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Multiparametric serological testing in autoimmune encephalitis using recombinant immunofluorescence

cell substrates and EUROTIDE technology

K.P. Wandinger1, C. Klingbeil1, P. Waters2, J. Dalmau3, S. Saschenbrecker1,

K. Borowski1, A. Vincent2, C. Probst1, and W. Stoecker1

1Institute for Experimental Immunology, affiliated to EUROIMMUN AG, Luebeck, Germany2Neurosciences Group, Department of Clinical Neurology, University of Oxford, UK

3Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA

Introduction

Recently, several new antigenic targets of autoantibodies associated with autoimmune forms of encephalitis were identified and found to be located on the neuronal cell surface: glutamate (Glu) receptors (type NMDA and type AMPA), GABAB receptors (GABABR), and the VGKC-complex antigens, LGI1 and CASPR2. Since the clinical features associated with these anti-neuronal surface antibodies (ANSA) often overlap, it is appro-priate to test for all of the ANSA as well as the classical paraneoplastic antibodies. Using a multiparametric BIOCHIP mosaic, the frequencies of ANSA and classical paraneoplastic antibodies were analyzed by indirect immunofluorescence assay (IIFA).

Methods

cDNAs for glutamate receptors (type NMDA; subunit NR1 and type AMPA; GluR1/GluR2), GABA receptor (B1), LGI1 and CASPR2 were inserted into eukaryotic expression vectors and transfected into HEK293 cells. The recombinant cells were grown and acetone-fixed on glass slides, which were fragmented to BIOCHIPs and used in mosa-ics additionally containing frozen sections of rat hippocampus, cerebellum and different control tissues. These mosaics were used for antibody detection in serum and CSF by IIFA using EUROTIDE incubation technology, in which reactions are accelerated and opti-mized by forced convection. Identification of classical paraneoplastic antibodies (i.e. anti-Hu, -Yo, -Ri, -PCA-2, -Ma, -CV2, -amphi-

physin) was based on the characteristic IIFA tissue pattern confirmed by a monospecific line immunoblot assay.

Results

Out of 2716 requests for anti-neuronal an ti-body testing received between October and December 2010, 108 patients were positive for specific antibodies*. ANSA were found in 63% of positive patients: anti-glutamate receptor (type NMDA) 38%, anti-glutamate receptor (type AMPA) 0%, anti-GABAB re-ceptor 3%, anti-LGI1 11%, and anti-CASPR2 11%. Classical paraneoplastic antibodies were detected in 31% of specimens: anti-Hu 6%, anti-Yo 8%, anti-Ri 9%, anti-Ma 2%, anti-PCA-2 1%, anti-CV2 4%, and anti-am-phiphysin 1%. Parallel detection of two dif-ferent anti-neuronal antibodies occurred in 7% of cases: anti-Hu / anti-CV2 3%, anti-glu-

tamate receptor (type NMDA) / anti-Hu 2%, anti-glutamate receptor (type NMDA) / anti-GABAB receptor 1%, anti-glutamate recep-tor (type NMDA) / anti-CASPR2 1%. In 31% of cases, the detected antibody was differ-ent from that requested.

Conclusion

Application of recombinant cell-based BIO-CHIP mosaics and EUROTIDE-based IIFA al-lows monospecific detection and differen-tiation of ANSA. The overall prevalence of ANSA was approximately twice the preva-lence of the classical paraneoplastic anti-bodies, with reactivity against glutamate receptor (type NMDA) being most frequent-ly detected in this panel. The findings sub-stantiate the importance of multiparametric serological testing in suspected cases of autoimmune-mediated encephalitis.

