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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: J Immunol Methods. 2018 Feb 8;455:80–87. doi: 10.1016/j.jim.2018.02.002

A sensitive and selective ELISA methodology quantifies a demyelination marker in experimental and clinical samples

Albert G Remacle 1, Jennifer Dolkas 2,4, Mila Angert 2,4, Swathi K Hullugundi 2,4, Andrei V Chernov 1, R Carter W Jones III 2,3,4, Veronica I Shubayev 2,4,*, Alex Y Strongin 1,*,#
PMCID: PMC5886741  NIHMSID: NIHMS942526  PMID: 29428829

Abstract

Sciatic nerve chronic constriction injury (CCI) in rodents produces nerve demyelination via proteolysis of myelin basic protein (MBP), the major component of myelin sheath. Proteolysis releases the cryptic MBP epitope, a demyelination marker, which is hidden in the native MBP fold. It has never been established if the proteolytic release of this cryptic MBP autoantigen stimulates the post-injury increase in the respective circulating autoantibodies. To measure these autoantibodies, we developed the ELISA that employed the cryptic 84-104 MBP sequence (MBP84-104) as bait. This allowed us, for the first time, to quantify the circulating anti-MBP84-104 autoantibodies in rat serum post-CCI. The circulating IgM (but not IgG) autoantibodies were detectable as soon as day 7 post-CCI. The IgM autoantibody level continually increased between days 7 and 28 post-injury. Using the rat serum samples, we established that the ELISA intra-assay (precision) and inter-assay (repeatability) variability parameters were 2.87% and 4.58%, respectively. We also demonstrated the ELISA specificity by recording the autoantibodies to the liberated MBP84-104 epitope alone, but not to intact MBP in which the 84-104 region is hidden. Because the 84-104 sequence is conserved among mammals, we tested if the ELISA was applicable to detect demyelination and quantify the respective autoantibodies in humans. Our limited pilot study that involved 16 female multiple sclerosis and fibromyalgia syndrome patients demonstrated that the ELISA was efficient in measuring both the circulating IgG- and IgM-type autoantibodies in patients exhibiting demyelination. We believe that the ELISA measurements of the circulating autoantibodies against the pathogenic MBP84-104 peptide may facilitate the identification of demyelination in both experimental and clinical settings. In clinic, these measurements may assist neurologists to recognize patients with painful neuropathy and demyelinating diseases, and as a result, to personalize their treatment regimens.

Keywords: ELISA, myelin, demyelination, myelin basic protein, multiple sclerosis, fibromyalgia, neuroimmune disease, nerve injury, experimental animals

INTRODUCTION

Myelin basic protein (MBP) is a major component of the myelin shield in the central and peripheral nervous systems (CNS/PNS). Because MBP is an intrinsically unstructured and abundant cationic protein, MBP and its fragments exhibit a multitude of physiological functions including, but not limited to, interactions with anionic lipids and actin and tubulin cytoskeletal proteins, regulation of myelin compaction and structural assembly of the axon-glia unit (1,2). Furthermore, MBP exhibits the cryptic epitopes that play a critical role in autoimmune demyelinating pathologies such as multiple sclerosis (MS) (3). These epitopes are also release in the course of PNS/CNS injury and neurodegenerative diseases.

Matrix metalloproteinase (MMP) proteolysis of MBP releases the fragments of MBP, including the cryptic epitope sequence that is hidden in the MBP native fold, into the injured nerve microenvironment (4–13). The centrally located 84-104 region of MBP [that is exemplified in our studies by a synthetic MBP84-104 peptide (ENPVVHFFKNIVTPRTPPPSQ)] is highly conserved in mammals (Figure 1a). This region includes the major immunodominant MBP84-95 T cell epitope sequence identified in patients with MS (14,15). This sequence is frequently used to induce experimental autoimmune encephalomyelitis (EAE), a murine model of human MS (14-16). Previous studies by us and other have suggested that neuropathic pain associated with both demyelinating sciatic nerve chronic constriction injury (CCI) and EAE depends on this centrally located MBP epitope (17-23). A single injection of the synthetic MBP84-104 peptide, the sequence of which correlates to the MBP cryptic epitope, into the intact sciatic nerve is sufficient to stimulate robust, long-lasting, T cell-dependent mechanical pain hypersensitivity and neuroinflammation (9,17,22–24). Because of the absence of a reliable methodology, it remained unknown whether an increase in the autoantibodies follows the release of the cryptic MBP84-104 epitope after painful peripheral mononeuropathy (25,26). Currently, there are no simple serum tests for detecting biomarkers of demyelination, and these tests remain an unmet clinical need, especially in MS (27). For example, in MS diagnosis the current blood tests, including ELISAs, help to rule out lookalike diseases that share some similar symptoms with MS, but which require distinct treatment regiments relative to MS (27–29).

