MOG cell-based assay detects non-MS patients with inflammatory neurologic disease

Abstract

Objective: To optimize sensitivity and disease specificity of a myelin oligodendrocyte glycoprotein (MOG) antibody assay.

Methods: Consecutive sera (n = 1,109) sent for aquaporin-4 (AQP4) antibody testing were screened for MOG antibodies (Abs) by cell-based assays using either full-length human MOG (FL-MOG) or the short-length form (SL-MOG). The Abs were initially detected by Alexa Fluor goat anti-human IgG (H + L) and subsequently by Alexa Fluor mouse antibodies to human IgG1.

Results: When tested at 1:20 dilution, 40/1,109 sera were positive for AQP4-Abs, 21 for SL-MOG, and 180 for FL-MOG. Only one of the 40 AQP4-Ab-positive sera was positive for SL-MOG-Abs, but 10 (25%) were positive for FL-MOG-Abs (p = 0.0069). Of equal concern, 48% (42/88) of sera from controls (patients with epilepsy) were positive by FL-MOG assay. However, using an IgG1-specific secondary antibody, only 65/1,109 (5.8%) sera were positive on FL-MOG, and AQP4-Ab- positive and control sera were negative. IgM reactivity accounted for the remaining anti-human IgG (H + L) positivity toward FL-MOG. The clinical diagnoses were obtained in 33 FL-MOG-positive patients, blinded to the antibody data. IgG1-Abs to FL-MOG were associated with optic neuritis (n = 11), AQP4-seronegative neuromyelitis optica spectrum disorder (n = 4), and acute disseminated encephalomyelitis (n = 1). All 7 patients with probable multiple sclerosis (MS) were MOG-IgG1 negative.

Conclusions: The limited disease specificity of FL-MOG-Abs identified using Alexa Fluor goat anti-human IgG (H + L) is due in part to detection of IgM-Abs. Use of the FL-MOG and restricting to IgG1-Abs substantially improves specificity for non-MS demyelinating diseases.

Classification of evidence: This study provides Class II evidence that the presence of serum IgG1- MOG-Abs in AQP4-Ab-negative patients distinguishes non-MS CNS demyelinating disorders from MS (sensitivity 24%, 95% confidence interval [CI] 9%-45%; specificity 100%, 95% CI 88%-100%).

Figure 1. Flow diagram of the assays and the samples that were evaluated

A total of 1,109 samples were initially screened at a serum dilution of 1:20 for antibodies to aquaporin-4 (AQP4), short-length MOG (SL-MOG), and full-length human MOG (FL-MOG). Different secondary antibodies were then evaluated on FL-MOG–positive serum samples by FL-MOG cell-based assay (CBA) or flow cytometry. When the assay was established, 2 patient cohorts with clinical diagnoses from Oxford, UK and Sendai, Japan were used to calculate assay metrics.

Figure 2. Antibodies to MOG detected with anti-human IgG (H + L) as the secondary antibody

(A) Schematic of the human MOG proteins tested. The extracellular and transmembrane domains are identical, but the short-length MOG (SL-MOG) is 73 amino acids shorter at the C-terminus than full-length MOG (FL-MOG). (B) Screening 1,109 consecutive samples sent for aquaporin-4 (AQP4) antibody testing. With anti-human IgG (H + L) as the secondary antibody, 21 SL-MOG– positive samples and 180 FL-MOG–positive samples were identified; however, a cohort of epilepsy sera demonstrates the striking lack of specificity in the FL-MOG assay. Comparing the AQP4 seropositivity in the 2 MOG assays, 1/38 AQP4-positive samples were also positive for SL-MOG antibodies (C), compared with 10/38 for FL-MOG antibodies (D). CBA = cell-based assay.

Figure 3. Antibodies to MOG using different secondary antibodies: Anti-human IgG (H + L), IgG1, or IgM

(A) Comparison of binding to full-length myelin oligodendrocyte glycoprotein (FL-MOG) using anti-human IgG (H + L), anti-IgM, or anti-IgG1 secondary antibodies with 3 different test sera (a-c) and a healthy control serum (con). (B) IgM and (C) IgG1 binding scores for patients and healthy controls (HC). (D.a) PIRES2-DsRed2-FL-MOG transiently transfected HEK cells are separated into cells that express MOG and DsRed2 well (in the upper section of the graph) or poorly or not at all (lowest section of the graph). (D.b) Healthy control sera (upper panels) causes a specific shift in the MOG-transfected cells compared to the untransfected cells when anti-human IgG (H + L) or anti-human IgM secondary antibodies are used (arrows), but not when anti-human IgG1 secondary antibodies are used. The lower panels show higher shifts in sera positive for FL-MOG antibodies compared to controls in the upper panel. (E) Fifteen samples that were IgG (H + L) positive and 5 healthy controls were tested on flow cytometry with anti-IgM or IgG1. A high cutoff is generated with anti-human IgM secondary antibody (ΔMFI of 270) vs a ΔMFI of 2.5 for the anti-human IgG1 antibody. Of note, one IgM-positive patient is IgG1 negative (blue circle). Ab = antibody; AQP4 = aquaporin-4; CBA = cell-based assay; MFI = mean fluorescence intensity; MS = multiple sclerosis.

Figure 4. Confirmatory cohorts to assess MOG-IgG1 assay

(A) All 81 aquaporin-4 (AQP4)- seropositive patients (blue) from the Oxford National neuromyelitis optica (NMO) service were negative for IgG1 antibodies to myelin oligodendrocyte glycoprotein (MOG); however, 23 AQP4-seronegative patients were identified as MOG-IgG1 seropositive (red). Of the 58 patients with NMO, 37 (63.4%) were AQP4 seropositive, 8 (13.8%) were MOG-IgG1 seropositive, and 13 (22%) were double seronegative. (B) A second cohort from Japan was screened blinded to clinical information. None of the 49 AQP4-seropositive patients (blue) or 27 patients with multiple sclerosis (MS) were positive for MOG antibodies, but 6/25 patients with acute disseminated encephalomyelitis (ADEM), transverse myelitis (TM), optic neuritis (ON), or AQP4-seronegative NMO were MOG antibody positive (red). CBA = cell-based assay.