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Introduction Autoantibodies against the M-type phospholipase A2 receptor (PLA2R) are important markers in the diagnosis and monitoring of primary membranous nephropathy (pMN). For the detection of anti-PLA2R autoantibodies, a standardized recombinant cell-based indirect immunofluorescence assay (RC-IFA) and enzyme-linked immunosorbent assay (ELISA) are widely used, the former providing higher sensitivity but lacking a finely graduated quantification of antibody titers. In this study, we evaluated the diagnostic performance characteristics of a novel standardized chemiluminescence immunoassay (ChLIA) by comparison with the established anti-PLA2R test systems. Methods Sera from 155 patients with biopsy-proven pMN and 154 disease controls were analyzed for autoantibodies against PLA2R by the novel ChLIA as well as by ELISA and RC-IFA. Results The clinical sensitivity of the ChLIA (83.9%) was higher compared with ELISA (73.5%) and equaled that of RC-IFA (83.2%), at similar specificities (≥99.4%). Among ELISA-negative pMN samples, ChLIA and RC-IFA yielded positive results in 39.0% and 36.6%, respectively. The qualitative agreement amounted to 94.5% (ChLIA vs. ELISA) and 99.4% (ChLIA vs. RC-IFA). Conclusion The novel anti-PLA2R ChLIA outperforms the ELISA in detecting patients with pMN and demonstrates almost perfect agreement with RC-IFA. It thus presents a promising alternative tool for accurate anti-PLA2R testing, with the advantage of rapid turnaround times and fully automated random-access processing., Graphical abstract

In the current COVID‐19 pandemic, a better understanding of the relationship between merely binding and functionally neutralizing antibodies is necessary to characterize protective antiviral immunity following infection or vaccination. This study analyzes the level of correlation between the novel quantitative EUROIMMUN Anti‐SARS‐CoV‐2 QuantiVac ELISA (IgG) and a microneutralization assay. A panel of 123 plasma samples from a COVID‐19 outbreak study population, preselected by semiquantitative anti‐SARS‐CoV‐2 IgG testing, was used to assess the relationship between the novel quantitative ELISA (IgG) and a microneutralization assay. Binding IgG targeting the S1 antigen was detected in 106 (86.2%) samples using the QuantiVac ELISA, while 89 (72.4%) samples showed neutralizing antibody activity. Spearman's correlation analysis demonstrated a strong positive relationship between anti‐S1 IgG levels and neutralizing antibody titers (r s = 0.819, p, Highlights A new quantitative anti‐S1 assay (EUROIMMUN Anti‐SARS‐CoV‐2 QuantiVac ELISA IgG) was compared to a microneutralization assay.The QuantiVac ELISA showed a high level of qualitative overall agreement and a strong quantitative correlation with the results of neutralization testing.The QuantiVac ELISA has the potential to predict protective immunity against SARS‐CoV‐2 folllowing infection or vaccination.

In flavivirus-endemic areas, serological testing of Zika virus (ZIKV) infections is complicated by (i) multiform reaction kinetics of ZIKV-specific antibodies, for example, flavivirus-primed secondary infections often present with low or undetectable titers of anti-ZIKV IgM and with early-positive anti-ZIKV IgG, and (ii) by previous flavivirus contact causing elevated cross-reactivities. Specific IgA antibodies can be a useful additional marker to provide laboratory evidence of acute ZIKV infection. A recent study about anti-ZIKV nonstructural protein 1 IgA, IgG, and IgM responses at different stages of acute infection in patients living in flavivirus-endemic countries showed that a combined detection of specific IgAM using enzyme-linked immunosorbent assay yielded a sensitivity of 100% and a specificity of > 95%. When the virus can no longer be detected by polymerase chain reaction, the detection of anti-ZIKV nonstructural protein 1 IgA in addition to IgM strongly supports accurate diagnosis of acute ZIKV infection, especially when IgM is absent.

Autoimmune bullous dermatoses (AIBD) encompass a variety of organ-specific autoimmune diseases that manifest with cutaneous and/or mucosal blisters and erosions. They are characterized by autoantibodies targeting structural proteins of the skin, which are responsible for the intercellular contact between epidermal keratinocytes and for adhesion of the basal keratinocytes to the dermis. The autoantibodies disrupt the adhesive functions, leading to splitting and blister formation. In pemphigus diseases, blisters form intraepidermally, whereas in all other disease types they occur subepidermally. Early identification of autoimmune bullous dermatoses is crucial for both treatment and prognosis, particularly as regards tumor-associated disease entities. The diagnosis is based on clinical symptoms, histopathology, direct immunofluorescence to detect antibody/complement deposits, and the determination of circulating autoantibodies. The identification of various target antigens has paved the way for the recent development of numerous specific autoantibody tests. In particular, optimized designer antigens and multiplex test formats for indirect immunofluorescence and ELISA have enhanced and refined the laboratory analysis, enabling highly efficient serodiagnosis and follow-up. This review elaborates on the current standards in the serological diagnostics for autoimmune bullous dermatoses.

