Neurology Mosaics

Instructions for the indirect immunofluorescence test

SPECIE FORMAT
ORDER NO. ANTIBODIES AGAINST SUBSTRATE
S SLIDES x FIELDS
monkey
Nerves
Medullated nerves monkey
Cerebellum
Grey and white matter, monkey
Intestinal tissue
Non-medullated nerves monkey
Liver
Cell nuclei (ANA) monkey
Cerebrum
Cerebrum antigens human
HEp-2 cells
Cell nuclei (ANA) monkey
Spinal cord
FA 1111-1 Spinal cord antigens monkey
Pons 10 x 03 (030)
to Brain antigens monkey
Temporal lobe 10 x 05 (050)
FA 1111-17 Brain antigens monkey
Pancreas
Pancreas islets monkey 10 x 10 (100)
FA 111m-3 Optic nerve
Optic nerve antibodies rat 10 x 50 (500)
FA 112d-1 Hippocampus
Hippocampus antigens rat 20 x 05 (100)
FA 112k-1 Cerebellum
Cerebellum antigens EU 90 20 x 10 (200)
(see p. 24) Transfected cells
Aquaporin-4 (AQP-4) EU 90
Transfected cells
Glutamate receptor (type NMDA) EU 90
Transfected cells
Glutamate receptor (type AMPA1) EU 90
Transfected cells
Glutamate receptor (type AMPA2) EU 90
Transfected cells
Contactin-associated protein 2 (CASPR2) EU 90
Transfected cells
Leucine-rich glioma-inactivated protein 1 (LGI1) EU 90
Transfected cells
GABA receptor B1 (GABARB1) EU 90

Indication: neurological diseases.

Test principle: This test kit is designed exclusively for the in vitro determination of human anti-
bodies in serum or plasma. The determination can be performed qualitatively or quantitatively.
Combinations of substrates are incubated with diluted patient sample. If the reaction is positive,
specific antibodies of classes IgA, IgG and IgM attach to the antigens. In a second step, the
attached antibodies are stained with fluorescein-labelled anti-human antibodies and made visible
with a fluorescence microscope.
Contents of a test kit for 50 determinations: FA 1111-1005-1 (IgG)
Description Format Symbol
1. Slides, with a mosaic of BIOCHIPs
10 slides .SLIDE.
(specifications: see page 24)
2. Fluorescein-labelled anti-human IgAGM (goat), ready for use 1 x 1.5 ml .CONJUGATE.
3. Positive control: anti-Purkinje cell cytoplasm (Yo), ready for use 1 x 0,1 ml .POS CONTROL.
4. Positive control: anti-neurone nuclei: Hu-antigen, ready for use 1 x 0.1 ml .POS CONTROL.
5. Negative control: autoantibody-negative, ready for use 1 x 0.1 ml .NEG CONTROL.
6. Salt for PBS pH 7.2 2 packs .PBS.
7. Tween 20 2 x 2.0 ml .TWEEN 20.
8. Embedding medium, ready for use 1 x 3.0 ml .GLYCEROL.
9. Cover glasses (62 mm x 23 mm) 12 pieces .COVERGLASS.
10. Instruction booklet 1 booklet ---
.LOT. Lot description Storage temperature
.IVD. In vitro diagnostics Unopened usable until
Single slides (e.g., EUROIMMUN order no. FB 1111-1005-1) are provided together with cover glasses.
Additional positive control (e.g., order no. CA 1113-0101: Yo-ab control and CA 1116-0101: Hu-ab
control) and negative control (e.g., order no. CA 1000-0101) can be ordered.
Performance of the test requires reagent trays TRAY, which are not provided in the test kits. They
are available from EUROIMMUN under the following order no.
- ZZ 9999-0105 Reagent trays for slides containing up to 5 fields (9x7 mm)
- ZZ 9999-0110 Reagent trays for slides containing up to 10 fields (5x5 mm)

FA_1111-1_A_UK_C09.doc
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Performing the test (reaction fields 5 x 5 mm)