Anti-GABAB receptorAnti-Glu receptor (type NMDA) Anti-LGI1 Anti-CASPR2

AntigenIIFA positive

n = 108

Glutamate receptor (type NMDA) 41 38%Glutamate receptor (type AMPA) 0 0%GABAB receptor 3 3%LGI1 12 11%CASPR2 12 11%Hu 6 6%Yo 9 8%Ri 10 9%Ma 2 2%PCA-2 1 1%CV2 4 4%Amphiphysin 1 1%Hu and CV2 3 1%Glutamate receptor (type NMDA) and Hu 2 1%Glutamate receptor (type NMDA) and GABABR 1 2%Glutamate receptor (type NMDA) and CASPR2 1 3%

Samples positive for anti-neuronal antibodies by IIFA (n = 108)*:

Anti-neuronal surface antibodies (n = 68)Classical paraneoplastic antibodies (n = 33)Parallel detection of two different anti-neuronal antibodies (n = 7)

n = 68(63%)

n = 33(31%)

n = 7(7%)

Anti-neuronal surface antibodies

Classical paraneoplastic antibodies

Parallel detection of two different anti-neuronal antibodies

* The sum of percentages is 101% due to rounding up

Scientific presentation at the 10th Dresden Symposium on Autoantibodies, Dresden, Germany, September 2011

Introduction

Bullous pemphigoid (BP) is characterized by autoantibodies against the NC16A domain of BP180 and the C-terminal globular do-main of BP230. In contrast, epidermolysis bullosa acquisita (EBA) is associated with autoantibodies against the non-collagenous domain 1 (NC1) of collagen VII*, and pem-phigus foliaceus and pemphigus vulgaris (PV) are characterized by autoantibodies against the ectodomains of desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3), respec-tively.

Methods

Transfected HEK293 expressing the C-ter-minal globular domain of BP230, the NC1 domain of collagen VII (Col7-NC1), the ectodomains of Dsg1 or Dsg3 were used in combination with bacterially expressed

BP180-NC16A and primate esophagus cryo-sections in a BIOCHIP format for indirect immunofluorescence (IIF). IgG autoantibod-ies were determined in patients with BP (n = 55), EBA (n = 57), PV (n = 24), as well as in 154 healthy blood donors (HBD).

Results

Anti-BP180-NC16A antibodies were detect-ed in all 55 (100%) BP sera (PV 0%, EBA 2%), and in 24 (44%) of these additional anti-BP230 reactivity was found (PV 0%, EBA 2%). Anti-Col7-NC1 antibodies were present in 50 (88%) of EBA sera (PV 0%, BP 2%). Anti-Dsg3 antibodies were detected in all 24 (100%) PV sera, and in 9 (28%) of these additional anti-Dsg1 reactivity was found. No autoantibodies against Dsg1 and 3 were found in EBA and BP sera. Lower prevalences were obtained with conven-tional IIF microscopy using esophagus:

21 (88%) PV sera produced a desmosomal pattern, whereas 39 (71%) BP and 43 (75%) EBA sera produced a basement membrane pattern. HBD sera did not show any of the analyzed IgG specificities.

Conclusion

The IIF BIOCHIP Mosaic based on a com-bination of transfected HEK293 and bacte-rially expressed BP180-NC16A is easy to interpret and represents a highly sensitive and specific tool for the parallel determi-nation of autoantibodies against BP180-NC16A, BP230, Col7-NC1, Dsg1, and Dsg3. It is a simple and time-saving alternative to conventional IIF employing cryosections of esophagus or salt-split human skin, which often produce equivocal patterns.

* Schmidt, Zillikens et al. Modern diagnosis of autoim-

mune blistering skin diseases. Autoimmun Rev 2010.

EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 451 58550 · Fax 5855591 · E-mail euroimmun@euroimmun.de

Detection of autoantibodies in bullous pemphigoid, epidermolysis bullosa acquisita, and pemphigus vulgaris by indirect immunofluorescence with a BIOCHIP Mosaic

C. Probst1, I.-M. Bloecker1, W. Stoecker1, E. Schmidt2,

D. Zillikens2, and L. Komorowski1

1Institute for Experimental Immunology, affiliated to EUROIMMUN AG, Luebeck, Germany2Department of Dermatology, University of Luebeck, Germany

Cohort nEsophagus

epid. BM pos.BP180-NC16A

pos.HEK293

BP230 pos.HEK293

Col7-NC1 pos.Esophagus

desmosomes pos.HEK293

Dsg1 pos.HEK293

Dsg3 pos.