Climate change, increased urbanization and international travel have facilitated the spread of mosquito vectors and the viral species they carry. Zika virus (ZIKV) is currently spreading in the Americas, while dengue virus (DENV) and chikungunya virus (CHIKV) have already become firmly established in most tropical and also many non-tropical regions. ZIKV, DENV and CHIKV overlap in their endemic areas and cause similar clinical symptoms, especially in the initial stages of infection. Infections with each of these viruses can lead to severe complications, and co-infections have been reported. Therefore, laboratory analyses play an important role in differential diagnostics. A timely and accurate diagnosis is crucial for patient management, prevention of unnecessary therapies, rapid adoption of vector control measures, and collection of epidemiological data.There are two pillars to diagnosis: direct pathogen detection and the determination of specific antibodies. Serological tests provide a longer diagnostic window than direct methods, and are suitable for diagnosing acute and past infections, for disease surveillance and for vaccination monitoring. ELISA and indirect immunofluorescence test (IIFT) systems based on optimized antigens enable sensitive and specific detection of antibodies against ZIKV, DENV and CHIKV in patient serum or plasma. In recent years, Euroimmun (Lübeck, Germany) has developed numerous test systems for the serological diagnosis of (re-)emerging diseases, including a very sensitive and specific anti-ZIKV ELISA.

Antinuclear autoantibodies (ANA) displaying a dense fine speckled pattern (DFS, ICAP AC-2) on HEp-2 cells are frequently observed in clinical laboratory referrals, often associated with anti-DFS70 specificity. Anti-DFS70 positive patients rarely develop systemic autoimmune rheumatic disease (SARD), especially in the absence of clinical evidence or additional anti-extractable nuclear antigen (ENA) antibodies, prompting suggestions that an isolated DFS70-specific ENA may be an exclusionary finding for SARD. In this study, the frequency and diagnostic significance of anti-DFS70 autoantibodies was investigated in a community hospital cohort of patients undergoing routine ANA testing. ANA screening was performed by HEp-20-10-based indirect immunofluorescence, followed by ENA profiling using a multiparametric line immunoassay (LIA). Of 6,511 patient samples tested for ANA in 2016, the DFS pattern was identified in 1,758 (27.0%), 720 (41.0%) of which were anti-DFS70 positive by LIA. Of these, 526 (73.1%) revealed isolated anti-DFS70 reactivity, while 194 (26.9%) showed additional ENA specificities. Among 1,038 anti-DFS70 negative or borderline samples, 778 (75.0%) were ENA profile negative, while the remaining 260 (25.0%) showed a varied presence of other ENA specificities. Chart reviews of patients with an isolated anti-DFS70 ANA affirmed that ANA-related SARD is rare in the absence of clinical evidence or other ENA specificities, there being no case thus far identified. Rheumatoid arthritis patients occasionally had an isolated anti-DFS70 ANA and were positive for rheumatoid factor and anti-cyclic citrullinated peptide antibodies. In conclusion, the recognition of a DFS ANA pattern using a mitotic-rich HEp-2 substrate, followed by confirmation of anti-DFS70 specificity should be a routine ANA testing service. Use of an expanded ENA profile and clinical correlation is necessary to affirm the “isolation” of anti-DFS70 as the cause of an ANA. Recognition of isolated anti-DFS70 ANA enables reassurance of patients that SARD is unlikely, thus avoiding referral for more extensive testing. The presence of significant elevations of other ENAs may reflect SARD and warrants close clinical correlation and follow-up.

Climate change, increased urbanization and international travel have facilitated the spread of mosquito vectors and the viral species they carry. Zika virus (ZIKV) is currently spreading in the Americas, while dengue virus (DENV) and chikungunya virus (CHIKV) have already become firmly established in most tropical and also many non-tropical regions. ZIKV, DENV and CHIKV overlap in their endemic areas and cause similar clinical symptoms, especially in the initial stages of infection. Infections with each of these viruses can lead to severe complications, and co-infections have been reported. Therefore, laboratory analyses play an important role in differential diagnostics. A timely and accurate diagnosis is crucial for patient management, prevention of unnecessary therapies, rapid adoption of vector control measures, and collection of epidemiological data.