The TITERPLANE Technique was developed by EUROIMMUN in order to standardize
immunological analyses: Samples or labelled antibodies are applied to the reaction fields of a
reagent tray. The BIOCHIP Slides are then placed into the recesses of the reagent tray, where all
BIOCHIPs of the slide come into contact with the fluids, and the individual reactions commence
simultaneously. Position and height of the droplets are exactly defined by the geometry of the
system. As the fluids are confined to a closed space, there is no need to use a conventional
“humidity chamber”. It is possible to incubate any number of samples next to each other and
simultaneously under identical conditions.
Prepare: The preparation of the reagents and of the serum and plasma samples is
described on page 4 of this test instruction.
Pipette: Apply 30 µl of diluted sample to each reaction field of the reagent tray, avoiding
air bubbles. Transfer all samples to be tested before starting the incubation (up to
200 droplets). Use a polystyrene pipetting template.
Incubate: Start reactions by fitting the BIOCHIP Slides into the corresponding recesses of
the reagent tray. Ensure that each sample makes contact with its BIOCHIP and
that the individual samples do not come into contact with each other. Incubate for
30 min at room temperature (+18°C to +25°C).
Wash: Rinse the BIOCHIP Slides with a flush of PBS-Tween using a beaker and immerse
them immediately afterwards in a cuvette containing PBS-Tween for at least 5
min. Shake with a rotary shaker if available. Wash max. 16 slides then replace
PBS-Tween with new buffer.
Pipette: Apply 25 µl of fluorescein labelled anti-human globulin to each reaction field of
a clean reagent tray. Add all droplets before continuing incubation. Use a stepper
pipette. The labelled anti-human serum should be mixed before use. To save time,
conjugate can be pipetted onto separate reagent trays during the incubation with
the diluted sample.
Incubate: Remove one BIOCHIP Slide from cuvette. Within five seconds blot only the back
and the long sides with a paper towel and immediately put the BIOCHIP Slide into
the recesses of the reagent tray. Do not dry the areas between the reaction fields.
Check for correct contact between the BIOCHIPs and liquids. Then continue with
the next BIOCHIP Slide. From now on, protect the slides from direct sunlight.
Incubate for 30 min at room temperature (+18°C to +25°C).
Wash: Fill cuvette with new PBS-Tween. Rinse the BIOCHIP Slides with a flush of PBS-
Tween using a beaker and put them into the cuvette filled with the new PBS-
Tween for at least 5 min. Shake with a rotary shaker if available. 10 drops of
Evans Blue for each 150 ml phosphate buffer can be added for counterstaining.
Wash max. 16 slides then replace PBS-Tween with new buffer.
Embed: Place embedding medium onto a cover glass - drops of max. 10 µl per reaction
field. Use a polystyrene embedding template. Remove one BIOCHIP Slide from
PBS Tween and dry the back, all four sides, as well as the surface around, but not
between the reaction fields with a paper towel. Put the BIOCHIP Slide, with the
BIOCHIPs facing downwards, onto the prepared cover glass. Check immediately
that the cover glass is properly fitted into the recesses of the slide. Correct the
position if necessary.
Evaluate: Read the fluorescence with the microscope.
General recommendation: Objective 20x (tissue sections, infected and transfected
cells), 40x (cell substrates).
Excitation filter: 488 nm, color separator: 510 nm, blocking filter: 520 nm.
Light source: mercury vapor lamp, 100 W, EUROIMMUN LED, EUROStar
Bluelight.

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BIOCHIP slide
TITERPLANE Technique
BIOCHIPs reagent tray

diluted samples
Pipette: 30 µl per field

Incubate: 30 min

Wash: 1 s flush
5 min cuvette PBS-Tween

labelled antibody
Pipette: 25 µl per field

Incubate: 30 min

Wash: 1 s flush
5 min cuvette PBS-Tween

Embed: max. 10 µl per field embedding medium

cover glass

20 x
40 x
Evaluate: fluorescence microscopy

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Preparation and stability of reagents

Note: After initial opening, the reagents are stable until the expiry date when stored between +2°C
and 8°C and protected from contamination, unless stated otherwise below.

