over 20 times more IgG were extracted from MS plaques than those from control brain [32]. Furthermore, significant higher amounts of bound IgG (oligoclonal in nature) were eluted from MS brains with both high and low pH buffer [30,32,33], and the consistent presence of complement, IgG antibodies, and Fcγ receptors (FcR) in phagocytic macrophages suggests that antibody- and complement-mediated myelin phagocytosis is the dominant mechanism of demyelination in established MS [34]. We have previously analyzed clinical laboratory parameters from 91 patients with MS and showed that in MS there was not a linear relationship between the numbers of OCBs and CSF IgG concentration [35]. The complex relationship between OCBs and other CSF parameters suggests that at certain concentrations, the IgG antibody in the CNS is being sequestered or aggregated to form IgG complexes (as bound IgG) and therefore unable to contribute to the number of OCBs, with the relationships become negatively associated [35].

IgG1 and IgG3 are the first 2 Ig classes after IgM. They have superior ability to activate effector functions. Both IgG1 and IgG3 subclasses were found to be present in the same OCB in MS CSF [36,37], and the elevation of IgG1 and IgG3 indices in MS were found more frequently than the elevation of the general IgG index [1]. Furthermore, patients with a relapse were significantly more frequently seropositive for anti-MOG and anti-MBP IgG3 than those in remission [38]. It would be interesting to investigate IgA, IgG2 and IgG4 in MS but we did not carry out these experiments due to the limitation of study scope. Using western blots analysis, we showed that the intrathecal phage peptides were recognized by IgG1 and IgG3 antibodies in both CSF and paired serum of MS patients, with equal band intensities, indicating that both subclasses could be important for disease. Both IgG1 and IgG3 subclasses have been found in the intrathecal IgG [39] and in the same oligoclonal bands [36], indicating that the OCBs may be consisted of at least IgG1 and IgG3 subclasses. Further, molecular sequencing data revealed that in MS clonally related and even the same IgG-VH sequences are found in multiple OCBs band [40], further supporting the notion that OCBs may be represented by multiple IgG subclasses.

The distinct sets of oligoclonal IgG-reactive peptides identified by individual MS CSF suggest that the elevated intrathecal antibodies may target patient-specific antigens, it also indicates the limitation of this approach. Other limitations include the relatively short nature of the linear phage display peptides that limits recognition of more extensive secondary or tertiary structures/epitopes. The discovered “mimotopes” might not be the real antigens, which prevents us from discovering concordant antibody binding between patients. Other advance technologies might be more successful. Nonetheless, further investigations are needed to determine characteristics and the role of the increased intrathecal antibodies in MS.

Supporting information

S1 Fig.

A. There is no shared and differential phage peptide binding between MS and IC CSF. IPCR was performed to screen 42 MS and 13 IC CSF with pooled phage peptides MS05-4A6, MS05-4A2, and MS04-3B1. p = 0.47. B. Summary of S1 Fig. A. There is no shared and differential phage peptide binding between MS and IC CSF.

https://doi.org/10.1371/journal.pone.0228883.s001

(TIF)

S1 Table. MS CSF used for IPCR screening.

https://doi.org/10.1371/journal.pone.0228883.s002

(DOCX)

S2 Table. Inflammatory control (IC) patients used for IPCR CSF screening.

https://doi.org/10.1371/journal.pone.0228883.s003

(DOCX)

S1 Raw Images. Original images for IEF and western blots.

https://doi.org/10.1371/journal.pone.0228883.s004

(PDF)

Acknowledgments

We dedicate this paper to Dr. Don Gilden for his lifelong passion and effort in trying to find the cause of MS.

