Myelin-associated glycoprotein (MAG) is a 100 kDa transmembrane glycoprotein expressed by Schwann cells and represents a target of monoclonal IgM autoantibodies (MAG-Abs). These antibodies recognize the human natural killer-1 (HNK-1) carbohydrate epitope, which is highly expressed on MAG. MAG-Abs are associated with a slowly progressive large-fiber distal symmetrical sensorimotor polyneuropathy with prominent sensory impairment, ataxia, and intention tremor (1–5). The pathogenic role of MAG-Abs has been confirmed by pathological studies on nerve biopsies, showing a relationship between the widening of myelin lamellae (with deposits of IgM M-protein and complement) and segmental demyelination (6–8). Antibodies to MAG can be detected in patient sera by enzyme-linked immunosorbent assay (ELISA) or Western blot using MAG or purified myelin (9–11). For both methods of MAG-Abs detection, a normal range should be defined, as low titers may be found in normal subjects (12). ELISA is often preferred to Western blot because it is more sensitive in detecting serum MAG-Abs, easier to use, and allows antibody titration, which is potentially useful for monitoring responses to therapies (13).

The aim of this study was to determine the clinical performance of a novel immunofluorescence fixed cell-based assay (CBA, EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) in comparison with the gold-standard technique used to identify MAG-Abs (ELISA, Bühlmann, Schönenbuch, Switzerland).

We analyzed 95 patients (F = 32, median age 74 years, range 49–92) with clinical and neurophysiological signs compatible with anti-MAG-associated neuropathy confirmed using the recommended ELISA (>1,000 BTU). All were positive at a 1:10 titration using EUROIMMUN’s fixed CBA. When performing further titrations, 94/95 were also positive at a 1:100 dilution, 74 at 1:1,000, 60 at 1:10,000, 20 at 1:100,000, and 11 at 1:1,000,000 (Figure 1).

Among cases with “weak positivity” (n = 33) according to the standardized ELISA technique (1:1,000–1:10,000 BTU), 28 had a titer of ≤1:1,000 using the fixed CBA (1:10 n = 1, 1:100 n = 18, 1:1,000 n = 9). Among cases with “strong positivity” (n = 62) according to the standardized ELISA technique (≥1:10,000 BTU), 55 had a titer ≥1:10,000 using the fixed CBA (1:10,000 n = 38, 1:100,000 n = 7, 1:1,000,000 n = 10).

All patients with other forms of peripheral neuropathy, included as controls (n = 55, F = 18, median age 67 years, range 7–91), were negative by ELISA. Of these, 6 were positive by CBA at a 1:10 dilution and none was positive at 1:100 or further titrations.

When comparing results of the fixed CBA with those of the reference ELISA technique, we observed that at a 1:10 dilution, the CBA had 100% (CI 96.2–100%) sensitivity and 89.1% specificity (CI 77.8–95.9%, PPV 94.1%, NPV 100%), while at a 1:100 dilution, the sensitivity was 98.9% (CI 94.3–100%) and the specificity 100% (CI 93.5–100%, PPV 100%, NPV 98.2%). There was a trend toward correlation between the titer observed using the two techniques (p = 0.07).

We herein provide preliminary comparative data of MAG-Abs testing using the recommended ELISA technique and a novel ready-to-use fixed CBA. For the assessment of anti-MAG positivity, our results support the use of the novel CBA at a dilution of 1:100, which has the highest specificity (100%) and a very good sensitivity (98.9%). Compared to the other methods, this easy technique has many advantages, including [i] the expression of natively folded antigen in the membranes of the cell substrate enabling the detection of Abs binding to conformational epitopes, [ii] flexibility in assay design (BIOCHIP mosaics), and [iii] fluorescence stability, which allows multiple persons to analyze the slides and exchange images among centers. This standardized technique minimizes inter-assay variability and enables efficient preparation of large, standardized batches in a short time. In addition, it allows both identification and quantification (using further titrations) of specific antibodies.

Italian guidelines for MAG-Abs testing were recently developed through a consensus process that also reported indications and limits of anti-MAG testing, instructions for results interpretation, and an agreed-upon laboratory protocol (14). This consensus supports the use of ELISA as the most appropriate diagnostic technique, although the best cut-off of positivity is still an open question. In accordance with a previous study (13), sera with values between 1,000 and 10,000 BTU are interpreted as “weak positive” and are prone to diagnostic issues. Furthermore, a recent study identified a grey area between 1,500 and 7,000 BTU in which the differential diagnosis between chronic inflammatory demyelinating polyneuropathy (CIDP) and anti-MAG-associated neuropathy is challenging (15). In this scenario, CBA allows monitoring of MAG-Abs titer levels through further titrations, which is important for clinical correlations (16). In addition, fluorescent CBA is based on an easy and standardized technique, that is already used by many laboratories to identify antibodies to central nervous system antigens, such as antibodies to neuronal surface antigens in patients with autoimmune encephalitis or to aquaporin-4 in subjects with neuromyelitis optica spectrum disorders (17). Although qualitative concordance between CBA and ELISA results is promising, titers and BTUs are not perfectly comparable. Specific studies examining MAG-Abs titers in relation with clinical activity will clarify the suitability of this novel assay for disease monitoring.

In conclusion, the novel immunofluorescence CBA analyzed herein has a good sensitivity/specificity for the detection of MAG-Abs and retains several practical advantages. Larger studies designed with the aim of comparing and monitoring MAG-Abs titers in parallel with clinical activity are mandatory to confirm and expand our data.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethical approval was not required for the studies involving humans because only leftover samples after completion of all diagnostic measures were included and patient data were processed anonymously. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements because only leftover samples after completion of all diagnostic measures were included and patient data were processed anonymously. All samples analyzed are part of Biob-Neu-DNA-2014.

SM: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. PG: Data curation, Investigation, Methodology, Writing – review & editing. DJ: Conceptualization, Supervision, Writing – review & editing. SH: Conceptualization, Methodology, Validation, Writing – review & editing. CF: Methodology, Validation, Writing – review & editing, Conceptualization. SS: Writing – review & editing. EL: Conceptualization, Methodology, Validation, Writing – review & editing. AD: Data curation, Formal analysis, Investigation, Writing – review & editing. SF: Conceptualization, Project administration, Supervision, Writing – review & editing.