Figure 1.

Figure 1

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ELISA to detect autoantibodies against the cryptic MBP peptide. (a) Sequence alignment of the evolutionary conserved cryptic central region of MBP. Human (Homo sapiens), chimpanzee (Pan troglodytes), pig (Sus scrofa), guinea pig (Cavia porcellus), rat (Rattus norvegicus), mouse (Mus musculus), cattle (Bos taurus), rabbit (Oryctolagus cuniculus), horse (Equus caballus) and chicken (Gallus gallus). Conserved residues are in bold. The cryptic MBP84-104 epitope, black square. Residues are numbered according to the human MBP sequence (GenBank #AAH08749). (b) Illustration of the ELISA. The biotin-labeled wild-type and scrambled MBP84-104 peptides (WT and SCR, respectively) were immobilized on the ExtrAvidin-coated wells of a 96-well plate. Serum aliquots were added to the wells to capture the circulating anti-MBP84-104 epitope IgG/IgM autoantibodies. The captured IgG/IgM autoantibodies were then detected and quantified using horseradish peroxidase (HRP)-conjugated species-specific IgG/IgM antibodies, and a HRP substrate.

To specifically quantify the serum concentrations of the autoantibodies against the cryptic 84-104 MBP epitope we developed a streamlined ELISA methodology. This ELISA employed the immobilized synthetic MBP84-104 peptide as bait for capturing the circulating anti-MBP84-104 IgG or IgM autoantibodies from the sample, and then the horseradish peroxidase (HRP)-labeled species-specific IgG or IgM antibodies for the detection of the captured complex. The scrambled peptide (SCR) was used as a control. Our multiple tests demonstrated the high reproducibility of the ELISA methodology with its low intra- and inter-assay coefficient of variability (CV) values of 2.87% and 4.58%, respectively. The ELISA we developed and then optimized was used to measure the anti-MBP84-104 autoantibody levels in rats post-CCI (8,9,17). We then employed the ELISA for testing human serum samples in order to identify a cohort with demyelinating conditions (MS) relative to fibromyalgia syndrome (FMS) patients and healthy volunteers (all females).

Overall, our results suggest that seropositivity for the IgM and IgG autoantibodies against the cryptic MBP epitope represents a valuable biomarker of nerve demyelination in both experimental and clinical settings. We further believe that the ELISA tests may facilitate the rational diagnosis of painful neuropathy patients with the earlier or ongoing demyelination and help to personalize their treatment regimen and to avoid the futility of continuous and widespread use of the addictive conventional opioid analgesics in which some patients may be opioid-resistant.

MATERIALS AND METHODS

General reagents and antibodies

All reagents were purchased from Millipore and Sigma unless indicated otherwise. The HRP-conjugated goat anti-rat IgM (#3020-05) and goat anti-rat IgG (#112-035-175) was from Southern Biotech and Jackson ImmunoResearch, respectively. A 3,3′,5,5′-tetramethylbenzidine substrate (TMB/E) and IgG & protease-free BSA (a 30% solution) were from Surmodics and US Biological, respectively.

MBP and peptides

The synthetic wild-type (WT; ENPVVHFFKNIVTPRTPPPSQ) and scrambled (SCR; EFPHIKVTVVTPRNGFPNSPP) MBP84-104 peptides (97–99% purity) were synthesized by GenScript and protected from exoprotease degradation by N-biotinylation and C-terminal amidation, respectively. Peptides are numbered according to the human MBP sequence (GenBank #AAH08749). Human intact MBP (an 18.5 kDa isoform) was from Meridian Life Science.

Animal model and serum sample collection

Eight adult female Sprague-Dawley rats (200–225 g) were obtained from Envigo Labs and housed in a temperature-controlled room (22°C) on a 12-h light/dark cycle with free access to food and water. Animals were anesthetized with 4% isoflurane in oxygen (Aerrane; Baxter) and then the common sciatic nerve was exposed unilaterally at the mid-thigh level. The nerve received three loosely constrictive chromic gut ligatures to produce CCI (9,17). At day 0 (prior injury) and days 7, 14 and 28 post-CCI blood samples (1–2 ml, each) were collected without anticoagulant from the same cohort of rats. Blood samples were allowed to clot for 30 min at ambient temperature, centrifuged (2,000 × g; 10 min; 4°C) and the supernatant serum was stored at −80°C. All animal procedures were performed according to the PHS Policy on Humane Care and Use of Laboratory Animals with the experimental protocol approved by the Institutional Animal Care and Use Committee at the VA San Diego Healthcare System, and complied with ethical guidelines of the International Association for the Study of Pain.