References

1. Caroscio JT, Kochwa S, Sacks H, Makuku S, Cohen JA, Yahr MD. Quantitative cerebrospinal fluid IgG measurements as a marker of disease activity in multiple sclerosis. Arch Neurol [Internet]. 1986 Nov;43(11):1129–31. pmid:3778245
- View Article
- PubMed/NCBI
- Google Scholar
2. Avasarala JR, Cross AH, Trotter JL. Oligoclonal band number as a marker for prognosis in multiple sclerosis. Arch Neurol. 2001;58(12):2044–5. pmid:11735778
- View Article
- PubMed/NCBI
- Google Scholar
3. Joseph FG, Hirst CL, Pickersgill TP, Ben-Shlomo Y, Robertson NP, Scolding NJ. CSF oligoclonal band status informs prognosis in multiple sclerosis: a case control study of 100 patients. J Neurol Neurosurg Psychiatry [Internet]. 2009 Mar;80(3):292–6. pmid:18829628
- View Article
- PubMed/NCBI
- Google Scholar
4. Calabrese M, Poretto V, Favaretto A, Alessio S, Bernardi V, Romualdi C, et al. Cortical lesion load associates with progression of disability in multiple sclerosis. Brain [Internet]. 2012 Oct;135(Pt 10):2952–61. pmid:23065788
- View Article
- PubMed/NCBI
- Google Scholar
5. Ferreira D, Voevodskaya O, Imrell K, Stawiarz L, Spulber G, Wahlund L-O, et al. Multiple sclerosis patients lacking oligoclonal bands in the cerebrospinal fluid have less global and regional brain atrophy. J Neuroimmunol [Internet]. 2014 Sep 15;274(1–2):149–54. pmid:24999245
- View Article
- PubMed/NCBI
- Google Scholar
6. Farina G, Magliozzi R, Pitteri M, Reynolds R, Rossi S, Gajofatto A, et al. Increased cortical lesion load and intrathecal inflammation is associated with oligoclonal bands in multiple sclerosis patients: a combined CSF and MRI study. J Neuroinflammation [Internet]. 2017;14(1):40. pmid:28222766
- View Article
- PubMed/NCBI
- Google Scholar
7. Blauth K, Soltys J, Matschulat A, Reiter CR, Ritchie A, Baird NL, et al. Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid cause demyelination of spinal cord explants. Acta Neuropathol [Internet]. 2015 Dec;130(6):765–81. pmid:26511623
- View Article
- PubMed/NCBI
- Google Scholar
8. Liu Y, Given KS, Harlow DE, Matschulat AM, Macklin WB, Bennett JL, et al. Myelin-specific multiple sclerosis antibodies cause complement-dependent oligodendrocyte loss and demyelination. Acta Neuropathol Commun [Internet]. 2017;5(1):25. pmid:28340598
- View Article
- PubMed/NCBI
- Google Scholar
9. Brändle SM, Obermeier B, Senel M, Bruder J, Mentele R, Khademi M, et al. Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins. Proc Natl Acad Sci U S A [Internet]. 2016;113(28):7864–9. pmid:27325759
- View Article
- PubMed/NCBI
- Google Scholar
10. Yu X., Burgoon M., Green M., Barmina O., Dennison K., Pointon T., Davis M and Gilden D. Intrathecally synthesized IgG in multiple sclerosis cerebrospinal fluid recognizes identical epitopes over time. J. Neuroimmunol. 240–241. 129–136. 2011. pmid:22079192
- View Article
- PubMed/NCBI
- Google Scholar
11. Yu X, Owens GP, Gilden DH. Rapid and efficient identification of epitopes/mimotopes from random peptide libraries. J Immunol Methods [Internet]. 2006 Oct 20;316(1–2):67–74. pmid:17010370
- View Article
- PubMed/NCBI
- Google Scholar
12. Yu X, Burgoon MP, Shearer AJ, Gilden DH. Characterization of phage peptide interaction with antibody using phage mediated immuno-PCR. J Immunol Methods. 2007 Sep 30;326(1–2):33–40. pmid:17669417
- View Article
- PubMed/NCBI
- Google Scholar
13. Yu X, Barmina O, Burgoon M, Gilden D. Identification of measles virus epitopes using an ultra-fast method of panning phage-displayed random peptide libraries. J Virol Methods [Internet]. 2009 Mar;156(1–2):169–73. pmid:19095007
- View Article
- PubMed/NCBI
- Google Scholar
14. Franciotta D, Salvetti M, Lolli F, Serafini B, Aloisi F. B cells and multiple sclerosis. Lancet Neurol [Internet]. 2008 Sep;7(9):852–8. pmid:18703007
- View Article
- PubMed/NCBI
- Google Scholar
15. Storch MK, Stefferl A, Brehm U, Weissert R, Wallström E, Kerschensteiner M, et al. Autoimmunity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of multiple sclerosis pathology. Brain Pathol [Internet]. 1998 Oct;8(4):681–94. pmid:9804377
- View Article
