PowerPoint-Prsentation

Latest innovations in autoantibody detection for better diagnosis of autoimmune rheumatic diseases

Kochi, India, November 2016

Winfried Stcker

1987: Life experience of IFT 5 years ?

Frozen tissue sections: Insufficient adherance on glass surfaces

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1983: Chemical activation(aminosilane, glutardialdehyde)

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1979: TITERPLANE-techniqueTITERPLANE

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2013: MERGITE !

Conventional washing: Sporadically unspecific background

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2009: EUROTide

EUROTide: Ebb and Flow

EUROTide Micro-Immunblot

EUROTidekonventionell

EUROTide

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EUROTide, mab 1:100manuell, mab 1:100EUROTide, mab 1:1.000manuell, mab 1:1.000

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TITERPLANE-slide

Cryosectioning (4 mikrometers)

1983: BIOCHIPs for indirect immunofluorescence

BIOCHIP slides

PANKREAS PRIM. / INTESTINUM PRIM.SACCHA. CEREV. / GRANULOC. EOHEUROIMMUN

BIOCHIP-Mosaic:Autoimmune hepatitis

2020

16 BIOCHIPs per reaction field!

EUROIMMUNMicrochip-Mosaic ANA

SS-AnucleosomesHEp-2010

Scl-70SmSm + RNPJo-1nDNASS-BPCNAIgAGMCrith.luc.

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22

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LED illumination: Coproduct ofCarl Zeiss & EUROIMMUN

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26Allgemeine berschriften,evtl. das Warum vor der Evaluierung ?

Regulated light emission (LED)

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27Allgemeine berschriften,evtl. das Warum vor der Evaluierung ?

EuroLabOffice - Result input at the microscope

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EURO-PATTERN- Microscope

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500 wells in 2 hours, walk-away

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ANA pattern recognition: Cell finder plus specific fluorescence

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ANA pattern recognition: Cell finder

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ANA pattern recognition:Specific fluorescence

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Computer-aided immunofluorescence microscopy (CAIFM)

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34Computer-aided immunofluorescence microscopy (CAIFM)

100 ANA more thananti-ENA and anti-DNA in

Autoimmune MyositisSystemic sclerosisDermatomyositisLupus erythematodes and vasculitisPrimary biliar liver cirrhosis

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Control

nRNP/SmSmSS-ASS-BScl-70Jo-1CENP B

dsDNAHistonesrib. P-Proteins

EUROLINE ENA PLUS

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Autoimmune Myositis

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37

Almost no overlap between antibodies!Jo-1Mi-2KuPM-SclPL-7SRPPL-12OJEJFor highest sensitivity, investigate antibodies in parallel !

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DermatomyositisMi2SAE1NXP2TIF1MDA5Mi2SAE2

PolymyositisJo-1PL-12SRPEJOJPL-7

OverlapPM-Scl100KuPM-Scl75

Necrotizing myopathyHMGCR

Inclusion Body MyositisMup44Autoimmune Inflammatory Myopathies- Subgroups and associated autoantigens -

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3939Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of autoimmune disorders predominantly affecting skeletal muscles, resulting in muscle inflammation and weakness. Along with the musculoskeletal manifestations, involvement of other organ systems is seen, including the skin, cardiac, gastrointestinal, and pulmonary systems. The 3 most common inflammatory myopathies are polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM). Several much rarer syndromes are also described under the broad spectrum of inflammatory myopathies.Necrotizing myopathy is related to statin use in majority; most patients are statin exposed, but myopathy also reported in a minority of statin-naive patients (antibody: HMG-CoA reductase)[Statins (or HMG-CoA reductase inhibitors) are a class of drugs used to lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase, which plays a central role in the production of cholesterol in the liver.]

Antibodies in myositis are categorized into myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs). MSAs are generally found only in patients with inflammatory myositis or in patients with ILD with subclinical myositis, whereas MAAs are also encountered in other connective tissue diseases without signs of myositis.

