IgG-specific cell-based assay detects potentially pathogenic MuSK-Abs in seronegative MG

Abstract

Objective: To increase the detection of MuSK-Abs using a CBA and test their pathogenicity.

Methods: Sera from 69 MuSK-RIA-positive patients with myasthenia gravis (MG) (Definite MuSK-MG), 169 patients negative for MuSK-RIA and AChR-RIA (seronegative MG, SNMG), 35 healthy individuals (healthy controls, HCs), and 16 NMDA receptor-Ab-positive (NMDAR-Ab) disease controls were tested for binding to MuSK on a CBA using different secondary antibodies.

Results: Initially, in addition to 18% of SNMG sera, 11% of HC and 19% of NMDAR-Ab sera showed positive binding to MuSK-transfected cells; this low specificity was due to anti-IgG(H+L) detection of IgM bound nonspecifically to MuSK. Using an IgG Fc gamma-specific secondary antibody, MuSK-Abs were detected by CBA in 68/69 (99%) of Definite MuSK-MG, 0/35 HCs, 0/16 NMDAR-Ab, and 14/169 (8%) of SNMG sera, providing increased sensitivity with high specificity. The RIA-negative, CBA-positive MuSK-IgG sera, but not IgM-MuSK-binding sera, reduced agrin-induced AChR clustering in C2C12 myotubes, qualitatively similar to RIA-positive MuSK-Abs.

Conclusions: An IgG-specific MuSK-CBA can reliably detect IgG MuSK-Abs and increase sensitivity. In the MuSK-CBA, IgG specificity is essential. The positive sera demonstrated pathogenic potential in the in vitro AChR-clustering assay, although less effective than Definite MuSK-MG sera, and the patients had less severe clinical disease. Use of IgG-specific secondary antibodies may improve the results of other antibody tests.

Classification of evidence: This study provides Class III evidence that an IgG-specific MuSK-CBA identifies patients with MG.

Figure 1. CBA with anti-IgG(H+L)

(A) Scatter plots of results from patients and controls. (B) Representative CBA images from SNMG and HC sera. Scale bar = 50 μM. (C) Colocalization of commercial MuSK-AF562 antibody (red) with anti-IgG(H+L) (green) in an SNMG serum. Scale bar = 20 μM. CBA = cell-based assay; HC = healthy control; MG = myasthenia gravis; SNMG = seronegative MG.

Figure 2. Improving the CBA with anti-IgG Fc(γ)

(A) Antibodies in 2 Definite MuSK-MG sera were mainly IgG subclasses, particularly IgG1 and IgG4. By contrast, 12 SNMG sera detected with anti-IgG(H+L) were also detected with anti-IgM secondary antibody but not with anti-IgG subclass secondary antibodies. (B) Representative CBA images of a Definite MuSK-MG serum detected with anti-IgG Fc(γ). Scale bar = 50 μM. (C) End-point titrations in 4 Definite MuSK-MG sera were higher with IgG Fc(γ) vs IgG(H+L). (D) Scatter plots of results from the SNMG patients and 51 disease and healthy controls. CBA = cell-based assay; MG = myasthenia gravis; SNMG = seronegative MG.

Figure 3. Functional effects of MuSK-IgG and IgM on agrin-LRP4-MuSK–clustering pathway

(A) Representative images of myotubes, fluorescent labeled for AChR clusters in the presence of different sera. Definite MuSK-MG and CBA MuSK-IgG–positive sera reduced the number of AChR clusters, but CBA MuSK-IgM sera did not reduce clusters. Scale bar = 50 μM. (B) Mean results from 2 experiments of CBA MuSK-IgG and control sera on AChR clusters. (C) Mean results of 2 experiments with CBA MuSK-IgM–positive and control sera. Values shown are mean + SEM. HC = healthy control; MG = myasthenia gravis.