- PubMed/NCBI
- Google Scholar
16. Lucchinetti C, Brück W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol [Internet]. 2000 Jun;47(6):707–17. pmid:10852536
- View Article
- PubMed/NCBI
- Google Scholar
17. Breij ECW, Heijnen P, van der Goes A, Teunissen CE, Polman CH, Dijkstra CD. Myelin flow cytometry assay detects enhanced levels of antibodies to human whole myelin in a subpopulation of multiple sclerosis patients. J Neuroimmunol [Internet]. 2006 Jul;176(1–2):106–14. pmid:16712958
- View Article
- PubMed/NCBI
- Google Scholar
18. Ingram G, Loveless S, Howell OW, Hakobyan S, Dancey B, Harris CL, et al. Complement activation in multiple sclerosis plaques: an immunohistochemical analysis. Acta Neuropathol Commun [Internet]. 2014 May 9;2:53. pmid:24887075
- View Article
- PubMed/NCBI
- Google Scholar
19. Tintoré M, Sastre-Garriga J. New treatment measurements for treatment effects on relapses and progression. J Neurol Sci [Internet]. 2008 Nov 15;274(1–2):80–3. pmid:18822433
- View Article
- PubMed/NCBI
- Google Scholar
20. Zipoli V, Hakiki B, Portaccio E, Lolli F, Siracusa G, Giannini M, et al. The contribution of cerebrospinal fluid oligoclonal bands to the early diagnosis of multiple sclerosis. Mult Scler [Internet]. 2009 Apr;15(4):472–8. pmid:19153171
- View Article
- PubMed/NCBI
- Google Scholar
21. Fitzner B, Hecker M, Zettl UK. Molecular biomarkers in cerebrospinal fluid of multiple sclerosis patients. Autoimmun Rev [Internet]. 2015 Oct;14(10):903–13. pmid:26071103
- View Article
- PubMed/NCBI
- Google Scholar
22. Thompson AJ, Banwell BL, Barkhof F, Carroll WM, Coetzee T, Comi G, et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteriaTHOMPSON, A. J. et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. The Lancet Neurology, v. 17, n. 2, p. 162–173, 2018. Lancet Neurol. 2018;17(2):162–73.
- View Article
- Google Scholar
23. McLaughlin KA, Wucherpfennig KW. Chapter 4 B Cells and Autoantibodies in the Pathogenesis of Multiple Sclerosis and Related Inflammatory Demyelinating Diseases. Vol. 98, Advances in Immunology. 2008. p. 121–49.
- View Article
- Google Scholar
24. Meinl E. Untapped targets in multiple sclerosis. J Neurol Sci [Internet]. 2011 Dec;311 Suppl 1:S12–5.
- View Article
- Google Scholar
25. Weber MS, Hemmer B, Cepok S. The role of antibodies in multiple sclerosis. Biochim Biophys Acta [Internet]. 2011 Feb;1812(2):239–45. pmid:20600871
- View Article
- PubMed/NCBI
- Google Scholar
26. Owens GP, Bennett JL, Lassmann H, O’Connor KC, Ritchie AM, Shearer A, et al. Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid. Ann Neurol. 2009 Jun;65(6):639–49. pmid:19557869
- View Article
- PubMed/NCBI
- Google Scholar
27. Willis SN, Stathopoulos P, Chastre A, Compton SD, Hafler DA, O’Connor KC. Investigating the Antigen Specificity of Multiple Sclerosis Central Nervous System-Derived Immunoglobulins. Front Immunol [Internet]. 2015;6:600. pmid:26648933
- View Article
- PubMed/NCBI
- Google Scholar
28. Navas-Madroñal M, Valero-Mut A, Martínez-Zapata MJ, Simón-Talero MJ, Figueroa S, Vidal-Fernández N, et al. Absence of antibodies against KIR4.1 in multiple sclerosis: A three-technique approach and systematic review. PLoS One [Internet]. 2017;12(4):e0175538. pmid:28414733
- View Article
- PubMed/NCBI
- Google Scholar
29. Bashford-Rogers RJM, Smith KGC, Thomas DC. Antibody repertoire analysis in polygenic autoimmune diseases. Immunology [Internet]. 2018;155(1):3–17. pmid:29574826
- View Article
- PubMed/NCBI
- Google Scholar
30. Gilden D, Tachovsky T. Immunoglobulin elution from multiple sclerosis brain. J Neurosci Methods [Internet]. 1979 Aug;1(2):133–42. pmid:544959
- View Article
- PubMed/NCBI
- Google Scholar
31. Ewan PW, Lachmann PJ. IgG synthesis within the brain in multiple sclerosis and subacute sclerosing panencephalitis. Clin Exp Immunol [Internet]. 1979 Feb;35(2):227–35. pmid:436336
- View Article
- PubMed/NCBI
- Google Scholar
32. Glynn P, Gilbert HM, Newcombe J, Cuzner ML. Analysis of immunoglobulin G in multiple sclerosis brain: quantitative and isoelectric focusing studies. Clin Exp Immunol [Internet]. 1982 Apr;48(1):102–10. pmid:7083640