ELISA to detect the anti-MBP84-104 epitope IgG and IgM autoantibodies in serum samples

The wells of a 96-well Maxisorp ELISA plate (Thermo Scientific) were coated for 18 h at 4°C with ExtrAvidin (3 μg/ml in 0.125 ml 15 mM bicarbonate buffer, pH 9.6). Non-specific binding was blocked for 1 h at 37°C using 1% IgG & protease-free BSA (0.4 ml) in 50 mM Tris-HCl buffer, pH 7.8, containing 1 M NaCl and 0.1% Tween-20 (TBS/T). After six washes (5 min, each; 500-700 rpm) in TBS/T at ambient temperature, the biotin-labeled WT and SCR MBP84-104 peptides (5 μg/ml in 0.1 ml TBS/T-1% BSA, each) were added to the wells. Incubation was continued at 4°C for an additional 16–18 h. The follow-on procedures were carried out at ambient temperature and gentle agitation (500–700 rpm). After six washes (5 min, each) in TBS/T, rat or human serum samples (diluted 1:50-150 in 0.1 ml TBS/T-1% BSA) were allowed to bind to the peptide-coated wells for 3 h. Following extensive washes in TBS/T (5 min, each), the secondary HRP-conjugated species-specific IgG or IgM antibodies (diluted 1:10,000 in 0.1 ml TBS/T-1% BSA, each) were added to the wells for 1 h. After extensive washes in TBS/T (5 min, each), and then with H2O, the TMB/E substrate (0.1 ml) was added to the wells for 15-60 min. The reaction was stopped by adding 1 M H2SO4 (0.1 ml). The resulting A450 value was measured using a plate reader. Data are means ± standard deviation (SD) from at least 3 individual experiments performed in two-four replicates.

Cuff-off and threshold value determination for the ELISA

The cut-off value for the ELISA measurements with the WT and SCR MBP84-104 peptides were determined using eight normal serum samples from female intact, healthy animals analyzed in four replicates. To reach insignificant experimental fluctuations that may occur in the course of the ELISA procedure, all serum samples (1:50 dilution, each) were used in the same ELISA plate to determine the A450 values for the WT and SCR peptides each. The threshold values for the WT and SCR were then calculated as 3 SD above the mean of the samples.

The ELISA assay specificity

The specificity of the ELISA was evaluated using serum samples collected from rats prior to CCI and at days 7, 14 and 28 post-CCI (n=4/group, each) and the full-length intact MBP as bait. The wells were coated with MBP (3 μg/ml in 0.125 ml 15 mM bicarbonate buffer, pH 9.6). The wells coated with BSA (3 μg/ml) served as a control. TBS/T-1% BSA (0.4 ml) was used to block the non-specific binding. The plates were incubated for 3 h at ambient temperature with the serum samples (diluted 1:50 in 0.1 ml TBS/T-1% BSA). The follow-on steps were as described above.

The ELISA assay reproducibility

The precision and repeatability of the ELISA were evaluated using serum samples collected from rats prior to CCI and at days 7, 14 and 28 post-CCI (n=4/group, each). The samples were analyzed for the IgM autoantibodies to determine the A450 values against the WT and SCR baits, and then to calculate the intra- and inter-assay CV. To calculate the intra-assay CV, two replicates of 48 serum samples (1:50–150 dilution) were tested in the same plate using the WT and SCR baits. To determine the inter-assay CV, four replicates of four serum samples with the different anti-MBP84-104 autoantibody levels (threshold, low, intermediate and high levels at day 0, 7, 14 and 28, respectively) were repeatedly and independently tested in the four individual plates against the WT and SCR baits.

Human serum samples

Serum samples were obtained from sixteen patients who were recruited from two different studies, both approved by the Institutional Review Board at UCSD, where the studies were conducted. Six patients with MS and two healthy volunteers (all females) were recruited from the Multiple Sclerosis Clinic, the Department of Neurology, UCSD, and eight female patients with FMS were enrolled from the primary care and Center for Pain Medicine clinics at UCSD. None of the subjects showed any clinical sign of active infection at the time of sample collection. Serum samples were handled using standard procedures and then stored at −80°C until use.

RESULTS

ELISA concept

The cryptic immunodominant epitope is hidden in intact MBP. In the course of demyelination and limited MMP proteolysis of MBP, this epitope is released and could be recognized by the immune system. The identification and then the quantification of the released MBP epitope in the serum is exceedingly difficult. Conversely, based on the studies by us and others (8,9,17,22,24,30–33), we speculated that an elevated level of the circulating antibodies to this epitope would be a reliable marker of demyelinating pathologies. However, whether the raise in the autoantibodies follows the release of the immunodominant MBP epitope in demyelinating neuropathy caused by traumatic nerve injury was not known.