Mi2alpha

Mi2beta

TIF1g

MDA5NXP2

SAE1

Ku

PM-Scl100

PM-Scl75

Jo-1

SRP

PL-7

PL-12

EJ

OJ

Control

DermatomyositisMi2SAE1NXP2TIF1MDA5Mi2

PolymyositisJo-1PL-12SRPEJOJPL-7

OverlapPM-Scl100KuPM-Scl75

DL 1530-4 GEUROLINEAutoimmune Inflammatory Myopathies

Mi2

Ku

PM-Scl100

Jo-1

PL-7

PL-12

Ro-52

Control

Mi2

Ku

PM-Scl100

PM-Scl75

Jo-1

SRP

PL-7

PL-12

EJ

OJ

Ro-52

Control

DL 1530-GDL 1530-3G

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4040We have nearly all.Mi2 alpha and Mi2beta had in our hands the same specificitySAE2 we did not include, it has the same sensitivity as SAE1 and there was no case with SAE2 allone, only SAE1 allone, thus we did not take SAE2

Systemic Sklerosis

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41

limitedTh/ToCenp BCenp A

diffuseRP155RP11Scl-70overlapPM-Scl75PM-Scl100Ku

otherNOR90FibrillarinPDGFRScl-70

Cenp A

Cenp B

RP11

RP155

Fibrillarin

NOR90

Th/To

PM-Scl100

PM-Scl75

Ku

PDGFR

Ro-52

Control

DL 1532-1601 GSystemic Sclerosis- Subgroups and associated autoantigens -

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4242

Autoimmune encephalitis

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Primate intestinePrimate cerebellumEncephalitisSensory / Autonomous NeuropathyAutoantibodies against neuronal nuclei: Hu (ANNA 1)

Primate cerebellumPrimate intestineOpsoclonus-Myoclonus SyndromeAutoantibodies against neuronal nuclei: Ri (ANNA 2)

Primate cerebellumPrimate intestineSubacute cerebellar degenerationAutoantibodies against Purkinje cell cytoplasm: Yo (PCA1)

Combination of native and rec. Antigens: EUROLINE-Westernblot* Recombinant Antigens are protected by patent in the USA

Cerebellar extract:Hu: 38 kDaRi: 80 kDa 55 kDaYo: 62 kDa 34 kDaplusrecombinant Hurecombinant Yorecombinant Ri

EUROLINE: Neuronal Antigens*

Hu (ANNA 1) Yo (PCA 1) Ri (ANNA 2) Ta / Ma 2 CV-2 (CRMP 5) AmphiphysinControl

* Recombinant Antigens are protected by patent in the USA

2004:Aquaporin-42008:NMDA-receptors 2009: AMPA-receptors 2010:GABAB-receptors 2010:LGI1; CASPR22012:DNER (Tr)

Newly discovered neurological autoantibodies (examples)

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49Rho GTPase activating protein 26 (ARHGAP26)

Serological Diagnosis of NMO

optic nerveNMO-IgG:

HEKcerebellum

Anti-AQP-4: AQP4 transf.

Subpiale und supepenymale Astrozytenfortstze

Anti-aquaporin aab in NMO,as tested using rec HEK293 cells

HEK293-cellsAQP4-transfectedHEK293-cellssensitivity: 78%specificity: 100%

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51AQP4 transfected HEK cells are a highly sensitive and specific tool for standardized detection of antibodies to AQP4

Short development phase

Authentic environment

No purification necessary

Combination of tissue and recombinant substrates possible (BIOCHIP mosaic)

Easy to read outRecombinant cell based IFA

Hippocampus rat, anti-glutamate receptor autoantibodies (type NMDA)

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Hippocampus rat, ex-vivo-culture, anti-glutamate receptor autoantibodies (type NMDA)

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Limbic encephalitis:Anti-glutamate receptor autoantibodies (type NMDA)

hippocampuscerebellum

NMDA receptor HEK

55

Encephalitis: Anti-Lgi1 (leucinerich glioma inactivated protein 1)hippocampuscerebellumLgi1 HEK-Cells

Neuromyotonia: Anti-Caspr2 (Contactin associated protein 2)hippocampuscerebellumCaspr2 HEK-Cells

IFA Neurology Mosaic

EURO-PATTERN- Microscope

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Anti-NMDAAnti-CASPR2Anti-DPPX

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61Neuronal PPT_Beirut 1011CIBD 03.2009_JA61

Pemphigus vulgaris

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6363

Bullous pemphigoid: Anti-BP180

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64

Pemphigus vulgaris: Anti-Desmoglein 3. Substrate: Transfected HEK-293-cells

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65

NC16A: Target antigen inBullous pemphigoid

Hemidesmosomal proteins BP180 / type XVII collagen.

