Analysis of anti-ganglioside antibodies by a line immunoassay in patients with chronic-inflammatory demyelinating polyneuropathies (CIDP)

Introduction

The last two decades of research have led to remarkable advances in the clinical and electrophysiological categorization of acute and chronic immune-mediated neuropathies (IN) that was paralleled with the identification of multiple novel autoantibodies (autoAb) and their corresponding targets. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare autoimmune disorder of the peripheral nervous system and can clinically distinguished between typical CIDP and atypical variants, such as distal acquired-demyelinating polyneuropathy (DADS), multifocal-acquired demyelinating sensory and motor polyneuropathy (MADSAM) and sensory-CIDP [1], [2]. Its heterogeneous clinical manifestation suggests different autoimmune targets. Previously, we and others detected autoreactive T-cell responses against myelin antigenic epitopes [3], [4]. Further, autoAbs against the paranodal protein neurofascin NF155 have been identified in patients suffering from combined central and peripheral demyelination [5] and in a subset of CIDP patients with distinct clinical features [6]. In addition, autoAbs against the nodal neurofascin NF186 have been found in CIDP [7].

In contrast to acute IN, the value of anti-ganglioside autoantibody (aGAAb) testing has been established for only a minority of chronic IN yet. This holds particularly true for autoAbs against GM1 (aGM1) in multifocal motor neuropathy (MMN) and to the myelin-associated glycoprotein (aMAG) in DADS [8], [9]. In addition, increased titers of anti-sulfatide (aSF) IgM are found in patients with neuropathy where they are often associated with a concomitant reactivity to MAG. A selective reactivity to sulfatide (SF), however, was identified only in 1% of CIDP patients [10] so that a diagnostic value is still in doubt. In a retrospective analysis of altogether 172 patients, aGM1 IgM was found in 46% of patients with MMN but in only 3% of CIDP patients [11].

Thus, in most IN, the diagnostic value of aGAAb is still elusive. Thin-layer chromatography is supposed to be the gold standard assay technique for aGAAb analysis, although it is not applicable for routine use. Enzyme immunoassays with highly purified gangliosides are not cost-effective. Dot or line immunoassays (LIA) may be a good alternative for the multiparametric determination of aGAAbs, provided that an optimal autoantigenic epitope-preserving binding of gangliosides on membranes can be achieved [12], [13]. We developed a novel semiquantitative evaluation method for aGAAb detection by LIA and tested its performance for the analysis of GAAb in patients with chronic IN.

Materials and methods

Results

Intra- and interassay variability of the line immunoassay (LIA)

Binding of aGAAbs was quantified for each target as OD values by the DotBlot Analyzer software using a grayscale calibration. The intra-assay (Figure 1A) and inter-assay (Figure 1B) coefficients of variation (CVs) for aGAAb testing by LIA were analyzed for three aGAAb-positive sera in accordance with the CLSI-protocol EP15-A2 (https://clsi.org/media/1431/ep15a3_sample.pdf). Thus, intra-assay CV was determined by eight measurements for each serum, whereas interassay CV was assessed by analyzing eight determinations for each serum on five different days. At the cutoff value of 20 OD, the intra-assay CV reached 18% (Figure 1A) and the interassay CV 32% (Figure 1B).

Figure 1:

Intra-assay (A) and interassay (B) coefficients of variations (CVs) for the analysis of anti-ganglioside antibodies (aGAAb) by line immunoassay.

Intra-assay CVs were determined by eight measurements for three aGAAb-positive sera within one assay run, whereas interassay CVs were obtained by analyzing eight determinations for each serum on five different days in accordance with the CLSI-protocol EP15-A2. Lines were fitted by regression analysis revealing for intra-assay and interassay CVs: CV coefficients of determination (R²)=0.6735 (y=1.7499x^−0.755) and 0.7288 (y=1.938x^−0.596), respectively. Accordingly, the intra-assay and interassay CVs reached 18% and 32% at the cutoff value of 20 OD, respectively.