To measure the level of these autoantibodies in the serum, we developed a robust, streamlined ELISA methodology to quantify both circulating IgM- and IgG-type autoantibodies against the cryptic immunodominant 84-104 MBP sequence region (exemplified in our study by a synthetic MBP84-104 peptide). The ELISA employed the biotin-labeled synthetic WT and SCR (control) MBP84-104 peptides as bait. The peptides were immobilized on the ExtrAvidin-coated wells of a 96-well plate. The baits were used to capture the circulating anti-MBP84-104 autoantibodies from the serum. The HRP-labeled species-specific IgG or IgM antibodies were then used to detect the captured complex (Figure 1a). Because demyelinating autoimmune diseases are prevalent in females (34–36), we used female subjects alone in our study.

ELISA optimization

The ELISA was designed and then optimized using serum samples obtained from the control and experimental female rats. The latter exhibited experimental mononeuropathy caused by CCI of sciatic nerve. Serum samples were collected prior to CCI and then at days 7, 14 and 28 post-CCI, and then used for the ELISA optimization.

Multiple tests were performed to optimize the ELISA performance, reproducibility and sensitivity. As a result, we established the optimal concentration of ExtrAvidin for plate coating (3 μg/ml), the requirements for the use of IgG & protease-free 1% BSA for blocking non-specific binding, the optimal concentration of the biotin-labeled MBP84-104 peptides for their immobilization on the ExtraAvidin-coated plastic (5 μg/ml), the optimal dilution range (1:50) of rat serum samples for the subsequent tests, the optimal dilution (1:10,000) of the secondary HRP-conjugated species-specific IgG or IgM antibodies, the optimal buffer composition used for sample incubation and plate washing (Tris-buffered saline containing 1 M NaCl and 0.1% Tween-20) and multiple additional experimental parameters.

Following the ELISA protocol optimization, we determined the cut-off value of the methodology we developed. For these purposes, we, first, used serum samples of eight female intact rats and the WT and SCR peptides as baits. To avoid plate-to-plate variations, the four replicates of each sample were analyzed for the IgG and IgM levels in the same 96-well plate. In these samples, the A450 values for the IgM autoantibodies were recorded for the SCR and WT peptides [A450=0.194±0.015, range, 0.165–0.213 and A450=0.235±0.021, range, 0.198–0.269, respectively] (Table 1 and Figure 2a, day 0). The threshold value that differentiates the positive and negative samples was calculated as 3 standard deviations above the mean of the samples. Thus, the threshold value for the IgM with the SCR peptide equaled 0.198+3×0.015=0.237. Any sample with A450≥0.237 was then considered positive (Table 1). Similarly, we calculated the ELISA IgM threshold value for the WT peptide (0.298), and then any sample with A450≥0.298 was considered positive. In contrast, in the intact rat serum samples the mean A450 values for the IgG were exceedingly low for both WT and SCR peptides (A450=0.022±0.002 and A450=0.021±0.003, respectively). The calculated threshold values for the WT and SCR peptides were 0.028 and 0.030, respectively (Figure 2a, day 0).

Table 1. The ELISA threshold values for IgM in rat serum.

Rat serum samples (1:50 dilution; n=8) from naïve animals were analyzed in four replicates using the ELISA with the immobilized WT and SCR MBP84-104 peptides. The mean value against the individual WT and SCR peptide was calculated for each animal, and then used to determine the overall mean (mean of means) and standard deviation (SD). The threshold value was calculated as 3 SD above the overall mean.

WT MBP84-104 SCR MBP84-104
Samples A450 A450 A450 A450 Means A450 A450 A450 A450 Means
1 0.252 0.247 0.243 0.238 0.245 0.208 0.203 0.203 0.198 0.203
2 0.220 0.224 0.230 0.234 0.227 0.210 0.199 0.201 0.200 0.202
3 0.244 0.249 0.264 0.269 0.257 0.165 0.185 0.185 0.181 0.179
4 0.229 0.246 0.242 0.258 0.244 0.192 0.196 0.193 0.197 0.194
5 0.205 0.209 0.198 0.214 0.207 0.192 0.196 0.193 0.197 0.194
6 0.241 0.243 0.248 0.250 0.245 0.190 0.192 0.182 0.184 0.187
7 0.243 0.247 0.256 0.261 0.252 0.209 0.213 0.203 0.207 0.208
8 0.204 0.189 0.207 0.200 0.200 0.197 0.199 0.197 0.196 0.197
Mean of Means 0.235 Mean of Means 0.196
SD of Means 0.021 SD of Means 0.009
Threshold 0.298 Threshold 0.223
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Figure 2.