Epitopes clustered within the 16th non-collagenous domain (NC16A)

Variante NC16A-4-X

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66

Bullous Pemphigoid and Pemphigus gestationis: Detection ofAnti-NC16A by ELISA or EUROPLUS

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Oesophagus (Prim.)EUROPLUS BP180-NC16A-4XTransfizierte HEK-ZellenBP230-gCSpalthaut (Prim.)Transfizierte HEK-Zellen EnvoplakinTransfizierte HEK-ZellenCollagen Typ VII NC1Leber (EmA)EUROPLUS GAF-3XTransfizierte HEK-Zellen Dsg 3Transfizierte HEK-Zellen Dsg 1

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Indirect immunofluorescence

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Detect

100 different autoantibodies

using one substrate and

with one incubation

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HEp-2 cells / primate liver:ANA homogeneous pattern

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HEp-2 cells / primate liver:Anti-nuclear dots (SP-100)

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HEp-2 cells, primate liver: Autoantibodies against actin

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HEp-2 cells / primate liver: Anti-GP 210

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One substrate (HEp-2) Screening of 100 different Aab

ANA homogenNuclear dotsCentromereANA nucleolar

Scl-70

Sm/RNP

SS-A/SS-B

Nuclear memb.RibosomesMitochondriaGolgi apparat.LysosomesActinVimentin

Negative result presence of all these antibodies excluded

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One method one SOP,1.000 different test parametersAnti-basement membr.Anti-GBM Anti-Treponema Anti-tTG/ gliadin

Anti-SARS

ANA (HEp-2)

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Method of choice where test antigensare not defined or cannot be isolated

Purkinje cells cANCA

Pancreas acini

Pancreas islets

N. opticus,anti-aquaporin Cerebellum,anti-DPPX (memb.) Cerebellum,anti-IgLON5

Cerebellum,anti-flotillin

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Serology in tropical fever diseases

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78Soll hier evtl erwhnt werden, da das RKI eine Durchseuchungsstudie plant ?

Visual identification of specific signalsSchilddrse, Affe: AAk gegen SD-PeroxidaseSchilddrse, Affe: AAk gegen Thyreoglobulin

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79

Unspecific background easily identifiable

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The universal EUROIMMUN dilution scheme for immunofluorescencein autoantibody diagnostics

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81

Dilution factor for IFT titration 1 : 3.2 (square root of 10)

Dilution steps: 1:3.2 1:32 1:320 1:3,200 etc. 1:10 1:100 1:1,000 1:10,000

Not overly exact (quadratic dilutions: 1:2, 1:4, 1:8 ...)

Not too inexact (four-fold dilutions: 1:4, 1:16, 1:64...)

No numerical acrobatics

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Suitable starting dilutions for indirect immunofluorescence (Aab) Group I: Diagnostically relevant from 1:10 Most organ-specific AAb, ANCA, anti-dsDNA parallel 1 : 10 and 1 : 100

Group II: Diagnostically relevant from 1:100 ANA, AMA, ASMA, anti-skeletal muscle Ab parallel 1 : 100 and 1 : 1,000

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Clinical significance of antibody titer 1:10 1:32 1:100 1:320 1:1,000 1:3,200 1:10,000Group I + ++ ++ +++ +++ ++++ ++++Group II + ++ +++ ++++ ++++

+ weak ++ moderate +++ high ++++ very high

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Two parallel initial dilutions: a) Avoid blocking at high antibody concentrationsAnti-Yo (cerebell.)

1:101:100Anti-bas. memb. (tongue)

1:101:100AMA (kidney)

1:1001:1,000pANCA (granulocytes)1:11:10

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Two parallel initial dilutions:b) Unmask relevant autoantibodies

Anti-ribosomes and anti-nucleoli

1:100

1:1,0001:1,000

ANA homogen and anti-centromere1:100

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Two parallel initial dilutions:c) Estimate Titer

Fluorescence at

1:100

1:1,000

AAb-titer

weakneg1:100

mediumneg1:320

strong weak1:1,000

strongmedium1:3,200

strongstrong1:10,000

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No photometry at just one dilution!

1:1,000

ANA homogeneous (titer 1:10,000)1:100

1:1,000

ANA homogeneous (titer 1:1,000)1:100

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Testing anti-ENA plus anti-DNA is sufficient.Indirect immunofluorescence using Hep-2-cells as substrate provides complete information.Combination of indirect immunofluorescence with monospecific profile testing is optimal.Indirect immunofluorescence is out of date.For detection of autoantibodies against cell nuclei ...

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