- Slides: Ready for use. Remove the protective cover only when the slides have reached room
temperature (+18°C to +25°C) (condensed water can damage the substrate). Mark with a felt-
tip pen. Do not touch the BIOCHIPs. After the protective cover has been opened, the slide
should be incubated within 15 minutes. If the protective cover is damaged, the slide must not
be used for diagnostics.

- Fluorescein-labelled secondary antibody (FITC): Ready for use. Before using for the first
time, mix thoroughly. The conjugate is sensitive to light. Protect from sunlight .

- Positive and negative controls: Ready for use. Before using for the first time, mix them
thoroughly.

- PBS-Tween: 1 pack of “Salt for PBS” should be dissolved in 1 liter of distilled water (optimal:
aqua pro infusione, aqua ad injectabilia) and mixed with 2 ml of Tween 20 (stir for 20 min until
homogeneous). The prepared PBS-Tween can be stored at +2°C to +8°C, generally for 1
week. PBS-Tween should not be used if the solution becomes cloudy or contamination
appears.

- Embedding medium: Ready for use.

- Reagent trays: Reaction fields of the reagent tray must be hydrophilic and surrounding area
hydrophobic. If necessary, wipe with Extran MA 01 (Merck) and rinse generously with water. To
disinfect: Immerse in Sekusept Extra (Henkel) (3% in water) for 1 hour. After disinfection rinse
generously with water and dry with absorbent paper.

Storage and stability: The slides and the reagents should be stored at a temperature between
+2°C and +8°C. Stability is guaranteed for 18 months after the date of manufacture if stored
properly.

Waste disposal: Patient samples, controls and slides are to be handled as potentially infectious
materials. All reagents are to be disposed in accordance with official disposal regulations.

Warning: The BIOCHIPs coated with antigen substrates have been treated with a disinfecting
fixing agent. Neither HBsAg nor antibodies against HIV-1, HIV-2, and HCV could be detected in the
control sera using appropriate ELISA or indirect immunofluorescence tests. Nevertheless, all test
system components should be handled as potentially infectious materials. Some of the
reagents also contain the toxic agent sodium azide. Avoid skin contact.

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Preparation and stability of samples

Samples: Human sera or EDTA, heparin or citrate plasma, CSF.

Stability: The patient samples to be investigated can generally be stored up to 14 days at a

temperature between +2°C and 8°C. Diluted samples must be incubated within one working day.

Recommended sample dilution for qualitative evaluation: The sample to be investigated is

diluted 1:10 in PBS-Tween. For example, dilute 11.1 µl sample in 100 µl PBS-Tween and mix
thoroughly, e.g. vortex for 4 seconds. EUROIMMUN recommends incubating samples from a
dilution of 1:10 and 1:100. CSF samples are used undiluted.

Recommended sample dilution for quantitative evaluation: The dilution of samples to be

investigated is performed using PBS-Tween. For each add 100 µl of PBS-Tween to the tube and
mix with 11.1 µl of the next highest concentration, e.g. vortex for 2 seconds. EUROIMMUN
recommends incubating samples from a starting dilution of 1:10, and analysing CSF samples
undiluted.

Dilution Dilution scheme

1:10 100 µl PBS-Tween + 11.1 µl undiluted sample

11.1 µl
After every two
1:100 100 µl PBS-Tween + 11.1 µl 1:10 diluted sample dilution steps, a new
pipette tip should be
11.1 µl used to prevent
carryover.
1:1000 100 µl PBS-Tween + 11.1 µl 1:100 diluted sample
...

...

Test evaluation
Fluorescence pattern (positive reaction): Different autoantibodies against neural structures
bind to the primate brain tissue. Antibodies against grey matter react with the stratum granulosum
and in a weaker form with the stratum moleculare of the cerebellum.