Objective 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. Methods Serum samples of healthy (n = 1,703) versus neuropsychiatrically ill subjects (schizophrenia, affective disorders, stroke, Parkinson disease, amyotrophic lateral sclerosis, personality disorder; total n = 2,533) were tested. For analysis based on indirect immunofluorescence, we used biochip mosaics of frozen brain sections (rat, monkey) and transfected HEK293 cells expressing respective recombinant target antigens. Results Seroprevalence of all screened AB was comparable in healthy and ill individuals. None of them, however, reached the abundance of NMDAR1 AB (again ∼10%; immunoglobulin [Ig] G ∼1%). Appreciable frequency was noted for AB against amphiphysin (2.0%), ARHGAP26 (1.3%), CASPR2 (0.9%), MOG (0.8%), GAD65 (0.5%), Ma2 (0.5%), Yo (0.4%), and Ma1 (0.4%), with titers and Ig class distribution similar among groups. All other AB were found in ≤0.1% of individuals (anti–AMPAR-1/2, AQP4, CV2, Tr/DNER, DPPX-IF1, GABAR-B1/B2, GAD67, GLRA1b, GRM1, GRM5, Hu, LGl1, recoverin, Ri, ZIC4). The predominant Ig class depended on antigen location, with intracellular epitopes predisposing to IgG (chi-square = 218.91, p = 2.8 × 10−48). Interpretation To conclude, the brain antigen-directed AB tested here are comparably detectable in healthy subjects and the disease groups studied here, thus questioning an upfront pathological role of these serum AB. Ann Neurol 2014;76:82–94

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.

Modern diagnostics for the determination of neurologically relevant autoantibodies are based on indirect immunofluorescence using tissue sections of the hippocampus, cerebellum and other tissues. For monospecific detection human embryonic kidney (HEK) cells transfected with different neurological antigens are used. Biochip mosaics are designed to give a quick overview and contain 20 or more substances positioned next to each other on a reaction field, which are incubated with the serum or cerebrospinal fluid (CSF) sample. Western blots based on cerebellum or hippocampus extracts or line blots containing defined recombinant antigens are used additionally. Initial investigations should always comprise the parallel analysis of all major antineural autoantibodies instead of performing only single parameter tests. Up until a few years ago autoantibodies against intracellular neuronal antigens were mainly investigated. Antibodies against structures of the neural cell surface, however, are much more frequently found, especially those against glutamate receptors (type NMDA).

Background This study aimed to evaluate autoantibodies against the native ribosomal P complex (anti-Rib-PC) and recombinant ribosomal P proteins (anti-Rib-P0, anti-Rib-P1, anti-Rib-P2) for their prevalence, diagnostic relevance and clinical associations in a Chinese cohort with systemic lupus erythematosus (SLE). Methods Anti-Rib-P, anti-dsDNA and anti-Smith antigen (Sm) antibodies were analyzed in sera from 198 patients with SLE, 33 with rheumatoid arthritis, 61 with Sjogren's syndrome and 70 healthy individuals by means of ELISA. Results Antibody prevalences were 29.8% (anti-Rib-PC), 33.3% (anti-Rib-P0), 42.9% (anti-Rib-P1) and 34.3% (anti-Rib-P2), at a specificity of 99%. Among SLE patients lacking anti-dsDNA and anti-Sm, 27.8% showed positive for at least one of the investigated anti-Rib-P types. The serological hit rate provided by anti-dsDNA/anti-Sm detection (72.7%) was increased upon parallel testing for anti-Rib-PC (77.3%) or anti-Rib-P0/P1/P2 (80.3%). Anti-Rib-P positivity was associated with disease activity, neuropsychiatric events, lupus nephritis, skin rash, lymphocytopenia, increased erythrocyte sedimentation rates, decreased complement C3/C4 and elevated IgA/IgG levels. Conclusion Based on these results, antibodies against ribosomal P proteins are important complementary parameters to anti-dsDNA and anti-Sm, and should be considered for inclusion in the classification criteria for SLE. The diagnostic value of anti-Rib-P0/P1/P2 is diagnostically superior to that of anti-Rib-PC. J. Clin. Lab. Anal. 27:87–95, 2013. © 2013 Wiley Periodicals, Inc.