Bullous pemphigoid 55 39 (71%) 55 (100%) 24 (44%) 1 (2%) 0 0 0

Epidermolysis bullosa acquisita

57 43 (75%) 1 (2%) 1 (2%) 50 (88%) 9 (16%) 0 0

Pemphigus vulgaris 24 0 0 0 0 21 (88%) 9 (28%) 24 (100%)

Healthy blood donors 154 0 0 0 0 0 0 0

BP180-NC16A BP180

Salt-split human skin

Monkey esophagus

Desmoglein 3

BP230 C-terminal Col7NC1 Envoplakin

Gliadin (GAF-3X)Desmoglein 1 ControlLaminin γ1

Scientific presentation at the 10th Dresden Symposium on Autoantibodies, Dresden, Germany, September 2011

Introduction

Idiopathic membranous nephropathy (IMN) is one leading cause of nephrotic syndrome in Caucasian adults. Up to 70% of patients with IMN exhibit autoan-tibodies of the IgG4 subclass directed against M-type phospholipase A2 recep-tor (PLA2R). The aim of the current study was to evaluate a cell-based immunofluo-rescence assay for the determination of anti-PLA2R.

Methods

A cDNA encoding full-length PLA2R iso-form 1 was used for transient transfection of HEK293 cells. 48 hours after transfection, cells were fixed and used as substrates for indirect immunofluorescence. Antibody ti-

ters of follow-up samples from 11 IMN pa-tients under therapy were monitored and results were compared with reactions of sera from healthy blood donors (n = 150).

Results

With the recombinant cell-based assay PLA2R-specific antibodies (IgG1-4) were detected in 6 of 11 IMN patients (specifi-city 100%). During a monitoring period of up to 9 months there was a decrease in antibody titers in five patients.

Conclusion

Detection of autoantibodies in patients with IMN may delineate those patients who need immunosuppressive therapy in

order to reduce proteinuria and prevent loss of renal function. The new substrate is suited for broad screening in the detection of anti-PLA2R antibodies in nephrology.

EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 451 58550 · Fax 5855591 · E-mail euroimmun@euroimmun.de

Detection of PLA2R specific autoantibodies in patients with idiopathic membranous nephropathy using

PLA2R producing HEK293 cells

E. Hoxha1, I.-M. Bloecker2, C. Probst2, L. Komorowski2, K.P. Wandinger2,

W. Schlumberger2, W. Stoecker2, and R. Stahl1

1University Medical Center Hamburg-Eppendorf, Hamburg, Germany2Institute for Experimental Immunology, affiliated to EUROIMMUN AG, Luebeck, Germany

HEK293 cells transfected with recombinant human PLA2R isoform 1

Non-transfected HEK293 cells

1:3200

1:320

1:1000

1:101:32

1:100

1 3 5 1096

Follow-up of the anti-PLA2R titer in 6 IMN patients

An

ti-P

LA2R

IIFT

(ti

ter)

Schematic illustration of PLA2R isoform 1

cysteine-rich domain

fibronectin type II domain

c-type lectin-like domains

transmembrane domain

intracellular tail

reco

mbi

nant

pro

tein

Scientific presentation at the 7th International Congress on Autoimmunity, Ljubljana, Slovenia, May 2010

EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 451 58550 · Fax 5855591 · E-mail euroimmun@euroimmun.de

EUROPattern Microscope Representation of results in EUROPattern

+++ Centromeres 1:3,200

Computer result

Visual microscopic result

Final result

Unofficial remark

Delete all

Pos.Neg.Borderline?

Pos.Neg.Borderline?Repeat

++++ Centromeres 1:10,000Addition:ENA EUROPLUS neg.

Pos.Neg.Borderline?