Antibodies against white matter react with the lamina alba of the cerebellum and of the cerebrum.
These serum antibodies correspond essentially with antibodies against myelin.

Antibodies against myelin react with the myelin sheath of the axons. They appear as fluorescing
hyaline cylinders on tissue sections of peripheral nerve, while a “drop-like”, ring-shaped
fluorescence pattern is observed on cross sections of nerves.

Antibodies against myelin-associated glycoprotein (MAG), a cell-membrane protein of the

myelin sheath, show a streaky shaped fluorescence pattern on nerve tissue and a mostly fine-
granular ring-shaped fluorescence on cross sections of nerves. In the cerebellum tissue
predominantly the whole surface of the white substance is stained.

Antibodies against MAG are frequently of the immunoglobulin class M. Therefore, for
comprehensive diagnostics, incubation with class IgM conjugate is essential.

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Autoantibodies against the enzyme glutamate decarboxylase (GAD) show a (stronger)

fluorescence similar to "leopard skin" in the granular layer of cerebellum tissue. The molecular
layer shows a weaker, even fluorescence, excluding the cell nucleus. GAD antibodies in higher
titers react with the endocrine part of the pancreas tissue and manifest themselves through a
smooth to granular cytoplasmic fluorescence of the islet cells.

Autoantibodies against Yo (PCA-1), PCA-2 or Tr give rise to a cytoplasmic fluorescence of the

Purkinje cells. While with Yo antibodies only the cytoplasm of the Purkinje cells and some cells in
the hilum of the hippocampus is stained, the fluorescence obtained with PCA-2 antibodies extends
into the dendrites. Antibodies against Tr show a fine fluorescence of the Purkinje cell cytoplasm
and a dot-like staining of the molecular layer.

If antibodies against Hu (ANNA-1) or Ri (ANNA-2) are present, all neurone nuclei of the
cerebellum and hippocampus show a granular fluorescence. While antibodies against HU stain the
cell nuclei of the plexus myentericus on frozen sections of primate intestine, antibodies against Ri
do not show a reaction.

Antibodies against neurofilaments show a fibrous structure in the stratum granulosum of the
cerebellum and, in the case of high titers, also in the stratum moleculare.

Furthermore, antibodies against neuroendothelia bind to their corresponding antigens in the

whole of the cerebellum and cerebrum.

Autoantibodies against non-medullated nerves react in different organs with autonomous nerve
segments. In the intestine antigens are detected in the myenteric plexuses and in the submucosal.

Antibodies against Ma (Ma1, Ma2/Ta) react with the nucleoli of the nerve cells in the cerebellum,
cerebrum and hippocampus.

Antibodies against CV2 show a sand-like fluorescence in the cerebellum, which is best visible in
the stratum moleculare.

Antibodies against amphiphysin show a fluorescence in the presynaptic nerve ends of the
cerebellum. The nerve dendrites of the stratum moleculare show a more intense fluorescence than
those of the stratum granulosum. The fluorescence pattern of the stratum granulosum resembles
the pattern found with antibodies against GAD.

Anti-glial nuclear antibodies (AGNA)/anti-SOX1 react with the cell nuclei of the Bergmann glia in
the Purkinje cell layer of the cerebellum.

Antibodies against GFAP (glial fibrillary protein) react with the astrocytes of the cerebellum.
Longitudinally cut peripheral nerves show a streaky shaped fluorescence.

Antibodies against synaptophysin show a fluorescence of the presynaptic nerve ends on the
substrates intestine and cerebellum.

Antibodies against aquaporin-4 (NMO-IgG) react specifically with the corresponding transfected
cells. In cytoplasm they form a flat, smooth, fine granular fluorescence, the cell nucleus is only
slightly stained. On the tissue substrates cerebellum and optic nerve the small cerebral vessels of
the pia, subpia and Virchow-Robin space fluoresce along the small arterioles.