Altres ajuts: This study was funded by the Eugene Devic European Network (EDEN) project (ERA-Net ERARE 2: http://www.erare.eu/financed-projects/eden). Euroimmun AG provided assay kits for groups from Denmark and Hungary at 50% discount, and at no cost to both Italian groups. The work in Oxford was supported by the National Health Service National Specialised Commissioning Group for Neuromyelitis Optica and the National Institute for Health Research Oxford Biomedical Research Centre. Competing interests: AS is funded by Fundació la Marató de TV3 (101610). FT is enrolled in the doctoral programme SPIN funded by the Austrian Science Fund (FWF; project W1206). ZI is funded by Lundbeckfonden and Scleroseforeningen of Denmark. AB has received a grant from Bayer Healthcare on NMO. LZ would like to thank RH for her excellent technical assistance with the assay. RH was supported by a Schrödinger fellowship funded by the Austrian Science Fund (FWF; project J3230). CC acknowledges funding from Fundació la Marató de TV3 (493/C/2012). FP acknowledges funding from the Bundesministerium für Bildung und Forschung (Competence Network Multiple Sclerosis KKNMS). BW acknowledges funding from the Dietmar-Hopp-Stiftung and from Merck Serono. RM and AR acknowledge Nathalie Dufay from NeuroBioTec-Banques (Hospices Civils de Lyon, France) and funding from Association pour la Recherche sur la Sclerose en Plaques (ARSEP). PW has received speaker honoraria from Biogen Idec and Euroimmun AG; holds a patent with Oxford University for LGI1/CASPR2 antibodies, licensed to Euroimmun AG; and for GABAAR antibodies. MR reports other from University Hospital of Innsbruck (TILAK), during the conduct of the study; personal fees from Euroimmun, outside the submitted work. AS has received compensation for consulting services and speaking from Bayer-Schering, Merck-Serono, Biogen-Idec, Sanofi-Aventis, Teva Pharmaceutical Industries Ltd and Novartis. HHN has received travel funding and speaker honoraria from Novartis Healthcare, Biogen Idec, Genzyme Denmark and Teva Denmark. ZI reports grants from Lundbeckfonden, Denmark; Scleroseforeningen, Denmark, during the conduct of the study; personal fees from compensation for consulting services and speaking from Bayer-Schering, Biogen-Idec, Merck-Serono, Novartis, Sanofi-Aventis, and Teva Pharmaceutical Industries Ltd, outside the submitted work. TB reports non-financial support from Euroimmun AG, during the conduct of the study. KR is shareholder and an employee of Euroimmun AG. AB reports grants from Bayer Heathcare, personal fees from Biogen Idec, personal fees from Merck Serono, personal fees from Teva, personal fees from Novartis, personal fees from Genzyme, other from Biogen Idec, other from Novartis, other from Genzyme, other from Roche, other from Alexion Pharmaceuticals, outside the submitted work. LG received congress and travel compensations from Euroimmun. AB received honoraria for serving in the scientific advisory boards of Almirall, Bayer, Biogen Idec, Genzyme, and received speaker honoraria from Biogen Idec, Genzyme, Novartis, TEVA with approval by the Director of AOU San Luigi University Hospital; his institution has received grant support from Bayer, BiogenIdec, Merck, Novartis, TEVA from the Italian Multiple Sclerosis Society, Associazione Ricerca Biomedica ONLUS and San Luigi ONLUS. LZ reports personal fees from Teva Pharmaceutical, personal fees from Novartis, outside the submitted work. MC has received compensation for consulting services and speaking honoraria from Bayer Schering Pharma, Merk Serono, Biogen-Idec, Teva Pharmaceuticals, Sanofi-Aventis, Genzyme, and Novartis. XM has received speaking honoraria and travel expenses for scientific meetings, has been a steering committee member of clinical trials or participated in advisory boards of clinical trials in the past years with Bayer Schering Pharma, Biogen Idec, EMD Merck Serono, Genentech, Genzyme, Novartis, Sanofi-Aventis, Teva Phramaceuticals, Almirall and Roche. MT has received compensation for consulting services and speaking from Bayer-Schering, Merk-Serono, Biogen-Idec, Teva, Sanofi-Aventis, and Novartis. AS reports receiving grants from the Scientific and Technological Research Council of Turkey-Health Sciences Research (grants numbers 109S070 and 112S052); and also unrestricted grants from Bayer-Schering AG and Merck-Serono to their Neurology Department Clinical Neuroimmunology Unit. AS also reports receiving honoraria or consultation fees Biogen Idec, Novartis and Teva. He had received travel and registration coverage for attending several national or international congresses or symposia, from Merck Serono, Biogen Idec, Novartis, Teva, Bayer-Schering and Allergan. AA received grants to her department from The Scientific and Technological Research Institute of Turkey-Health Sciences (grants numbers 109S070 and 112S052), and she also received honoraria, research and travel grants from Teva, Merck-Serono, Gen Ilac, Bayer-Schering, Novartis. FP has received travel funding, speaker honoraria and personal compensation for activities on advisory boards and steering committees from Bayer, Biogen Idec, Alexion, Chugai, MedImmune, MerckSerono, Novartis, Teva, and Sanofi Genzyme. He has received grants from the Guthy Jackson Charitable Foundation and the National Multiple Sclerosis Society of the USA. OA reports grants from German Ministry for Education and Research (BMBF), during the conduct of the study; German Research Foundation (DFG); Hertie Foundation; Biogen; Novartis; Bayer; outside the submitted work; and Honoraria for consultancy and speaking by Biogen, Novartis, Bayer Schering, Teva, Chugai, Genzyme, MedImmune, and Merck Serono. BW reports personal fees from Bayer Healthcare, grants and personal fees from Biogen, Merck Serono; Genzyme, a Sanofi Company; Novartis Pharmaceuticals; TEVA Pharma; grants from Biotest, German Ministry of Education and Research; Dietmar Hopp Foundation; outside the submitted work. H-PH reports personal fees from MedImmune, outside the submitted work. MP is an employee of and holds stock in Euroimmun AG. PT reports non-financial support from Roche Pharmaceuticals, outside the submitted work. SS is an employee of Euroimmun AG. AV is on the Advisory Board for Neurology; was an associate editor for Brain; holds patents with Oxford University for MuSK, LGI1/CASPR2, and GABAAR antibodies, and receives a proportion of royalties. RM reports personal fees from MedImmune, personal fees and non-financial support from Biogen, non-financial support from Merck-Serono, non-financial support from Teva, non-financial support from Novartis, non-financial support from Sanofi, outside the submitted work. 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). 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 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. 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.

