Sandra Saschenbrecker

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.

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.

Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016

Serological diagnosis of Zika virus (ZIKV) infections is challenging due to high cross-reactivity between flaviviruses. We evaluated the diagnostic performance of a novel anti-ZIKV ELISA based on recombinant ZIKV non-structural protein 1 (NS1). Assay sensitivity was examined using sera from 27 patients with reverse transcription (RT)-PCR-confirmed and 85 with suspected ZIKV infection. Specificity was analysed using sera from 1,015 healthy individuals. Samples from 252 patients with dengue virus (n = 93), West Nile virus (n = 34), Japanese encephalitis virus (n = 25), chikungunya virus (n = 19) or Plasmodium spp. (n = 69) infections and from 12 yellow fever-vaccinated individuals were also examined. In confirmed ZIKV specimens collected ≥ 6 days after symptom onset, ELISA sensitivity was 58.8% (95% confidence interval (CI): 36.0–78.4) for IgM, 88.2% (95% CI: 64.4–98.0) for IgG, and 100% (95% CI: 78.4–100) for IgM/IgG, at 99.8% (95% CI: 99.2–100) specificity. Cross-reactivity with high-level dengue virus antibodies was not detected. Among patients with potentially cross-reactive antibodies anti-ZIKV positive rates were 0.8% (95% CI: 0–3.0) and 0.4% (95% CI: 0–2.4) for IgM and IgG, respectively. Providing high specificity and low cross-reactivity, the NS1-based ELISA has the potential to aid in counselling patients, pregnant women and travellers after returning from ZIKV-endemic areas.

Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica

Objective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (AQP4-Ab) assays in neuromyelitis optica spectrum disorders (NMOSD). Methods Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4), immunohistochemistry (n=3) and ELISA (n=1). Results Results of tests on 92 controls identified 12assays as highly specific (0–1 false-positive results). 32 samples from 50 (64%) NMO sera and 34 from 51 (67%) NMOSD sera were positive on at least two of the 12 highly specific assays, leaving 35 patients with seronegative NMO/spectrum disorder (SD). On the basis of a combination of clinical phenotype and the highly specific assays, 66 AQP4-Ab seropositive samples were used to establish the sensitivities (51.5–100%) of all 21 assays. The specificities (85.8–100%) were based on 92 control samples and 35 seronegative NMO/SD patient samples. Conclusions The cell-based assays were most sensitive and specific overall, but immunohistochemistry or flow cytometry could be equally accurate in specialist centres. Since patients with AQP4-Ab negative NMO/SD require different management, the use of both appropriate control samples and defined seronegative NMOSD samples is essential to evaluate these assays in a clinically meaningful way. The process described here can be applied to the evaluation of other antibody assays in the newly evolving field of autoimmune neurology.

Automated Indirect Immunofluorescence Evaluation of Antinuclear Autoantibodies on HEp-2 Cells

Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability.

Epitope mapping of BP230 leading to a novel enzyme-linked immunosorbent assay for autoantibodies in bullous pemphigoid.

Background  Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by circulating autoantibodies against BP180 and BP230. For BP180, the NC16A domain has previously been identified as the main antigenic target in BP, while data about the diagnostic value of epitopes on BP230 were inconclusive.

Anti-neuronal autoantibodies: Current diagnostic challenges.

The spectrum of neurological autoimmune diseases has expanded substantially in the last 15 years due to the discovery of new anti-neuronal antibodies. There are at present numerous technical challenges for developing and improving standardized serological test systems for the detection of these autoantibodies, some of which occur very rarely. In particular, the determination of autoantibodies against complex cell surface structures generally requires authentically presented target antigens. Finally, research into syndrome associations benefits from multiplex analyses and accelerates the understanding of the complex autoimmune processes, forming an important basis for the development of novel therapy concepts.

Limbic encephalitis with mGluR5 antibodies and immunotherapy-responsive prosopagnosia

In 1982, Dr. Ian Carr described personality changes and memory loss in his 15-year-old daughter, who had limbic encephalitis and Hodgkin lymphoma (HL).1 He assumed “a circulating neurotransmitter-like molecule produced by the neoplasm” causing the brain disease, and noted that it “may be reversible and can be remembered as the Ophelia syndrome.” Almost 30 years later, Lancaster et al.2 identified antibodies against the metabotropic glutamate receptor 5 (mGluR5) as Carrs neurotransmitter-like molecule in 2 patients with limbic encephalitis and HL. Encephalitis was reversible in both patients receiving tumor treatment. The clinical spectrum is largely unknown and the role of immunotherapy is unclear.

Specific immunoadsorption of pathogenic autoantibodies in pemphigus requires the entire ectodomains of desmogleins.

Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are life‐threatening autoimmune blistering skin diseases. They are characterized by circulating autoantibodies which bind to the ectodomains of desmoglein (Dsg) 1 and Dsg3. These antibodies induce acantholysis in skin and mucous membranes. In severe cases of pemphigus, immunoadsorption is applied to remove total IgG from patient plasma using protein A or other ligands. To develop a specific adsorber for anti‐Dsg antibodies, epitope mapping studies of Dsg1 and Dsg3 ectodomains were conducted. Dsg variants were expressed on the surface of HEK‐293 cells and analysed for reactivity with pemphigus and control sera by indirect immunofluorescence technique. For Dsg1, a construct consisting of domain 1 directly fused to domain 5, seemed to be suitable for specific immunoadsorption of anti‐Dsg1 antibodies. The recognized epitopes were mainly conformation‐dependent. However, adsorption of pemphigus foliaceus IgG using this protein coupled to a Sepharose matrix did not completely remove pathogenicity from the sera, as proven by a keratinocyte dissociation assay. In contrast, full‐length Dsg1 and Dsg3 ectodomains were able to specifically adsorb anti‐Dsg antibodies and to efficiently eliminate pathogenicity. Therefore, the complete and correctly folded ectodomains of both desmogleins are required for therapeutic immunoadsorption.

The glycoproteins C and G are equivalent target antigens for the determination of herpes simplex virus type 1-specific antibodies

Seroreactivity to the glycoproteins C and G of herpes simplex virus type 1 (HSV-1) was compared in 310 serum samples using a Western blot assay containing a whole antigen extract of HSV-1 and an ELISA employing gC1 isolated from HSV-1. The prevalence of reactivity to gC1 was 75.8% by Western blot and 73.9% by ELISA, while antibody responses to gG1 were detected in 72.9% of sera by Western blot. An absolute correlation of 96.1% between the reactivity to gC1 and gG1 was demonstrated using the Western blot. The gC1-based ELISA correlated with Western blot detection of anti-gC1 and anti-gG1 antibodies in 95.2 and 97.7% of samples, respectively. 3.2% of all sera were reactive with gC1 in Western blot and/or ELISA, but were negative for anti-gG1. For analysis of cross-reactivity, antibodies against HSV-2, Epstein-Barr virus, varicella-zoster virus and cytomegalovirus were determined. The prevalence of antibodies against each individual virus was identical in the groups of sera reactive with gC1 or gG1. These findings indicate that gC1 and gG1 are equivalent antigenic targets for the type-specific serodiagnosis of HSV-1 infections.