The application of glycosphingolipid arrays to autoantibody detection in neuroimmunological disorders

Serum autoantibodies directed towards a wide range of single glycosphingolipids, especially gangliosides, in humans with autoimmune peripheral neuropathies have been extensively investigated since the 1980s and these are widely measured both in clinical practice and research. It has been recently appreciated that glycosphingolipid and lipid complexes, formed from 2 or more individual components, can interact to create molecular shapes capable of being recognised by autoantibodies that do not bind the individual components. Conversely, 2 glycosphingolipids may interact to form a heteromeric complex that inhibits binding of an antibody known to bind one of the partners. As a result of this, previously undiscovered autoantibodies have been identified, providing substantial new insights into disease pathogenesis and diagnostic testing. In particular, this newly-termed ‘combinatorial glycomic’ approach has provided the impetus to redesigning the assay methodologies traditionally used in the neuropathy-associated autoantibody field. Combinatorial glycoarrays can be readily constructed in house using any lipids and glycosphingolipids of interest, and as a result many new antibody specificities to gangliosides and other glycosphingolipid complexes are being discovered in neuropathy subjects. Herein we also highlight the role of the neutral lipids cholesterol and galactocerebroside in modifying glycosphingolipid orientation as two critical components of the molecular topography of target membranes in nerves that might favour or inhibit autoantibody binding

Anti-GQ1b ganglioside positive Miller Fisher syndrome - evidence of paranodal pathology on nerve biopsy

BACKGROUND: Miller Fisher syndrome is a regional variant of Guillain-Barre syndrome with a characteristic clinical triad of ophthalmoplegia, areflexia and ataxia and occasionally distal limb sensory loss. 90% of patients have associated antibodies to the GQ1b ganglioside. The pathophysiology of antibody-mediated peripheral nerve impairment remains uncertain. This report includes the first description of a peripheral sensory nerve biopsy in Miller Fisher syndrome. RESULTS: A single case report is described of a 46 year old woman who presented with 2 weeks of distal glove and stocking sensory loss to both deep and superficial sensory modalities, areflexia and weight loss. This was followed by rapid onset of ataxia, ophthalmoplegia, and bulbar impairment. Peripheral neurophysiology showed reduced sensory nerve amplitudes with preserved conduction velocities in keeping with an axonal pattern of impairment. Clinical concerns of a systemic inflammatory disorder led to a diagnostic peripheral nerve biopsy from the sensory branch of the radial nerve. However she subsequently made a complete recovery over 5 weeks. Combinatorial glycoarrays confirmed restricted serum binding for GQ1b in acute serum which later resolved in a convalescent sample. The nerve biopsy showed lengthening of nodes of Ranvier, myelin splitting and macrophage internodal axonal invasion without any features of demyelination. CONCLUSIONS: The pathological features were strikingly similar to those found in acute motor axonal neuropathy and indicate the region of the node of Ranvier to be a primary focus of GQ1b induced damage in Miller Fisher syndrome, at least in this particular overlap syndrome with prominent sensory nerve involvement

The diagnostic utility of determining anti-GM1: GalC complex antibodies in multifocal motor neuropathy: a validation study

Background: Multifocal motor neuropathy (MMN) is associated with IgM antibodies to GM1 ganglioside. The importance of the lipid milieu that might facilitate or inhibit antibody binding to GM1 in immunoassays is well recognised. Existing studies, using a range of different approaches, generally concur that anti-GM1 IgM antibody detection rates are improved by the addition of galactocerebroside (GalC) to the GM1 assay. Objective: The current study sought to formally evaluate the clinical utility of the GM1:GalC complex assay in the diagnosis of MMN. Methods: Anti-GM1 and -GM1:GalC antibodies were examined using ELISA and glycoarray (dot blot) in a fully blinded study design, consisting of 100 MMN patients, 100 ALS cases and 100 healthy controls. Results: The detection of anti-GM1 Abs using glycoarray was 67% sensitive and 85% specific. The addition of GalC to GM1, (1:1 weight to weight ratio), increased the sensitivity to 81% , whilst dropping specificity to 80% . Increasing the GalC content to a 1:5 ratio (or higher) further decreased specificity, and in doing so limited the usefulness of the GM1:GalC assay to the level of GM1 alone. The addition of GalC to the ELISA method also significantly increased sensitivity compared with GM1 alone, albeit with a significant decrease in specificity. Conclusions: This study indicates that the GM1:GalC assay is an advantageous assay adaptation for detecting anti-GM1 antibodies in MMN, using either glycoarray or ELISA, and warrants introduction into clinical diagnostic practice

Antibodies to GM1: galactocerebroside complexes in multifocal motor neuropathy: it takes two to tango

No abstract available

Improving the detection of IgM antibodies against glycolipids complexes of GM1 and galactocerebroside in multifocal motor neuropathy using glycoarray and ELISA assays

Antibodies against complexes of GM1:GalC are detected in multifocal motor neuropathy. Previous studies used different techniques, explaining disparities in the results. Antibodies against GM1 and GM1:GalC with different proportions of GalC were measured with both glycoarray and ELISA in 20 multifocal motor neuropathies, and 45 controls. The 1:5 ratio and the 1:1 ratio of GM1:GalC (weight ratio) were respectively the most effective for glycoarray and for ELISA. Testing for anti-GM1:GalC antibodies increased the sensitivity from 40% with anti-GM1 antibodies to 65% with array and 60% with ELISA without loss in specificity (above 91%). Anti-GM1:GalC antibodies are effective biological tools to diagnose multifocal motor neuropathy

Prospective study comparing enzyme-linked immunosorbent assay and glycoarray assay to detect antiglycolipid antibodies in a routine diagnostic neuroimmunology laboratory setting

Objective: Enzyme-linked immunosorbent assay (ELISA) is the conventional technique for antiglycolipid antibody testing in inflammatory neuropathy sera. Miniaturized array-based assays (glycoarrays) have also been used to detect these antibodies. As previous studies have focused on specific disease categories, such as Guillain–Barré syndrome, the array has never been tested on an unselected population in a routine diagnostic laboratory setting. Methods: In the present prospective study, we compared the results of the glycoarray with data obtained with a standardized inflammatory neuropathy cause and treatment-ELISA. A total of 300 sera sent to the Glasgow Neuroimmunology Laboratory for routine antiglycolipid antibody testing during a 6-month period were tested both with ELISA and glycoarray. Results: The two techniques were significantly correlated and showed good agreement. By ELISA, six sera were positive for immunoglobulin G antibodies against GM1 or GD1a, 11 for immunoglobulin G anti-GQ1b antibodies, five for immunoglobulin M anti-GM1 antibodies and three for immunoglobulin M antibodies against disialosyl gangliosides. The glycoarray had a sensitivity of 92% to detect ELISA-positive sera with a specificity above 92% for all the different ELISA patterns. Conclusions: The glycoarray allows testing of large panels of antibodies against single glycolipids and complexes of glycolipids on a very small scale. Its technical characteristics make it suitable as a diagnostic screening test. As data provided by the glycoarray and ELISA were reliably correlated in the present study, the glycoarray can be used in a routine setting to detect antiglycolipid antibodies. Further studies, including more positive samples, are required to clarify the future position of the array in the biological investigation of inflammatory neuropathies