Purpose Sporadic inclusion body myositis (sIBM) is an autoimmune degenerative disease of the muscle, with inflammatory infiltrates and inclusion vacuoles. Its pathogenesis is not fully understood and the diagnosis is hampered by its imprecise characteristics, at times indistinguishable from other idiopathic inflammatory myopathies such as polymyositis and dermatomyositis. The diagnosis may be assisted by the detection of autoantibodies targeting Mup44, a skeletal muscle antigen identified as cytosolic 5′-nucleotidase 1A (cN-1A, NT5C1A). A novel standardized anti-cN-1A IgG ELISA was developed and its diagnostic performance was evaluated by two reference laboratories. Methods Recombinant human full-length cN-1A was expressed and purified, and subsequently utilized to set up a standardized ELISA. To evaluate the novel assay, laboratory A examined sera from North American patients with clinically and pathologically diagnosed definite sIBM (n = 17), suspected sIBM (n = 14), myositis controls (n = 110), non-myositis autoimmune controls (n = 93) and healthy subjects (n = 52). Laboratory B analyzed a Dutch cohort of definite sIBM patients (n = 51) and healthy controls (n = 202). Results Anti-cN-1A reactivity was most frequent in definite sIBM (39.2–47.1%), but absent in biopsy-proven classic polymyositis or dermatomyositis. Overall diagnostic sensitivity and specificity amounted to 35.5 and 96.1% (laboratory A) and 39.2 and 96.5% (laboratory B). Conclusions Anti-cN-1A autoantibodies were detected by ELISA with moderate sensitivity, but high specificity for sIBM and may therefore help diagnose this infrequent and difficult-to-diagnose myopathy. The novel anti-cN-1A IgG ELISA can improve and accelerate the diagnosis of sIBM using sera where muscle biopsy is delayed or unfeasible.

Summary 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. Objectives To identify the most appropriate epitopes on BP230 to be applied in a simple, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for routine detection of serum autoantibodies. Methods Ten overlapping linear fragments covering the whole length of BP230 were expressed in Escherichia coli. Based on Western blot analysis with sera from patients with BP (n = 49) and healthy controls (n = 94), the diagnostic performance of the fragments was compared by receiver operating characteristics curve analysis. The BP230-C3 fragment comprising the C-terminal portion (amino acids 2326–2649) was subsequently applied in a novel ELISA. The operating characteristics of this ELISA were analysed by probing sera from patients with BP (n = 118), pemphigus vulgaris (n = 50), rheumatoid arthritis and other inflammatory arthritides (n = 170), and systemic lupus erythematosus (n = 56), and from healthy blood donors (n = 483). Results Among all the fragments, BP230-C3 provided the best efficiency in serologically diagnosing BP by Western blot. An ELISA employing BP230-C3 revealed a diagnostic sensitivity of 56·8% and specificity of 97·6%. Its diagnostic added value amounted to 4·2% compared with the anti-BP180-NC16A-4X ELISA alone. Conclusions Recombinant BP230-C3 is a suitable target antigen for the detection of serum autoantibodies against BP230.