- View Article
- PubMed/NCBI
- Google Scholar
33. Mehta PD, Frisch S, Thormar H, Tourtellotte WW, Wisniewski HM. Bound antibody in multiple sclerosis brains. J Neurol Sci [Internet]. 1981 Jan;49(1):91–8. pmid:7205323
- View Article
- PubMed/NCBI
- Google Scholar
34. Breij ECW, Brink BP, Veerhuis R, van den Berg C, Vloet R, Yan R, et al. Homogeneity of active demyelinating lesions in established multiple sclerosis. Ann Neurol [Internet]. 2008 Jan;63(1):16–25. pmid:18232012
- View Article
- PubMed/NCBI
- Google Scholar
35. Beseler C, Vollmer T, Graner M, Yu X. The complex relationship between oligoclonal bands, lymphocytes in the cerebrospinal fluid, and immunoglobulin G antibodies in multiple sclerosis: Indication of serum contribution. PLoS One [Internet]. 2017;12(10):e0186842. pmid:29059249
- View Article
- PubMed/NCBI
- Google Scholar
36. Losy J, Mehta PD, Wisniewski HM. Identification of IgG subclasses’ oligoclonal bands in multiple sclerosis CSF. Acta Neurol Scand [Internet]. 1990 Jul;82(1):4–8. pmid:2239136
- View Article
- PubMed/NCBI
- Google Scholar
37. Grimaldi L. M. E., Maimone D., Reggio A., Raffaele R. IgG1,3 and 4 oligoclonal bands in multiple sclerosis and other neurological diseases. The Italian Journal of Neurological Sciences. 1986. 7 (5), 507–513. pmid:3542897
- View Article
- PubMed/NCBI
- Google Scholar
38. García-Merino A, Persson MA, Ernerudh J, Díaz-Gil JJ, Olsson T. Serum and cerebrospinal fluid antibodies against myelin basic protein and their IgG subclass distribution in multiple sclerosis. J Neurol Neurosurg Psychiatry. 1986 Sep; 49(9):1066–70. pmid:2428940
- View Article
- PubMed/NCBI
- Google Scholar
39. Greve B, Magnusson CG, Melms A, Weissert R. Immunoglobulin isotypes reveal a predominant role of type 1 immunity in multiple sclerosis. J Neuroimmunol [Internet]. 2001 Dec 3;121(1–2):120–5. pmid:11730948
- View Article
- PubMed/NCBI
- Google Scholar
40. Bankoti J, Apeltsin L, Hauser SL, Allen S, Albertolle ME, Witkowska HE, et al. In multiple sclerosis, oligoclonal bands connect to peripheral B-cell responses. Ann Neurol. 2014;75(2):266–76. pmid:24375699
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Caroscio JT, Kochwa S, Sacks H, Makuku S, Cohen JA, Yahr MD. Quantitative cerebrospinal fluid IgG measurements as a marker of disease activity in multiple sclerosis. Arch Neurol [Internet]. 1986 Nov;43(11):1129–31. pmid:3778245
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Avasarala JR, Cross AH, Trotter JL. Oligoclonal band number as a marker for prognosis in multiple sclerosis. Arch Neurol. 2001;58(12):2044–5. pmid:11735778
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Joseph FG, Hirst CL, Pickersgill TP, Ben-Shlomo Y, Robertson NP, Scolding NJ. CSF oligoclonal band status informs prognosis in multiple sclerosis: a case control study of 100 patients. J Neurol Neurosurg Psychiatry [Internet]. 2009 Mar;80(3):292–6. pmid:18829628
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Calabrese M, Poretto V, Favaretto A, Alessio S, Bernardi V, Romualdi C, et al. Cortical lesion load associates with progression of disability in multiple sclerosis. Brain [Internet]. 2012 Oct;135(Pt 10):2952–61. pmid:23065788
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Ferreira D, Voevodskaya O, Imrell K, Stawiarz L, Spulber G, Wahlund L-O, et al. Multiple sclerosis patients lacking oligoclonal bands in the cerebrospinal fluid have less global and regional brain atrophy. J Neuroimmunol [Internet]. 2014 Sep 15;274(1–2):149–54. pmid:24999245
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Farina G, Magliozzi R, Pitteri M, Reynolds R, Rossi S, Gajofatto A, et al. Increased cortical lesion load and intrathecal inflammation is associated with oligoclonal bands in multiple sclerosis patients: a combined CSF and MRI study. J Neuroinflammation [Internet]. 2017;14(1):40. pmid:28222766
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Blauth K, Soltys J, Matschulat A, Reiter CR, Ritchie A, Baird NL, et al. Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid cause demyelination of spinal cord explants. Acta Neuropathol [Internet]. 2015 Dec;130(6):765–81. pmid:26511623
