Christian Probst

N-Methyl-d-Aspartate Receptor Antibodies in Herpes Simplex Encephalitis

To determine the presence and kinetics of antibodies against synaptic proteins in patients with herpes simplex virus encephalitis (HSE).

Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen

BACKGROUND Recently, a highly specific serum autoantibody was discovered in patients with neuromyelitis optica, called NMO-IgG, and aquaporin-4, the most abundant water channel in the CNS, was identified as the target antigen. Several assays for the detection of NMO-IgG/AQP4-Ab have been described. Tests based on recombinant human AQP4 have been repeatedly demonstrated to be more sensitive than the previous gold standard assay, i.e. immunohistochemistry (IHC) on mouse brain tissue. However, the sophisticated techniques applied restrict their availability to few laboratories worldwide. OBJECTIVE To develop an easy-to-use, recombinant immunofluorescence assay (rIFA) suitable for standardized and high-throughput detection of NMO-IgG/AQP4-Ab. METHODS HEK293 cells seeded on cover glasses were transfected with full-length recombinant human AQP4 at large scale. Cover glasses with the immobilized cells were cut into millimetre-sized fragments and transferred to microscopy slides. 151 serum samples from patients with NMO spectrum disorders (NMOSD) and controls were analysed both in the standard IHC assay and in the newly developed rIFA. RESULTS 25/32 (78.1%) patients with clinically definite NMO and 36/51 (70.6%) of total patients with NMOSD were positive for NMO-IgG/AQP4-Ab in the rIFA compared to 65.6% and 58.8%, respectively, in the IHC assay. CONCLUSION The recombinant IFA presented here provides laboratories familiar with indirect immunofluorescence microscopy with a highly sensitive and reproducible diagnostic tool for standardized detection of antibodies to AQP4. This new approach could make AQP4-Ab testing, which is of high clinical relevance, more widely available.

Seroprevalence of autoantibodies against brain antigens in health and disease.

We previously reported an unexpectedly high seroprevalence (∼10%) of N‐methyl‐D‐aspartate‐receptor subunit‐NR1 (NMDAR1) autoantibodies (AB) in healthy and neuropsychiatrically ill subjects (N = 2,817). This finding challenges an unambiguous causal relationship of serum AB with brain disease. To test whether similar results would be obtained for other brain antigen‐directed AB previously connected with pathological conditions, we systematically screened serum samples of 4,236 individuals.

Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance

BackgroundIn 70-80% of cases, neuromyelitis optica (NMO) is associated with highly specific serum auto-antibodies to aquaporin-4 (termed AQP4-Ab or NMO-IgG). Recent evidence strongly suggests that AQP4-Ab are directly involved in the immunopathogenesis of NMO.ObjectiveTo assess the frequency, syndrome specificity, diagnostic relevance, and origin of cerebrospinal fluid (CSF) AQP4-Ab in patients with NMO spectrum disorders (NMOSD).Methods87 CSF samples from 37 patients with NMOSD and 42 controls with other neurological diseases were tested for AQP4-Ab in a cell based assay using recombinant human AQP4. Twenty-three paired CSF and serum samples from AQP4-Ab seropositive NMOSD patients were further analysed for intrathecal IgG synthesis to AQP4.ResultsAQP4-Ab were detectable in 68% of CSF samples from AQP4-Ab seropositive patients with NMOSD, but in none of the CSF samples from AQP4-Ab seronegative patients with NMOSD and in none of the control samples. Acute disease relapse within 30 days prior to lumbar puncture, AQP4-Ab serum titres >1:250, and blood-CSF barrier dysfunction, but not treatment status, predicted CSF AQP4-Ab positivity. A positive AQP4-specific antibody index was present in 1/23 samples analysed.ConclusionsAQP4-Ab are detectable in the CSF of most patients with NMOSD, mainly during relapse, and are highly specific for this condition. In the cohort analysed in this study, testing for CSF AQP4-Ab did not improve the sensitivity and specificity of the current diagnostic criteria for NMO. The substantial lack of intrathecal AQP4-Ab synthesis in patients with NMOSD may reflect the unique localisation of the target antigen at the blood brain barrier, and is important for our understanding of the immunopathogenesis of the disease.