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.

Zusammenfassung In jüngster Zeit wurden mehrere Autoantikörper neu identifiziert, die eng mit verschiedenen Formen der Autoimmun-Enzephalitis assoziiert sind. Die Antikörper richten sich gegen Strukturen der Zelloberfläche von Neuronen: Glutamat-Rezeptoren (Typen NMDA und AMPA), GABAB-Rezeptoren sowie die Kaliumkanal(VGKC)-assoziierten Proteine LGI1 und CASPR2. Sie sind doppelt so häufig wie die klassischen paraneoplastischen Antikörper (Anti-Hu, -Yo, -Ri, -Ma, -CV2, -Amphiphysin), weniger eng mit einem Tumor assoziiert und die korrespondierenden klinischen Syndrome sprechen deutlich besser auf eine Immuntherapie an. Der monospezifische Nachweis dieser Autoantikörper in Serum oder Liquor der Patienten erfolgt vorwiegend durch indirekte Immunfluoreszenz, unter Verwendung transfizierter HEK293-Zellen, welche die Membran-assoziierten Zielantigene rekombinant exprimieren. Aufgrund symptomatischer Überlappungen der verschiedenen Krankheitsentitäten ist es sinnvoll, alle diese Parameter bei jedem Patienten parallel zu bestimmen (Autoantikörper-Profile). Eine rasche Diagnosestellung (maßgeblich unterstützt durch das serologische Labor), der unmittelbare Beginn immuntherapeutischer Intervention und, im Falle einer paraneoplastischen Ätiologie, die Entfernung des Tumors entscheiden die Prognose. Nach eigenen Untersuchungen findet man bei den neu identifizierten Autoimmun-Enzephalitis-Formen Antikörper gegen Glutamat-Rezeptoren (Typ NMDA) mit 42% am häufigsten. Ein Drittel der positiven Reaktionen wurde in der Laborpraxis durch einen Autoantikörper verursacht, dessen Bestimmung vom Kliniker nicht angefordert war: Das unterstreicht die Notwendigkeit, für diesen Diagnostik-Bereich wegen der gebotenen Eile multiparametrische serologische Testsysteme einzusetzen.

Reliable serodiagnosis of rubella virus (RV) infections requires discrimination of specific IgM induced by primary rubella from persistent, reactivated or non-specific IgM reactivity. Sera from 130 pregnant women with recent or past RV infection/vaccination, persistent IgM or negative rubella serology, 26 patients with other acute infections and 5 patients with rheumatoid factor-positivity were analyzed for RV-specific IgM by ELISA coated with whole-virus lysate or native glycoprotein, followed by determination of IgG avidity and E2-specific IgG using lysate-coated ELISA and non-reducing immunoblot. Compared to a reference μ-capture IgM ELISA, the sensitivity for diagnosing recent rubella infection/vaccination was 90.0% and 100% for the lysate-based and glycoprotein-based IgM ELISA, respectively. With respect to women with past RV infections or negative histories of RV infection/vaccination, both assays were 97.5-100% specific, whereas for patients with other acute infections the glycoprotein substrate provided a specificity of 92.3% compared to only 80.8% using whole-virus antigen. Analyzing anti-RV IgG avidity and anti-E2 IgG reactivity allowed the time point of primary infection to be determined unambiguously in >86% of samples. In conclusion, using RV glycoprotein antigen improves the specificity of indirect IgM ELISA. In cases of RV-specific IgM reactivity, recent primary rubella infection can be confirmed or excluded efficiently by specific IgG avidity and immunoblot analysis.

The objective was to examine the prevalence of Borrelia antibodies among symptomatic individuals with recent and past Lyme disease in endemic communities using standard assays and novel assays employing next-generation antigenic substrates. Single- and two-tiered algorithms included different anti-Borrelia ELISAs and immunoblots. Antibody prevalence was examined in sera from 32 individuals with recent erythema migrans (EM), 335 individuals with persistent symptoms following treatment for Lyme disease (PTLS), and 41 community controls without a history of Lyme disease. Among convalescent EM cases, sensitivity was highest using the C6 ELISA (93.8%) compared to other single assays, specificity was 92.7% for the C6 ELISA vs. 85.4&ndash, 97.6% for other assays. The two-tiered ELISA-EUROLINE IgG immunoblot combinations enhanced case detection substantially compared to the respective ELISA-IgG Western blot combinations (75.0% vs. 34.4%) despite similar specificity (95.1% vs. 97.6%, respectively). For PTLS cohorts, two-tier ELISA-IgG-blot positivity ranged from 10.1% to 47.4%, depending upon assay combination, time from initial infection, and clinical history. For controls, the two-tier positivity rate was 0&ndash, 14.6% across assays. A two-tier algorithm of two-ELISA assays yielded a high positivity rate of 87.5% among convalescent EM cases with specificity of 92.7%. For convalescent EM, combinations of the C6 ELISA with a second-tier ELISA or line blot may provide useful alternatives to WB-based testing algorithms.