+++ Centromeres 1:3,200

X Reset

Furtherdiagnostics Verify synthesis

15/28Serum No.: 127Macros

94%

Homogeneous patternGranular patternNucleolar pattern

1:3,200

98%

Nuclear dotsMitosis patternCytoplasmic patternNegative

KuPCNAMitosin (CENP F)Coilin (Few nuclear dots)Spindle apparatusCentriolesJo-1Other

?

Nuclear membrane

Centromeres

Introduction

Indirect immunofluorescence (IIF) has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Whereas there are some au-tomated technical solutions for IIF incuba-tion about to appear on the market, the performance of result evaluation is still in its infancy. The patterns are predominantly recorded and interpreted by visual micro-scopic examination and the results docu-mented in paper or electronic form.

The user is forced to permanently alternate between the microscopic image and the re-cords – and to shift his point of focus is tiring and often leads to incorrect allocation of results, especially since the evaluation is generally performed in a dark room. CAIFM was developed to support the laboratory experts in diagnosing antibodies.

Microscope configuration

A motorised camera-microscope with spe-cial IIF relevant functions was designed (EUROPattern by EUROIMMUN), contain-ing a magazine with a capacity for 50 slides, each with 10 reaction fields, or 10 slides, each with 50 reaction fields. A ma-trix code scanner enables slide identifica-tion and an incremental encoder identifies the field position.

Interactive microscopy

Starting with any of the 500 reaction fields by entering an ID or by mouse click, the sub-strates are selectively or consecutively visu-alized without eye-pieces at the computer screen. The slides are moved and focussed using a 3D actuator. Results are interpreted by the expert, they are entered via mouse and keyboard, images are recorded at the push of a button and automatically allocated and archived together with the results. A dark room is not required because the images on the screen are very bright. Owing to the cas-ing around magazine and microscope stage, sunlight is kept out and the fluorescence in the substrates is protected from fading.

Automatic pattern recognition

The 500 reaction fields are examined and interpreted automatically. The system auto-focuses and takes an adjustable number of images by means of a camera, followed by visual or software-based diagnosis. The EUROPattern software allows automated assessment of IIF patterns, at present for anti-nuclear antibodies on HEp-2/HEp-2010 cells. The software performs a positive/neg-ative differentiation and identifies deposit-ed patterns, including many pattern combi-nations. If a sample has been incubated at different dilutions, the software merges the results of the individual analyses into one

report form, which shows the recognised pattern and the antibody titer. Fluorescence image and computer interpretation are dis-played together on the computer screen to be confirmed with one mouse click or mod-ified if deemed necessary. In an optional two-step screening approach, all images defined as negative are sorted out, and the expert individually confirms or reclassifies only the remaining positive results.

Laboratory management software

The EUROPattern Microscope is integrated into EUROLabOffice (EUROIMMUN) which supports IIF processing by automatic proto-col generation, interconnection with further laboratory devices (e.g. dilution/incubation systems) or analytical techniques (ELISA, Immunoblot, RIA), data exchange with the pattern recognition software, merging of test results and archiving IIF images in elec-tronic report forms.

Conclusion

CAIFM reinforces the practical suitability of IIF. Results are interpreted at the office PC, the hands stay clean. The clinical pathologist can view all available IIF images by a simple mouse click and does not need to consult the microscope again in the event that a re-sult must be checked for confirmation.

Computer-Aided Immunofluorescence Microscopy (CAIFM) in the diagnosis of autoimmune and infectious diseases

W. Stoecker1, M. Rateike1, M. Morrin1, J. Voigt1, K. Rentzsch1, C. Krause1,2, K. Ens1,

H. Fauer1, E. Barth2, C. Feirer1, D. Wuttig1, K. Fechner1, S. Kloth1, and T. Martinetz2

1Institute for Experimental Immunology, affiliated to EUROIMMUN AG, Luebeck, Germany 2Institute for Neuro- and Bioinformatics, University of Luebeck, Germany

Scientific presentation at the 10th Dresden Symposium on Autoantibodies, Dresden, Germany, September 2011