Elevated frequencies of aSF, aGM1 and aGD1b IgM in INs

In order to evaluate whether aGAAbs were more frequent in IN compared to the control groups of OND, MS and HC, we screened the sera for aGAAbs IgG as well as IgM against 11 gangliosides and SF. For IgG aGAAbs, there were low frequencies in OND and IN (Figure 2A) with a positive aGAAb against at least one IgG aGAAb in 2/61 (3.3%) of IN patients, in 0/40 (0%) of OND, in 0/29 (0%) of MS patients compared with 7/54 (12.9%, p=0.01) of HC. By contrast, aGAAb IgM was generally more frequent and diverse in IN compared to OND, MS and HC (Figure 2B). Positive IgM reactivity against at least one ganglioside/SF was found in 17/61 (27.9%) of IN patients compared to 2/40 (5.0%) of OND, 2/29 (6.9%) of MS and 4/54 (7.4%) of HC, which was statistically relevant (p=0.002, χ²-test). Analyzing IgM aGAAbs, there were statistically higher frequencies of aSF IgM in IN compared to OND (Figure 3A; p=0.08). Anti-GM1 IgM was elevated in IN compared to OND and HC (Figure 3B; p=0.009). aGD1b IgM was statistically elevated in IN compared to HC (Figure 3C; p=0.04). The prevalence of aGM1 IgM in CIDP was lower (12%) compared to MMN (60%, p=0.027, Fisher’s exact test) (Figure 4).

Figure 2:

Proportions (%) of individuals with anti-ganglioside antibodies (AGAs) and diversity of AGAs within each study group: inflammatory neuropathies (IN, n=61), other neuromuscular disease (OND, n=40), multiple sclerosis (MS, n=29) as well as healthy controls (HC, n=54).

(A) IgG AGAs reactivity within each of the groups IN, OND, MS and HC. (B) More frequent and diverse IgM AGA reactivity in IN compared to OND and HC.

Figure 3:

Cutoff level was defined as OD≥20.

IgM sulfatide (A) are significantly increased in IN (n=61) compared to controls OND (n=40). (B) GM1 antibodies are significantly increased compared to OND and HC (n=54). (C) Anti-GD1b was significantly increased compared to HC. *p<0.05, **p<0.01, ***p<0.001 as measured by the Kruskal-Wallis test and Dunns multiple comparison test. Shown are scatter dot plot with line at cut off at 20.

Figure 4:

Prevalence of IgM GM1 in CIDP (n=56) vs. MMN (n=5).

The asterisk (*) indicates a significant (p<0.05) difference between the groups.

Discussion

By using a novel multiplex LIA for the analysis of aGAAb, we found an elevated frequency of at least one aGAAb IgM in patients suffering from CIDP or MMN compared to controls including patients with OND and HC. Of all tested autoAbs, aSF, aGM1 as well as aGD1b IgM were most frequently detected in IN compared to the control groups.

Anti-SF autoAbs were associated with different subtypes of peripheral neuropathy most of them of axonal type. However, also a demyelinating type with a lower prevalence was identified [21]. A meta-analysis of MS patients identified higher aSF titers in patients suffering from secondary axonal degeneration [22]. In accordance, MS control patients tested in the present study did not show a significantly increased frequency of aSF IgM antibodies, and most of the patients remained below cut off levels.

The assessment of the assay variability based on OD values obtained in a standardized scanning procedure revealed reliable data supporting the usability of quantitative autoAb analysis by LIA as demonstrated for antiphospholipid antibody testing by LIA, too [23]. The obtained interassay CV of 32% at the cutoff of 20 OD units appears satisfactory and confirms earlier data of antibody analysis for sera with higher OD units by LIA [23]. Further, the hydrophobic LIA-solid phase has already proven its usefulness for the specific analysis of auto/Abs to amphiphatic molecules like lipopolysaccharides and glycolipids exhibiting similar physicochemical characteristics [24], [25], [26].

Interestingly, aSF-positive patients in our cohort were younger and showed typical manifestation of clinical symptoms compared to the negative tested group. However, we found no association with axonal degeneration as described [21], [22]. We also did not find any association with hematological disease such as monoclonal gammopathy IgM nor combined with aMAG autoAbs as described recently [10], [27].