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. Abstract: Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are intraepidermal blistering skin diseases. PV is characterised by autoantibodies directed against desmoglein (Dsg) 3 and in patients with the mucocutaneous variant also against Dsg 1, whereas in PF, only Dsg 1 is targeted. Here, ectodomains of Dsg 3 and Dsg 1 were recombinantly expressed in a human cell line (HEK293) and applied as authentic solid phases in ELISA test systems. Autoantibodies against Dsg 3 and/or Dsg 1 could be detected in 71 (100%) of 71 PV sera and against Dsg 1 in 48 (96%) of 50 PF sera. Control sera showed reactivity with Dsg 3 and Dsg 1 in 0.2% and 0.7%, respectively, of 401 healthy blood donors and in 2.1% of 48 randomly selected patients with bullous pemphigoid. No reactivity with Dsg 1 and 3 was detected in 21 patients with linear IgA disease. For both pemphigus variants, a statistically significant correlation between clinical severity and autoantibody levels was observed as demonstrated for 10 PV and 5 PF patients. In conclusion, the use of the ectodomains of Dsg 3 and 1 as target antigens expressed in a human cell line resulted in sensitive and specific ELISA systems for both diagnosis and monitoring of PV and PF.

identified follicle-like structures containing B cells close to vessels and PDGFRb+ cells forming a reticular network expanding to the parenchyma. To find out if these structures could provide sufficient homing locations which support plasma cells to become long-lived, we performed 5-ethynyl-2′-deoxyuridine (EdU)-pulse experiments. During pulse, EdU is incorporated by dividing cells like plasma blasts. The detection of EdU-positive cells up to seven weeks after pulse suggests that niches in the CNS can favor survival of plasma cells. We then further analyzed the migration of plasma blasts to the CNS. Since the sphingosine-1-phosphate analogon FTY720 (fingolimod) is known to suppress immune cell trafficking, we performed EAE experiments under accompanying treatment with the drug. FTY720treated animals were resistant to EAE in contrast to control animals. Immunofluorescent histological findings confirmed that no immune cell infiltration took place in FTY720-treated animals and the protective effect of FTY720 was not due to retention of immune cells in spleen, lymph nodes or bone marrow, as proved by quantitative FACS analysis. Control mice displayed massive immune cell infiltration in the perivascular space and parenchyma of the CNS, some evidently of long-lived phenotype. These results can be useful to evaluate the therapeutic potential of targeting plasma cells in chronic neuroinflammation.

NMDA receptor (NMDAR) antibody encephalitis is the second most common autoimmune encephalitis after acute disseminated encephalomyelitis, presenting with a multistage clinical progression with prodromal signs, psychiatric abnormalities, reduced levels of consciousness, epileptic seizures, dyskinesias, and autonomic dysfunction.1 Pathogenic NMDAR autoantibodies downregulate NMDARs in cultured hippocampal neurons2 and after injection into the rat brain.3 Teratomas are common in adult women and are believed to trigger the antibody response against NMDARs by ectopically expressing neuronal antigens.1 Causal factors in tumor-negative patients are largely unknown. Recent studies showed that infectious encephalitides with herpes simplex virus (HSE) are associated with the generation of NMDAR antibodies4 and that antibodies cause immunotherapy-responsive relapsing symptoms post-HSE.5 It is unknown, however, whether further types of neuronal damage can trigger a secondary immune response leading to NMDAR encephalitis.