Figure 2

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ELISA measurements in rat serum samples for the circulating autoantibodies against the cryptic MBP peptide. (a) ELISA of the anti-MBP84-104 IgG and IgM autoantibodies in the serum of rats with mononeuropathy inducred by CCI. The biotin-labeled wild-type (WT, blue and green) and scrambled (SCR, red and purple) MBP84-104 peptides were immobilized on the ExtrAvidin-coated wells as antigen targets, respectively. Serum aliquots from females (n=4–8/group) collected at day 0 (prior injury) and at days 7, 14 and 28 post-CCI were allowed to bind to the peptides. The bound autoantibodies were detected using HRP-conjugated anti-rat IgM or IgG, and a TMB/E substrate. The threshold values for the ELISA for each antigen target were determined as 3 standard deviations above the mean of the rat serum samples collected prior injury (n=8). Dotted lines, the threshold values for the WT and SCR peptides were A450 = 0.298 and A450 = 0.223, respectively. (b) ELISA of the IgG and IgM autoantibodies against full-length MBP in rat serum. Intact MBP with the hidden cryptic MBP epitope (red and purple) and BSA (control; blue and green) were immobilized onto the wells as antigen targets, respectively. Serum aliquots (n=4/group) were collected and analyzed as described in (a).

We next measured the levels of circulating anti-MBP84-104 IgG and IgM autoantibodies in the intact rat serum samples (day 0) and also in the samples obtained at days 7, 14 and 28 post-CCI. The IgM autoantibodies against the WT peptide were readily detected in the serum at day 7 (an average A450=0.065 above the threshold). The IgM levels continued to increase at days 14 and 28 with an average A450=0.134 and A450=0.323 above the threshold, respectively. In turn, the levels of the IgM autoantibodies against the SCR peptide did not change significantly over time and remained similar to the threshold value. We then calculated the levels of the specific circulating IgM autoantibodies against the WT epitope as the difference in the average A450 value between the WT and the SCR peptides. Thus, relative to the threshold level in the intact rats, the specific anti-MBP84-104 IgM autoantibody pool increased 2.4-, 3.5- and 6.8-fold at days 7, 14 and 28 post-CCI, respectively (Figure 2a). Contrary to the seropositivity for the IgM autoantibodies, seronegativity was recorded for the anti-MBP84-104 IgG autoantibodies, likely because of the short-time nature of our experiments and the insufficient time to raise the IgG pool against the newly exposed immunodominant antigen (Figure 2a). Overall, our data support our suggestion that MBP is proteolysed shortly after nerve injury, and then its cryptic epitope is released, inducing as a result, the anti-epitope IgM autoantibodies, the first antibodies produced by the immune system exposed to a novel antigen. We have to emphasize that the ELISA methodology represents a novel diagnostic tool to detect the circulating autoantibodies against the cryptic MBP peptide associated with demyelination. It remains undetermined if a correlation between the autoantibody levels and the pain status exists.

ELISA reproducibility and specificity

To assess the reproducibility and specificity of the ELISA, we used the rat serum samples with the increasing level of the anti-MBP84-104 IgM autoantibody (at days 0, 7, 14 and 28 post-CCI). To establish the reproducibility of the ELISA, we determined the intra-assay CV (precision) and inter-assay CV (repeatability). Thus, using the WT and SCR baits, we measured the IgM levels in 48 serum samples (two replicates each) in the same plate to calculate the intra-assay CV (2.87%; a 0.16% to 6.68% range) (Table 2). To establish the inter-assay CV for the IgM antibodies, we analyzed the same four serum samples (four replicates each) on four different plates using both the WT and SCR baits. The inter-assay CV was then calculated to equal 4.58% (a 2.96% to 7.20% range) (Table 3). Intra- and inter-assay CV below 10% are normally considered acceptable for diagnostic ELISA tests (37,38) indicating the compliance of the ELISA we developed with the generally accepted standards.

Table 2. The ELISA intra-assay coefficient of variability (CV) for the IgM measurements in rat serum.

Two replicates of 48 rat serum samples (1:50–150 dilution) collected at the indicated time (0, 7, 14 and 28 post-CCI) were analysed using the ELISA with the individual WT and SCR MBP84-104 immobilized on the same plate. To determine the % CV we calculated, for each sample, the mean and standard deviation (SD) of the two A450 values of the replicates. The intra-assay CV (2.87%) is reported as the average of the individual % CV for the samples (a 0.16–6.68 range). Acceptable range for intra-assay CV for an ELISA was determined to be below <10%.