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Liu Y, Given KS, Harlow DE, Matschulat AM, Macklin WB, Bennett JL, et al. Myelin-specific multiple sclerosis antibodies cause complement-dependent oligodendrocyte loss and demyelination. Acta Neuropathol Commun [Internet]. 2017;5(1):25. pmid:28340598
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Brändle SM, Obermeier B, Senel M, Bruder J, Mentele R, Khademi M, et al. Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins. Proc Natl Acad Sci U S A [Internet]. 2016;113(28):7864–9. pmid:27325759
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Yu X., Burgoon M., Green M., Barmina O., Dennison K., Pointon T., Davis M and Gilden D. Intrathecally synthesized IgG in multiple sclerosis cerebrospinal fluid recognizes identical epitopes over time. J. Neuroimmunol. 240–241. 129–136. 2011. pmid:22079192
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Yu X, Owens GP, Gilden DH. Rapid and efficient identification of epitopes/mimotopes from random peptide libraries. J Immunol Methods [Internet]. 2006 Oct 20;316(1–2):67–74. pmid:17010370
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Yu X, Burgoon MP, Shearer AJ, Gilden DH. Characterization of phage peptide interaction with antibody using phage mediated immuno-PCR. J Immunol Methods. 2007 Sep 30;326(1–2):33–40. pmid:17669417
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Yu X, Barmina O, Burgoon M, Gilden D. Identification of measles virus epitopes using an ultra-fast method of panning phage-displayed random peptide libraries. J Virol Methods [Internet]. 2009 Mar;156(1–2):169–73. pmid:19095007
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Franciotta D, Salvetti M, Lolli F, Serafini B, Aloisi F. B cells and multiple sclerosis. Lancet Neurol [Internet]. 2008 Sep;7(9):852–8. pmid:18703007
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Storch MK, Stefferl A, Brehm U, Weissert R, Wallström E, Kerschensteiner M, et al. Autoimmunity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of multiple sclerosis pathology. Brain Pathol [Internet]. 1998 Oct;8(4):681–94. pmid:9804377
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Lucchinetti C, Brück W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol [Internet]. 2000 Jun;47(6):707–17. pmid:10852536
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Breij ECW, Heijnen P, van der Goes A, Teunissen CE, Polman CH, Dijkstra CD. Myelin flow cytometry assay detects enhanced levels of antibodies to human whole myelin in a subpopulation of multiple sclerosis patients. J Neuroimmunol [Internet]. 2006 Jul;176(1–2):106–14. pmid:16712958
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Ingram G, Loveless S, Howell OW, Hakobyan S, Dancey B, Harris CL, et al. Complement activation in multiple sclerosis plaques: an immunohistochemical analysis. Acta Neuropathol Commun [Internet]. 2014 May 9;2:53. pmid:24887075
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Tintoré M, Sastre-Garriga J. New treatment measurements for treatment effects on relapses and progression. J Neurol Sci [Internet]. 2008 Nov 15;274(1–2):80–3. pmid:18822433
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Zipoli V, Hakiki B, Portaccio E, Lolli F, Siracusa G, Giannini M, et al. The contribution of cerebrospinal fluid oligoclonal bands to the early diagnosis of multiple sclerosis. Mult Scler [Internet]. 2009 Apr;15(4):472–8. pmid:19153171
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Fitzner B, Hecker M, Zettl UK. Molecular biomarkers in cerebrospinal fluid of multiple sclerosis patients. Autoimmun Rev [Internet]. 2015 Oct;14(10):903–13. pmid:26071103
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Thompson AJ, Banwell BL, Barkhof F, Carroll WM, Coetzee T, Comi G, et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteriaTHOMPSON, A. J. et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. The Lancet Neurology, v. 17, n. 2, p. 162–173, 2018. Lancet Neurol. 2018;17(2):162–73.
View Article
Google Scholar