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.Serum samples of healthy (n = 1,703) versus neuropsychiatrically ill subjects (schizophrenia, affective disorders, stroke, Parkinson disease, amyotrophic lateral sclerosis, personality disorder; total n = 2,533) were tested. For analysis based on indirect immunofluorescence, we used biochip mosaics of frozen brain sections (rat, monkey) and transfected HEK293 cells expressing respective recombinant target antigens.Seroprevalence of all screened AB was comparable in healthy and ill individuals. None of them, however, reached the abundance of NMDAR1 AB (again ~10%; immunoglobulin [Ig] G ~1%). Appreciable frequency was noted for AB against amphiphysin (2.0%), ARHGAP26 (1.3%), CASPR2 (0.9%), MOG (0.8%), GAD65 (0.5%), Ma2 (0.5%), Yo (0.4%), and Ma1 (0.4%), with titers and Ig class distribution similar among groups. All other AB were found in ≤0.1% of individuals (anti-AMPAR-1/2, AQP4, CV2, Tr/DNER, DPPX-IF1, GABAR-B1/B2, GAD67, GLRA1b, GRM1, GRM5, Hu, LGl1, recoverin, Ri, ZIC4). The predominant Ig class depended on antigen location, with intracellular epitopes predisposing to IgG (chi-square = 218.91, p = 2.8 × 10(-48) ).To conclude, the brain antigen-directed AB tested here are comparably detectable in healthy subjects and the disease groups studied here, thus questioning an upfront pathological role of these serum AB.

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 Carr's 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.

Recently, several novel autoantibodies have been identified which are closely associated with different subtypes of autoimmune encephalitis. These antibodies are directed against structures located on the neuronal cell surface: glutamate receptors (types NMDA and AMPA), GABAB receptors, as well as the voltage-gated potassium channel-associated proteins LGI1 and CASPR2. They are much more common than the classical paraneoplastic antibodies (anti-Hu, -Yo, -Ri, -Ma, -CV2, -amphiphysin), less frequently associated with a tumor, and the corresponding clinical syndromes respond significantly better to immunotherapy. Monospecific detection of these autoantibodies in the serum or cerebrospinal fluid of patients is primarily performed by indirect immunofluorescence using transfected HEK293 cell lines recombinantly expressing the membrane-associated target antigens. Owing to the symptom overlap of the respective disorders, it is highly appropriate to determine these parameters in parallel for each patient (autoantibody profiles). Early diagnosis (substantially supported by the serological laboratory), the immediate initiation of immunotherapeutic intervention and, in cases of paraneoplastic etiology, tumor resection are crucial for prognosis. In our own investigations, antibodies against glutamate receptors (type NMDA) are most frequently found among the newly identified forms of autoimmune encephalitis, accounting for 42% of cases. In laboratory practice, one-third of positive reactions were caused by an autoantibody whose determination was not requested by the clinician. Considering the urgency for therapeutic measures in positive cases, these findings substantiate the need to implement multiparametric serological test systems in this diagnostic area.

The prevalence of herpes simplex virus (HSV) type-specific IgG was determined in sera taken in 1999 to 2006 from 1,100 children aged 0–18 years, 800 blood donors and 200 pregnant women in Thuringia, Germany, using tests based on the HSV glycoproteins (g) gG. By the age of 10–12 years, HSV-1 IgG prevalence reached 57.3%, rising to 69.3% by the age of 16–18 years and to 78.0% by the age of 28–30 years. Between 2.7% and 4.7% of the children aged up to 15 years had HSV-2 antibodies, increasing to 7.3% at the age of 16–18 years and to 13.6% among adults. The prevalence of HSV-1 antibodies among girls was significantly lower than among boys and a significantly higher prevalence of HSV-2 IgG in women than in men was detected. The reduced incidence of HSV-1 infections during childhood, especially in girls, has to be followed up since a higher number of primary HSV-2 infections may result. Between 2.7% and 4.7% of all children tested seemed to acquire HSV-2 by intrauterine or neonatal infection. We also compared the use of gG-1 with gC-1: the agreement of 97.2% between the two ELISAs suggests that gG-1 and gC-1 can be considered equivalent antigenic targets.

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.

The prevalence of herpes simplex virus (HSV) type-specific IgG was determined in sera taken in 1999 to 2006 from 1,100 children aged 0–18 years, 800 blood donors and 200 pregnant women in Thuringia, Germany, using tests based on the HSV glycoproteins (g) gG. By the age of 10–12 years, HSV-1 IgG prevalence reached 57.3%, rising to 69.3% by the age of 16–18 years and to 78.0% by the age of 28–30 years. Between 2.7% and 4.7% of the children aged up to 15 years had HSV-2 antibodies, increasing to 7.3% at the age of 16–18 years and to 13.6% among adults. The prevalence of HSV-1 antibodies among girls was significantly lower than among boys and a significantly higher prevalence of HSV-2 IgG in women than in men was detected. The reduced incidence of HSV-1 infections during childhood, especially in girls, has to be followed up since a higher number of primary HSV-2 infections may result. Between 2.7% and 4.7% of all children tested seemed to acquire HSV-2 by intrauterine or neonatal infection. We also compared the use of gG-1 with gC-1: the agreement of 97.2% between the two ELISAs suggests that gG-1 and gC-1 can be considered equivalent antigenic targets.