In contrast to the aforementioned studies, we included patients fulfilling diagnostic criteria of CIDP or MMN. The fact, that aSF-positive patients were younger and responded well to immunomodulatory treatment may support the autoimmune relevance of SF, which is predominantly expressed within the non-compact myelin [28]. Beside aSF IgM, we detected higher levels (16%) of aGM1 IgM in CIDP and MMN patients compared to controls. This rate is higher than the one shown recently (7%) where IgM to glycolipid complexes containing GM1 and SF were the most frequently observed autoAbs of CIDP patients using combinatorial thin layer chromatography [28]. Interestingly, those patients demonstrated more frequently motor disturbances than antibody-negative ones did [29]. Accordingly, patients of our study demonstrated typical clinical manifestation with predominantly distal and motor disturbances more frequently in aSF and, by trend, aGM1-IgM-positive patients. Moreover, patients with positive aSF autoAbs showed a higher rate of conduction blocks in nerve conduction studies (Table 2). The affection of primarily motor functions may be explained by the experimental model that SF depletion leads to dramatically reduction of myelin proteins, especially in the paranodal region affecting neurofascin 155 [28]. It is also in accordance with the fact that the GM1 epitope is highly expressed on the membranes of motor nerves and on the surface of Schwann cells. Additionally, binding of the antibodies to the axon at the nodes of Ranvier on to Schwann cells may cause complement activation and disruption of sodium channel clusters, resulting in conduction abnormalities [30], [31].

In the present study, aGD1b IgM was found in patients with the highest diagnostic certainty fulfilling EFNS criteria of definite CIDP. GD1b is a disialoganglioside with two sialic acids in its structure. Because they share a same terminal Gal-GalNAc residue, cross-reactivity is often seen between autoAbs to GM1, GD1b and GD1a [11], [29]. Here, we also found cross-reactivity between GD1b and GM1 in 80% of all positive tested patients.

In contrast to acute immune neuropathies such as Guillain-Barré syndrome, here we found aGAAbs of IgM isotype. This might be due to the fact that these autoantibodies are secondary to a predominant T cell-mediated demyelination. Indeed, previous studies of our working group found evidence for a T-cell-driven pathomechanism in CIDP (Klehmet et al. 2015). In order to exclude an unspecific secondary effect, we recruited MS patients as a control for demyelinating disease. Using LIA as novel assay technique, we did not find increased frequencies of IgM nor IgG aGAAbs in MS compared to IN which was all the more remarkable because MS patients were younger and less frequently treated with immunotherapy.

Patients included in this study are part of a very well characterized cohort. We therefore correlated a range of clinical features with antibody specificity but were not able to characterize a distinct subtype as shown in recently published neurofascin NF155-specific CIDP subtype [6].

The early diagnosis of CIDP and treatment initiation is essential in order to prevent irreversible axonal damage and thus disability. However, the correct diagnosis of CIDP is still challenging due to its very heterogeneous manifestation. A diagnostic marker could help to support early diagnosis. In addition, biomarkers could help to predict treatment response and differentiate between clinical phenotypes. It would be therefore of high relevance to validate our findings in larger multicentric studies.

Advantages of our study are a well-defined patient cohort with high diagnostic certainty and age-matched controls, which is essential to assess IgM reactivity. However, our study has also certain limitations. Even though this study is comparatively large with a total number of 130 patients, it is still too small to firmly identify autoAb specificities in correlation with clinical features or specific CIDP subtypes. Another limitation may be the fact that the included patients were not treatment-naive and had rather long disease duration.

In conclusion, we provide evidence of a pathogenetic relevance of aSF, aGM1 and aGD1b IgM in CIDP and MMN. Patients with at least two aGAAbs showed a strong association with fulfilling diagnostic EFNS criteria. Anti-SF, aGM1 and aGM2 IgM were associated with younger age supporting the autoimmune relevance. In addition, aSF and aGM1 IgM were associated with predominant motor disturbances. Further multicentric studies are required to validate these promising biomarkers with reliable cutoffs as useful diagnostic tools for CIDP, its subtypes and for treatment guidance.