Samples Peptides Days Dilutions A450 A450 Means SD % CV
1 WT 0 50 0.241 0.263 0.252 0.015 6.14
2 WT 0 50 0.268 0.290 0.279 0.015 5.56
3 WT 0 50 0.279 0.291 0.285 0.008 2.85
4 WT 0 50 0.284 0.272 0.278 0.008 2.92
5 WT 7 50 0.364 0.386 0.375 0.016 4.18
6 WT 7 50 0.351 0.355 0.353 0.002 0.64
7 WT 7 50 0.359 0.355 0.357 0.003 0.79
8 WT 7 50 0.397 0.390 0.393 0.005 1.26
9 WT 14 50 0.421 0.425 0.423 0.003 0.64
10 WT 14 50 0.450 0.454 0.452 0.003 0.59
11 WT 14 50 0.465 0.446 0.455 0.014 3.07
12 WT 14 50 0.405 0.408 0.407 0.002 0.43
13 WT 28 50 0.623 0.625 0.624 0.001 0.16
14 WT 28 50 0.629 0.630 0.630 0.001 0.16
15 WT 28 50 0.547 0.559 0.553 0.009 1.56
16 WT 28 50 0.608 0.596 0.602 0.009 1.43
17 SCR 0 50 0.210 0.202 0.206 0.006 2.78
18 SCR 0 50 0.182 0.200 0.191 0.013 6.62
19 SCR 0 50 0.185 0.201 0.193 0.011 5.71
20 SCR 0 50 0.228 0.215 0.222 0.009 4.15
21 SCR 7 50 0.187 0.196 0.192 0.006 3.03
22 SCR 7 50 0.192 0.196 0.194 0.002 1.17
23 SCR 7 50 0.175 0.165 0.170 0.007 4.28
24 SCR 7 50 0.201 0.200 0.200 0.001 0.49
25 SCR 14 50 0.219 0.223 0.221 0.003 1.22
26 SCR 14 50 0.193 0.197 0.195 0.003 1.38
27 SCR 14 50 0.200 0.215 0.207 0.011 5.32
28 SCR 14 50 0.199 0.194 0.197 0.004 1.87
29 SCR 28 50 0.163 0.164 0.163 0.001 0.61
30 SCR 28 50 0.197 0.199 0.198 0.001 0.50
31 SCR 28 50 0.199 0.216 0.207 0.012 5.59
32 SCR 28 50 0.186 0.193 0.189 0.005 2.76
33 WT 0 150 0.093 0.098 0.096 0.003 3.55
34 WT 0 150 0.080 0.073 0.076 0.005 6.68
35 WT 0 150 0.107 0.102 0.104 0.003 3.25
36 WT 0 150 0.082 0.080 0.081 0.001 1.49
37 WT 7 150 0.124 0.124 0.124 0.000 0.34
38 WT 7 150 0.142 0.156 0.149 0.010 6.54
39 WT 7 150 0.124 0.129 0.126 0.004 2.80
40 WT 7 150 0.122 0.112 0.117 0.008 6.48
41 WT 14 150 0.166 0.174 0.170 0.006 3.25
42 WT 14 150 0.164 0.164 0.164 0.000 0.30
43 WT 14 150 0.169 0.177 0.173 0.006 3.30
44 WT 14 150 0.159 0.160 0.160 0.000 0.18
45 WT 28 150 0.264 0.245 0.255 0.013 5.22
46 WT 28 150 0.249 0.223 0.236 0.018 7.82
47 WT 28 150 0.242 0.262 0.252 0.015 5.78
48 WT 28 150 0.245 0.249 0.247 0.003 1.09
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Intra-assay % CV 2.87

Table 3. The ELISA inter-assay CV for the IgM measurements in rat serum samples with the threshold, low, intermediate and high levels of the anti-MBP84-104 autoantibodies.

Four replicates of four rat serum samples (1:50 dilution) with the threshold, low, medium and high levels of the IgM autoantibodies were each analysed on four individual plates, using the individual WT and SCR MBP84-104 immobilized on the same plate. For each plate and each IgM autoantibody level, we determined the A450 WT-SCR value, using the average A450 values of the replicates against the WT and SCR peptides. To determine the % CV for the four individual IgM autoantibodies levels, we calculated the mean and standard deviation (SD) of the four A450 WT-SCR mean values recorded on each of the individual plates. The inter-assay CV (4.58%) is reported as the average of the individual %CV for the respective IgM autoantibodies levels (a 2.96–7.20 range). Acceptable range for inter-assay CV for an ELISA was determined to be below <10%.

Mean values (n=4)
IgM levels		 A450 WT A450 SCR A450 WT-SCR
Plate
Threshold 0.262 0.191 0.071 1
Threshold 0.269 0.193 0.076 2
Threshold 0.277 0.206 0.071 3
Threshold 0.276 0.207 0.069 4
Low 0.375 0.192 0.184 1
Low 0.353 0.194 0.159 2
Low 0.357 0.170 0.187 3
Low 0.383 0.200 0.183 4
Medium 0.423 0.197 0.227 1
Medium 0.452 0.221 0.231 2
Medium 0.455 0.207 0.248 3
Medium 0.437 0.195 0.242 4
High 0.644 0.207 0.437 1
High 0.630 0.198 0.432 2
High 0.623 0.163 0.460 3
High 0.622 0.189 0.433 4
Threshold Low Medium High
Mean of Means 0.072 0.178 0.237 0.440
SD of Means 0.003 0.013 0.010 0.013
% CV of Means 3.97 7.20 4.19 2.96
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Inter-assay CV = average % CV, 4.58