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Antibodies against glutamate receptor (type NMDA) react specifically with the corresponding
transfected cells of the test substrate and cause a cytoplasmic fluorescence, with the cell nuclei
being only weakly stained. On neurological tissue sections fluorescence is observed in the stratum
moleculare of the hippocampus (neuropil staining) and in the neuronal nuclei of the stratum
granulosum of the cerebellum.
Autoantibodies against AMPA receptors 1 und 2 (GluR1/GluR2) react with the stratum
moleculare of hippocampus (neuropil staining) and the hilum of the hippocampus, while the
stratum granulosum does not show a reaction. Tissue sections of the cerebellum show a fine-
granular fluorescence of the stratum moleculare. Antibodies against AMPA receptors react
specifically with the cytoplasm of the corresponding transfected cells.

Autoantibodies against contactin-associated protein 2 (CASPR2) react specifically with the

transfected cells of the test substrate. They induce a cytoplasmic fluorescence, with some
fluorescence of the cell membrane, while the cell nuclei are only slightly stained. On neurological
tissue sections fluorescence is observed in the stratum moleculare of the hippocampus (neuropil
staining) and the cerebellum.
Antibodies directed against leucine-rich glioma-inactivated protein 1 (LGI1) react specifically
with the cytoplasm of the corresponding transfected cells. On frozen sections of hippocampus
(neuropil staining) and cerebellum a fine-granular fluorescence is observed in the stratum
moleculare. There is additionally a blotchy fluorescence in the stratum granulosum of the
cerebellum. On hippocampus the outer molecular layer shows a stronger reaction than the inner.

Autoantibodies against GABA receptors B1 (GABARB1) show a course granular fluorescence in

the stratum moleculare of the hippocampus (neuropil staining) and the cerebellum. There is
additionally a blotchy fluorescence in the stratum granulosum of the cerebellum. On specifically
transfected cells, antibodies against GABA receptors B1 show a fine-granular to smooth
cytoplasmic fluorescence.

If the nucleus or the cytoplasm of all cells on neural tissue fluoresce, the comparison of the
fluorescence pattern with intestinal or liver tissue and HEp-2 cells helps differentiate other
autoanibodies without neural specificity. If the cell nuclei or the cytoplasm of all cells are stained,
antinuclear antibodies or antibodies against mitochondria and other cell antigens are present.
If the positive control shows no specific fluorescence pattern or the negative control shows a clear
specific fluorescence, the results are not to be used and the test is to be repeated.
A large range of fluorescence images can be found on the EUROIMMUN website
(www.euroimmun.com)

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Qualitative evaluation:
Anti-cerebellum, Evaluation
-cerebrum, (Clinical significance see page 12)
-hippocampus,
-spinal cord reactivity
No reaction at 1:10 Negative. No antibodies against neuronal antigens detected in the
patient sample.
Positive reaction at 1:10 Positive. For IF types MAG GAD, Yo, PCA-2, Tr, Hu, Ri, Ma, CV2,
Amphiphysin, AGNA, GFAP, Synaptophysin, AQP-4, NMDA, AMPA,
LGI1, GABARB1: If corresponding symptoms are present, indication
of different neurological diseases. Antibodies against GAD:
indication of diabetes mellitus type I.
Positive reaction at 1:100 Positive. For IF types neuroendothelium, neurofilaments: unspecific
result.For IF type myelin: If corresponding symptoms are present,
indication of different neurological diseases (multiple sclerosis),
diagnostic value questionable.

Anti-peripheral nerves Evaluation

reactivity
No reaction at 1:10 Negative. No antibodies against peripheral nerves are detectable in
the patient sample.
Positive reaction at 1:10 Positive. For IF type MAG: If corresponding symptoms are present,
indication of different neurological diseases (paraproteinaemic
neuropathy).
Positive reaction at 1:100 Positive. For IF type myelin: If corresponding symptoms are present,
indication of different neurological diseases (multiple sclerosis),
diagnostic value questionable.

