what is new in detection of autoantibody - latest innovations in autoantibody detection for better...
TRANSCRIPT
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
EUROIMMUN
EUROIMMUN
1983: Chemical activation(aminosilane, glutardialdehyde)
EUROIMMUN
EUROIMMUN
1979: TITERPLANE-techniqueTITERPLANE
EUROIMMUN
2013: MERGITE !
Conventional washing: Sporadically unspecific background
EUROIMMUN
EUROIMMUN
2009: EUROTide
EUROTide: Ebb and Flow
EUROTide Micro-Immunblot
EUROTidekonventionell
EUROTide
EUROIMMUN
EUROTide, mab 1:100manuell, mab 1:100EUROTide, mab 1:1.000manuell, mab 1:1.000
EUROIMMUN
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.
EUROIMMUN
22
EUROIMMUN
EUROIMMUN
LED illumination: Coproduct ofCarl Zeiss & EUROIMMUN
EUROIMMUN
26Allgemeine berschriften,evtl. das Warum vor der Evaluierung ?
Regulated light emission (LED)
EUROIMMUN
27Allgemeine berschriften,evtl. das Warum vor der Evaluierung ?
EuroLabOffice - Result input at the microscope
EUROIMMUN
EURO-PATTERN- Microscope
EUROIMMUN
500 wells in 2 hours, walk-away
EUROIMMUN
ANA pattern recognition: Cell finder plus specific fluorescence
EUROIMMUN
ANA pattern recognition: Cell finder
EUROIMMUN
ANA pattern recognition:Specific fluorescence
EUROIMMUN
Computer-aided immunofluorescence microscopy (CAIFM)
EUROIMMUN
34Computer-aided immunofluorescence microscopy (CAIFM)
100 ANA more thananti-ENA and anti-DNA in
Autoimmune MyositisSystemic sclerosisDermatomyositisLupus erythematodes and vasculitisPrimary biliar liver cirrhosis
EUROIMMUN
EUROIMMUN
Control
nRNP/SmSmSS-ASS-BScl-70Jo-1CENP B
dsDNAHistonesrib. P-Proteins
EUROLINE ENA PLUS
EUROIMMUN
Autoimmune Myositis
EUROIMMUN
37
Almost no overlap between antibodies!Jo-1Mi-2KuPM-SclPL-7SRPPL-12OJEJFor highest sensitivity, investigate antibodies in parallel !
EUROIMMUN
DermatomyositisMi2SAE1NXP2TIF1MDA5Mi2SAE2
PolymyositisJo-1PL-12SRPEJOJPL-7
OverlapPM-Scl100KuPM-Scl75
Necrotizing myopathyHMGCR
Inclusion Body MyositisMup44Autoimmune Inflammatory Myopathies- Subgroups and associated autoantigens -
EUROIMMUN
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
EUROIMMUN
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
EUROIMMUN
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 -
EUROIMMUN
4242
Autoimmune encephalitis
EUROIMMUN
43
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)
EUROIMMUN
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%
EUROIMMUN
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)
EUROIMMUN
Hippocampus rat, ex-vivo-culture, anti-glutamate receptor autoantibodies (type NMDA)
EUROIMMUN
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
EUROIMMUN
Anti-NMDAAnti-CASPR2Anti-DPPX
EUROIMMUN
61Neuronal PPT_Beirut 1011CIBD 03.2009_JA61
Pemphigus vulgaris
EUROIMMUN
6363
Bullous pemphigoid: Anti-BP180
EUROIMMUN
64
Pemphigus vulgaris: Anti-Desmoglein 3. Substrate: Transfected HEK-293-cells
EUROIMMUN
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
EUROIMMUN
66
Bullous Pemphigoid and Pemphigus gestationis: Detection ofAnti-NC16A by ELISA or EUROPLUS
EUROIMMUN
67
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
EUROIMMUN
68
Indirect immunofluorescence
EUROIMMUN
Detect
100 different autoantibodies
using one substrate and
with one incubation
EUROIMMUN
HEp-2 cells / primate liver:ANA homogeneous pattern
EUROIMMUN
EUROIMMUN
HEp-2 cells / primate liver:Anti-nuclear dots (SP-100)
EUROIMMUN
EUROIMMUN
HEp-2 cells, primate liver: Autoantibodies against actin
EUROIMMUN
EUROIMMUN
HEp-2 cells / primate liver: Anti-GP 210
EUROIMMUN
EUROIMMUN
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
EUROIMMUN
One method one SOP,1.000 different test parametersAnti-basement membr.Anti-GBM Anti-Treponema Anti-tTG/ gliadin
Anti-SARS
ANA (HEp-2)
EUROIMMUN
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
EUROIMMUN
Serology in tropical fever diseases
EUROIMMUN
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
EUROIMMUN
79
Unspecific background easily identifiable
EUROIMMUN
The universal EUROIMMUN dilution scheme for immunofluorescencein autoantibody diagnostics
EUROIMMUN
EUROIMMUN
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
EUROIMMUN
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
EUROIMMUN
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
EUROIMMUN
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
EUROIMMUN
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
EUROIMMUN
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
EUROIMMUN
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
EUROIMMUN
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 ...
EUROIMMUN