[86] View Article

[87] Google Scholar

[ref23] 23. McLaughlin KA, Wucherpfennig KW. Chapter 4 B Cells and Autoantibodies in the Pathogenesis of Multiple Sclerosis and Related Inflammatory Demyelinating Diseases. Vol. 98, Advances in Immunology. 2008. p. 121–49.
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref24] 24. Meinl E. Untapped targets in multiple sclerosis. J Neurol Sci [Internet]. 2011 Dec;311 Suppl 1:S12–5.
View Article
Google Scholar

[92] View Article

[93] Google Scholar

[ref25] 25. Weber MS, Hemmer B, Cepok S. The role of antibodies in multiple sclerosis. Biochim Biophys Acta [Internet]. 2011 Feb;1812(2):239–45. pmid:20600871
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref26] 26. Owens GP, Bennett JL, Lassmann H, O’Connor KC, Ritchie AM, Shearer A, et al. Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid. Ann Neurol. 2009 Jun;65(6):639–49. pmid:19557869
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref27] 27. Willis SN, Stathopoulos P, Chastre A, Compton SD, Hafler DA, O’Connor KC. Investigating the Antigen Specificity of Multiple Sclerosis Central Nervous System-Derived Immunoglobulins. Front Immunol [Internet]. 2015;6:600. pmid:26648933
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref28] 28. Navas-Madroñal M, Valero-Mut A, Martínez-Zapata MJ, Simón-Talero MJ, Figueroa S, Vidal-Fernández N, et al. Absence of antibodies against KIR4.1 in multiple sclerosis: A three-technique approach and systematic review. PLoS One [Internet]. 2017;12(4):e0175538. pmid:28414733
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref29] 29. Bashford-Rogers RJM, Smith KGC, Thomas DC. Antibody repertoire analysis in polygenic autoimmune diseases. Immunology [Internet]. 2018;155(1):3–17. pmid:29574826
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref30] 30. Gilden D, Tachovsky T. Immunoglobulin elution from multiple sclerosis brain. J Neurosci Methods [Internet]. 1979 Aug;1(2):133–42. pmid:544959
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref31] 31. Ewan PW, Lachmann PJ. IgG synthesis within the brain in multiple sclerosis and subacute sclerosing panencephalitis. Clin Exp Immunol [Internet]. 1979 Feb;35(2):227–35. pmid:436336
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref32] 32. Glynn P, Gilbert HM, Newcombe J, Cuzner ML. Analysis of immunoglobulin G in multiple sclerosis brain: quantitative and isoelectric focusing studies. Clin Exp Immunol [Internet]. 1982 Apr;48(1):102–10. pmid:7083640
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref33] 33. Mehta PD, Frisch S, Thormar H, Tourtellotte WW, Wisniewski HM. Bound antibody in multiple sclerosis brains. J Neurol Sci [Internet]. 1981 Jan;49(1):91–8. pmid:7205323
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref34] 34. Breij ECW, Brink BP, Veerhuis R, van den Berg C, Vloet R, Yan R, et al. Homogeneity of active demyelinating lesions in established multiple sclerosis. Ann Neurol [Internet]. 2008 Jan;63(1):16–25. pmid:18232012
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