To evaluate the specificity of the ELISA, we used the full-length MBP in which the epitope is hidden. Thus we analyzed the rat serum samples (collected prior injury and at days 7, 14 and 28 post-CCI) using intact MBP, instead of the WT or SCR peptides, as bait. There was no significant difference between full-length MBP and a BSA control for neither IgM nor IgG autoantibodies (Figure 2b). Based on these results, we concluded that the level of the autoantibodies against full-length MBP was insignificant, if any, in the rat serum post-CCI. Conversely, these data suggest again that native MBP is proteolyzed after nerve injury and that the released cryptic epitope, rather than the full-length MBP protein, is recognized as a novel antigen that engages a response by the immune system resulting in the specific pool of the anti-MBP84-104 epitope autoantibodies. Overall, the ELISA methodology we established allowed us to assess, both quantitatively and in a precise and repeatable fashion, the level of the circulating specific IgM autoantibodies against the cryptic MBP84-104 epitope fragment released post-CCI in the rat serum.

Evaluation of human samples

Because the MBP84-104 sequence is conserved in rodents and humans (Figure 1), we next determined if the ELISA was applicable to human serum samples. For these purposes, we performed a limited study that included sixteen serum samples. These samples were obtained from six patients with MS (a demyelinating disease characterized by the autoantibody presence to this immunodominant MBP epitope (14–16,25)), from eight patients with FMS (a presumably, non-demyelinating disease in which the presence of autoantibodies to this MBP epitope was not known) and two healthy, control, volunteers (all females).

One MS patient who received Copaxone, a drug known to interfere with immune system(39), exhibited confusing measurements and, as a result, was excluded from our analysis. Our ELISA measurements showed that in the serum from MS patients, the anti-MBP84-104 IgG autoantibodies were readily detectable (average A450=0.417, a 0.147–0.854 range), whereas the healthy volunteers were seronegative (average A450=0.012) (Figure 3a). Relative to healthy control, the circulating IgG autoantibody level was elevated 12.6–73.3-fold (average=35.8-fold). Relative to healthy volunteers, a less noticeable, 4.4–12.1-fold increase (average=6.5-fold) in the IgM autoantibodies was also recorded in MS patients. Because MS is a long-term demyelinating disease with episodic relapses, the predominant presence of the IgG antibodies against the cryptic epitope is not surprising.

Figure 3.

Figure 3

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ELISA measurements for the circulating autoantibodies against the cryptic MBP peptide in human serum. (a) ELISA in MS patients (demyelinating conditions). The biotin-labeled WT and SCR MBP84-104 peptides were immobilized on the ExtrAvidin-coated wells of a 96-well plate. Serum aliquots from healthy volunteers (averaged values, CTR) and MS patients (M-1, M-2, M-3, M-4 and M-5) were allowed to bind to the peptides. The bound IgG (black rectangles) and IgM (grey rectangles) antibodies were then detected using HRP-conjugated antihuman IgG or IgM, and a TMB/E substrate. Left, the specific A450 values for the WT peptide are calculated relative to the SCR peptide (A450 WT-SCR). Right, the IgG- and IgM-fold difference in the specific A450 WT-SCR values for the MBP84-104 peptide in MS patients relative to CTR. (b) ELISA in FMS patients (presumably, non-demyelinating conditions). Serum samples from eight patients (F-1, F-2, F-3, F-4, F-5, F-6, F-7 and F-8) were analyzed using the ELISA as described in (a). The average A450 WT-SCR values for the serum of healthy volunteers (CTR) and MS patients (MS) were used for comparison. The specific A450 WT-SCR values for the WT peptide were calculated relative to the SCR peptide. Black and grey rectangles, the specific IgG and IgM levels against the MBP84-104 peptide, respectively. (a–b) Data are means ±SD from at least three individual experiments performed in triplicate.

In contrast with MS, demyelination is not common in FMS. Some studies indicate that FMS does not involve nerve demyelination, whereas a few others claim otherwise and, as a result, there is no consensus as yet (40–44). To detect if the autoantibodies to MBP84-104 were present in FMS, we analyzed the serum samples from FMS patients. Our measurements recorded that, in contrast to MS, FMS patients exhibited a significantly lower level of the circulating IgG autoantibodies. On average, the anti-MBP84-104 IgG level was ~13-fold lower in FMS patients than in MS patients (Figure 3b). Intriguingly, four from eight FMS patients exhibited the measurable, albeit very low, IgG seropositivity that was ~5.5-fold higher relative to the healthy volunteers, while the other four patients were seronegative. The level of the specific IgM autoantibodies was exceedingly low or even below detection level in all FMS samples.