Enzyme-linked immunosorbent assay using multimers of the 16th non-collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies.

Abstract:  Bullous pemphigoid (BP) and pemphigoid gestationis (PG) are acquired autoimmune subepidermal blistering diseases characterized by autoantibodies against the hemidesmosomal proteins BP180/type XVII collagen and BP230. In the vast majority of BP and PG patients, these autoantibodies bind to epitopes clustered within the 16th non‐collagenous domain of BP180. An ELISA system for the detection of these autoantibodies was developed and evaluated using 16th non‐collagenous domain (NC16A) tetramers instead of monomers. In contrast to antigens fused to large proteins used in the past for the detection of autoantibodies against type XVII collagen, tetrameric antigen fragments bearing a small hexahistidine tag allow for high expression levels without the need to cleave off the fusion partner. Using tetrameric BP180 NC16A, positive reactions were found in 106 (89.8%) of 118 randomly selected BP sera and in all of 20 (100%) randomly selected PG sera, whereas only 2.2% of a large cohort of control subjects were positive in this assay, including patients with rheumatoid arthritis (two of 107), progressive systemic sclerosis (two of 50), systemic lupus erythematosus (one of 72), and healthy blood donors (10 of 494). Thus, the sensitivity and specificity of the new anti‐tetrameric NC16A ELISA were 89.9% and 97.8% respectively. Levels of circulating autoantibodies against BP180 paralleled disease activity in the pemphigoid patients. In conclusion, the use of tetrameric NC16A in ELISA results in a sensitive and specific tool for diagnosis and monitoring of BP and PG.

DPPX potassium channel antibody Frequency, clinical accompaniments, and outcomes in 20 patients

Objective: To describe the detection frequency and clinical associations of immunoglobulin G (IgG) targeting dipeptidyl-peptidase-like protein-6 (DPPX), a regulatory subunit of neuronal Kv4.2 potassium channels. Methods: Specimens from 20 patients evaluated on a service basis by tissue-based immunofluorescence yielded a synaptic immunostaining pattern consistent with DPPX-IgG (serum, 20; CSF, all 7 available). Transfected HEK293 cell-based assay confirmed DPPX specificity in all specimens. Sixty-nine patients with stiff-person syndrome and related disorders were also evaluated by DPPX-IgG cell-based assay. Results: Of 20 seropositive patients, 12 were men; median symptom onset age was 53 years (range, 13–75). Symptom onset was insidious in 15 and subacute in 5. Twelve patients reported prodromal weight loss. Neurologic disorders were multifocal. All had one or more brain or brainstem manifestations: amnesia (16), delirium (8), psychosis (4), depression (4), seizures (2), and brainstem disorders (15; eye movement disturbances [8], ataxia [7], dysphagia [6], dysarthria [4], respiratory failure [3]). Nine patients reported sleep disturbance. Manifestations of central hyperexcitability included myoclonus (8), exaggerated startle (6), diffuse rigidity (6), and hyperreflexia (6). Dysautonomia involved the gastrointestinal tract (9; diarrhea [6], gastroparesis, and constipation [3]), bladder (7), cardiac conduction system (3), and thermoregulation (1). Two patients had B-cell neoplasms: gastrointestinal lymphoma (1), and chronic lymphocytic leukemia (1). Substantial neurologic improvements followed immunotherapy in 7 of 11 patients with available treatment data. DPPX-IgG was not detected in any of the stiff-person syndrome patients. Conclusions: DPPX-IgG is a biomarker for an immunotherapy-responsive multifocal neurologic disorder of the central and autonomic nervous systems.

Functional and structural brain changes in anti-N-methyl-D-aspartate receptor encephalitis.