[ref35] 35. Beseler C, Vollmer T, Graner M, Yu X. The complex relationship between oligoclonal bands, lymphocytes in the cerebrospinal fluid, and immunoglobulin G antibodies in multiple sclerosis: Indication of serum contribution. PLoS One [Internet]. 2017;12(10):e0186842. pmid:29059249
View Article
PubMed/NCBI
Google Scholar

[135] View Article

[136] PubMed/NCBI

[137] Google Scholar

[ref36] 36. Losy J, Mehta PD, Wisniewski HM. Identification of IgG subclasses’ oligoclonal bands in multiple sclerosis CSF. Acta Neurol Scand [Internet]. 1990 Jul;82(1):4–8. pmid:2239136
View Article
PubMed/NCBI
Google Scholar

[139] View Article

[140] PubMed/NCBI

[141] Google Scholar

[ref37] 37. Grimaldi L. M. E., Maimone D., Reggio A., Raffaele R. IgG1,3 and 4 oligoclonal bands in multiple sclerosis and other neurological diseases. The Italian Journal of Neurological Sciences. 1986. 7 (5), 507–513. pmid:3542897
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref38] 38. García-Merino A, Persson MA, Ernerudh J, Díaz-Gil JJ, Olsson T. Serum and cerebrospinal fluid antibodies against myelin basic protein and their IgG subclass distribution in multiple sclerosis. J Neurol Neurosurg Psychiatry. 1986 Sep; 49(9):1066–70. pmid:2428940
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref39] 39. Greve B, Magnusson CG, Melms A, Weissert R. Immunoglobulin isotypes reveal a predominant role of type 1 immunity in multiple sclerosis. J Neuroimmunol [Internet]. 2001 Dec 3;121(1–2):120–5. pmid:11730948
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref40] 40. Bankoti J, Apeltsin L, Hauser SL, Allen S, Albertolle ME, Witkowska HE, et al. In multiple sclerosis, oligoclonal bands connect to peripheral B-cell responses. Ann Neurol. 2014;75(2):266–76. pmid:24375699
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Patients

Identification of high affinity phage peptides with MS CSF IgG

Dose–response phage-mediated real-time Immuno-PCR

Isoelectric focusing (IEF) immunoblotting

Western blots determining phage peptide reactivity to CSF/serum IgG antibodies of IgG1/IgG3 subclasses

Quantifying band intensity of western blots

DNA sequencing and database searches

Results

Specific phage peptides were identified by CSF from 14/20 OCB-positive MS patients, but no common peptide sequences were found

Phage peptides target intrathecally synthesized oligoclonal IgG and were recognized by IgG1 and IgG3 subclasses

Screening large number of MS CSF using phage-IPCR did not reveal any shared binding specificity of the intrathecal phage peptides

Protein database searches with selected MS peptides

Discussion

Supporting information

S1 Fig.

S1 Table. MS CSF used for IPCR screening.

S2 Table. Inflammatory control (IC) patients used for IPCR CSF screening.

S1 Raw Images. Original images for IEF and western blots.

Acknowledgments

References