In conclusion, our current study demonstrated as a proof-of-concept that the ELISA methodology we developed delivers the reproducible measurements of the circulating IgG and IgM autoantibodies against the cryptic MBP epitope that is released as a result of nerve demyelination in both experimental and clinical settings.

DISCUSSION

MBP is an established autoantigen in patients with autoimmune demyelinating MS and the respective experimental EAE models (3,14,16,45). In rodent models of traumatic peripheral nerve injury, including CCI, the cryptic central MBP epitope is released via MMP proteolysis (8,9,17). It is important to emphasize that the MBP84-104 sequence region is conserved in humans and rodents. This cryptic central MBP epitope has been implicated in neuropathic pain associated with both CCI and EAE (17–21). Further, a single bolus, adjuvant-free injection of the MBP84-104 epitope peptide into intact sciatic nerve is sufficient to produce robust mechanical pain hypersensitivity in female rats lasting for weeks, in the absence of overt neuropathology or widespread neuroinflammation (9,23).

Whereas circulating autoantibodies against the algesic MBP epitope are believed to contribute to MS and EAE, whether their levels are elevated in painful peripheral demyelinating neuropathy was not known. To generate a tool that may help to answer these questions, we developed the robust, sensitive and reproducible ELISA methodology. This ELISA measures the circulating anti-MBP84-104 cryptic epitope autoantibodies in serum samples. Using the ELISA we developed, we demonstrated, for the first time, that the level of the IgM, but not IgG, autoantibodies continuously increased in female rats after nerve injury. The upregulation of the IgM-type antibodies, the first antibodies isotype B cells produce in response to an novel foreign antigen exposure (28), may relate to the short time-frame of our neurotrauma experiments. It is well established that the avidity (accumulated strength of multiples affinities) of the pentameric IgM antibodies with 10 antigen-binding sites are superior relative to monomeric IgG that only contains 2 antigen binding sites.

To provide, ultimately, an additional tool for diagnosis of demyelinating pathologies, we tested if the ELISA we developed and validated in rats is applicable for the analysis of human serum. We determine that the ELISA readily discriminated MS patients from healthy volunteers and recorded the high level of the anti-MBP84-104 IgG autoantibodies in MS. In turn, the autoantibody level was low or nonexistent in FMS patients, a disease in which widespread nerve demyelination is uncommon (46). Neuropathic pain featuring allodynia, lancinating and burning pain is common in MS (47). Females constitute ~80% of patients with autoimmune conditions and are more common sufferers of MS, FMS and chronic pain in general (34–36,48-51). The MBP84-104 ELISA we developed is applicable for analyzing blood samples. This ELISA employs widely available and inexpensive reagents, and provides rapid measurements with a diagnostic value for demyelinating diseases, a capacity that other current blood tests are lacking.

Overall, we believe that the cryptic immunodominant 84-104 epitope of MBP represents a reliable nerve demyelination marker and that the ELISA we developed is a promising test to facilitate the rational diagnosis of demyelinating pathologies and to supplement the currently existing diagnostic protocols in neuropathy. The present study provides the first evidence for the presence of the circulating autoantibodies against the cryptic MBP epitope in the model of painful traumatic nerve injury. This is consistent with the emerging role of neural antigens in nociceptive pathways (25,26,52–56). Furthermore, because neuropathic pain in demyelinating pathologies, including MS, is poorly responsive to opioid analgesics (29,57), the use of this ELISA may also help to avoid opioid overuse.

HIGHLIGHTS.

  • The cryptic epitope of MBP is released in demyelinating diseases

  • This cryptic autoantigen stimulates the production of autoantibodies

  • To quantify these antibodies, we developed a robust and sensitive ELISA methodology

  • ELISA identifies demyelination pathologies in experimental and clinical settings

Acknowledgments

This work was supported by NIH R01 DE022757 (to VIS and AYS), the Department of Veterans Affairs 5I01BX000638 (to VIS), National Multiple Sclerosis Society PR-1503-03449 (to VIS and AYS) and the UCSD Clinical and Translational Science Program UL1TR001442 (to AGR) grants. We are grateful to Lucinda Martin, Katie Lam, Drs. Sabrina Oukil, Laura Muehl, Rip Kinkel and Tobias Moeller-Bertram for their help in collecting human serum samples.

Abbreviations

CCI

chronic constriction injury

FMS

fibromyalgia syndrome

HRP

horseradish peroxidase

MBP

myelin basic protein

WT and SCR MBP84-104

the wild-type and scrambled 84-104 peptide of MBP

MS

multiple sclerosis

TBS/T

50 mM Tris-HCl buffer, pH 7.8, containing 1 M NaCl and 0.1% Tween-20

Footnotes

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Conflict of Interest. The authors report no conflict of interest.

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