Anti–N‐methyl‐D‐aspartate receptor (NMDAR) encephalitis is an autoimmune encephalitis with a characteristic neuropsychiatric syndrome and severe and prolonged clinical courses. In contrast, standard clinical magnetic resonance imaging (MRI) remains normal in the majority of patients. Here, we investigated structural and functional brain changes in a cohort of patients with anti‐NMDAR encephalitis.

Autoantibodies against aquaporin-4 in patients with neuropsychiatric systemic lupus erythematosus and primary Sjögren's syndrome.

Neurologic manifestations occur in up to 70% of patients with systemic lupus erythematosus (SLE) and in 20% of patients with Sjögren’s syndrome (SS) and are associated with significant morbidity. There is as yet no biologic marker that specifically indicates neurologic involvement in rheumatic diseases. In the literature to date, the association between nervous system manifestations of SLE and autoantibodies against ribosomal P proteins and N-methyl-Daspartate receptor has been inconsistently demonstrated (1). Transverse myelopathy is a rare but serious condition reported in patients with SLE and SS (2,3). In sera from some of these patients, an IgG autoantibody marker, called NMOIgG, has been recently detected (4,5). NMO-IgG was initially identified by immunohistochemistry using brain tissue in sera from patients with neuromyelitis optica (NMO; also known as Devic’s disease), a severe demyelinating disorder of the central nervous system (CNS) that primarily affects the spinal cord and the optic nerves (6). In patients presenting with isolated longitudinally extensive transverse myelitis (LETM) involving at least 3 vertebral segments, as revealed using magnetic resonance imaging, and in patients with recurrent optic neuritis (ON), the presence of NMO-IgG indicates severe disease course with frequent relapses (7,8). NMO-IgG is not detectable in the sera of patients with multiple sclerosis (MS) (6). The antigenic target of NMO-IgG is aquaporin-4 (AQP-4), the most abundant water channel in the CNS (9). To detect these autoantibodies, assays employing recombinant AQP-4 have been repeatedly shown to be more sensitive than immunohistochemistry in CNS tissue (10). In order to evaluate the significance of NMO-IgG/ AQP-4 antibodies as potential markers of neurologic involvement in rheumatic conditions, we collected serum samples from patients with SLE (n 48) and SS (n 44), 28 and 22 of whom showed neurologic manifestations, respectively (Table 1). All patients fulfilled the American College of Rheumatology (ACR) classification criteria for SLE and SS (11,12). SLE patients with neurologic involvement fulfilled the ACR case definitions for neuropsychiatric lupus syndromes (13). Blood was drawn from 3 SLE patients (2 with LETM and 1 with LETM and recurrent ON) within 7 days of onset of a relapse. In the other SLE subsets, the time intervals between the serum sampling and the onset of the first neurologic symptoms or, in cases of relapses, the onset of the last relapse were as follows: TM, 1 year; LETM, 1–7 years (median 4 years); recurrent ON, 1 year; chorea, 1 year; psychosis/depression, 4–20 years (median 9.5 years); seizure disorders, 13 and 20 years. The duration of symptoms in SLE patients with polyneuropathy was 2–12 years (median 5 years). In patients with SS, time intervals were 7 and 8 years in 2 patients with TM and concomitant monophasic ON, 5 years in 1 patient with TM alone, and 13 years in both patients with LETM. Symptoms were present for 1–8 years (median 3 years) in SS patients with polyneuropathy. Samples were analyzed both by immunohistochemistry using native primate cerebellum, cerebrum, and optic nerve tissue sections and, for the first time in patients with SLE and SS, by means of a recombinant immunofluorescence assay (rIFA) using AQP-4–transfected HEK cells fixed in formalin. In direct comparison with the original procedure for the detection of NMO-IgG as described by Lennon et al (6), the rIFA exhibited an increase in sensitivity of 12.5% (overall sensitivity 78.1%; specificity 100%) in an earlier study based on 183 samples from NMO patients and relevant neurologic disease controls, including MS (14). Serum samples were classified as being positive or negative for NMO-IgG/AQP-4 antibodies by 2 independent investigators who were unaware of the clinical data. Testing was performed for diagnostic purposes in all cases. In SLE and SS, NMO-IgG/AQP-4 antibodies were found exclusively in patients with neurologic involvement comprising LETM or recurrent ON (Table 1). In the SLE cohort, autoantibodies against AQP-4 were detected in all of these patients, whereas NMO-IgG was found in 6 of 7 individuals, once more indicating a higher sensitivity of the recombinant cell substrate compared with immunohistochemistry. Antibody titers as determined by AQP-4–transfected HEK cells ranged from 1:10–1:1,000 in the 5 patients with LETM. The titer was 1:100 in the patient with recurrent ON and 1:3,200 in the patient with LETM and recurrent ON. In the SS group, 1 of the 2 patients with LETM was positive for NMO-IgG/AQP-4 antibodies, at a titer of 1:1,000. None of the SLE and SS patients without neurologic involvement or symptoms other than LETM or recurrent ON tested positive for NMO-IgG/AQP-4 antibodies (i.e., at a starting dilution of 1:10, antibodies were not detected in the serum samples from these patients). Our data demonstrate that autoantibodies against AQP-4 generally are not a marker for neurologic involvement in SLE and SS. Consistent with the findings of previous reports, our observations strongly support the notion that the presence of autoantibodies against AQP-4 is highly correlated with a distinct clinical phenotype, i.e., LETM and recurrent ON, which together are typical for NMO (6–8). Apparently, autoantibodies against AQP-4 are a marker for NMO that may occur either as an isolated syndrome or as part of a rheumatologic disease like SLE or SS. Of particular interest is the fact that 2 of the AQP-4–positive SLE patients (1 with LETM and 1 with recurrent ON) also had myasthenia gravis. This observation further supports the concept of coexisting independent, antibody-mediated autoimmune disorders in the same patient. It can be argued that the long intervals between the onset of neurologic symptoms and the time of blood sampling in most of the patients might have influenced the serologic findings. However, it is known from NMO patients with long-term followup that AQP-4 antibodies are detectable in the serum during relapse as well as during remission, suggesting that AQP-4 antibody testing can be of diagnostic relevance independently of disease activity (15). In clinical practice, myelitis and ON occurring in patients with SLE and SS can be

Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients

Please cite this paper as: Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients. Experimental Dermatology 2010; 19: 458–463.

New ELISA for Detecting Primary Biliary Cirrhosis–Specific Antimitochondrial Antibodies

BACKGROUND Antimitochondrial antibodies specific for primary biliary cirrhosis (PBC) target the E2 subunits of 2-oxo acid dehydrogenase complexes, in particular the pyruvate dehydrogenase complex (PDC)-E2. Their antigen-specific detection relies on conventional ELISA using purified PDC. More recent assays have employed a hybrid containing the 3 E2-subunits (MIT3). Some PBC sera react with one or the other preparation, suggesting the presence of nonoverlapping epitopes. METHODS We have developed an ELISA (anti-M2-3E) using a mixture of purified PDC and MIT3 as antigenic targets. We compared this assay to anti-MIT3 alone, conventional anti-PDC, and indirect immunofluorescence using 173 PBC and 247 disease controls. RESULTS The anti-M2-3E ELISA showed a 93.6% diagnostic sensitivity compared with 91.3%, 83.8%, and 87.3% for MIT3, purified PDC, or indirect immunofluorescence, respectively, when all specificities are set to 98.8%. By immunoblotting, anti-M2-3E-positive sera unreactive to purified PDC recognized recombinant E2-subunits of the other 2 complexes, whereas those with no reactivity to MIT3 immunofixed PDC subunits E1alpha or E1beta. CONCLUSIONS The diagnostic accuracy of the anti-M2-3E ELISA for detection of antibodies to 2-oxo acid dehydrogenase complexes exceeds that of conventional ELISA and IFL; its novelty derives from the combination of the MIT3 hybrid and purified PDC.