Anti-non-medullated Evaluation
nerves reactivity
No reaction at 1:10 Negative. No antibodies against peripheral nerves detected in the
patient sample.
Positive reaction at 1:10 Positive. For IF types non-medullated nerves, Hu: If corresponding
symptoms are present, indication of different neurological diseases.

Anti-optic nerve reactivity Evaluation

No reaction at 1:10 Negative. No antibodies against optic nerve detected in the patient
sample.
Positive reaction at 1:10 Positive. Indication of NMO or NMO spectrum diseases (LETM,
NETM or rON).

Anti-islet cell reactivity Evaluation

No reaction at 1:10 Negative. No antibodies against islet cells of the pancreas detected
in the patient sample.
Positive reaction at 1:10 Positive. Indication of insulin-dependent diabetes mellitus; with
simultaneous specific fluorescence of grey matter, indication of Stiff
person syndrome.

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Anti-transfected cells Evaluation

reactivity
No reaction at 1:10 Negative. No antibodies against antigens of specifically transfected
cells detected in the patient sample.
Positive reaction at 1:10: Positive. indication of NMO or NMO spectrum diseases (LETM,
AQP-4 NETM or rON).
NMDAR Positive. Indication of NMDAR encephalitis.
AMPA 1 and 2 Positive. Indication of limbic encephalitis
CASPR2 Positive. Indication of limbic encephalitis, neuromyotonia and
Morvan´s syndrome.
LGI1 Positive. Indication of limbic encephalitis
GABARB1 Positive. Indication of limbic encephalitis
The HEp-2 cells on the Mosaic FA 1111-####-16 serve only for the differentiation of anti-nuclear
antibodies. An ANA-positive result with a titer of 1: < 100 should not be evaluated.

Qualitative evaluation of CSF samples:

The normal value for neurological autoantibodies in CSF is negative. A positive reaction in the
undiluted sample should be evaluated as positive. Positive detection is not an indication of
intrathecal synthesis. This can be determined by calculating the CSF-serum quotient using a
quantitative test system (ELISA).

Quantitative evaluation: The titer is defined as the sample dilution factor for which specific
fluorescence is just identifiable. This should be compared with the reaction obtained using an
equivalently diluted negative serum.

Antibody titers can be determined according to the following table from the fluorescence of the
different sample dilutions.

Fluorescence at
antibody titer
1:10 1:100 1:1000 1:10000
weak negative negative negative 1:10
moderate negative negative negative 1:32
strong weak negative negative 1:100
strong moderate negative negative 1:320
strong strong weak negative 1:1000
strong strong moderate negative 1:3200
strong strong strong weak 1:10000
...

...

...

...

...

For diagnosis the clinical symptoms of the patient should always be taken into account along with
the serological results.

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Test characteristics
Antigens: For the determination of various neuronal autoantibodies by indirect
immunofluorescence, primate cerebellum, primate nerves, rat cerebellum and rat
hippocampus are used as standard substrate.
To supplement the spectrum of neural antigens primate cerebrum, primate spinal marrow,
pons, temporal lobes and optic nerve are used.
The parallel use of primate intestine permits the secure differentiation of various antibodies with
neural specificity from other autoantibodies (e.g. Hu antibodies to Ri antibodies and ANA,
antibodies against neuroendothelia to vascular endothelia).
The use of a substrate combination of primate cerebellum and primate liver or HEp-2 cells
permits the secure differentiation of various antibodies with neuronal specificity from other
autoantibodies, especially antinuclear antibodies (ANA) in the same test procedure

For the detection of autoantibodies against islet cells in diabetes mellitus (type I: direct-line
relatives; metabolic state during pregnancy) by indirect immunofluorescence, primate pancreas is
used as standard substrate. Combined with primate cerebellum GAD antibodies can be detected,
but compared to RIA and ELISA with decreased sensitivity.

Specific transfected cells are used as standard substrates for the monospecific detection of
neuronal antibodies by indirect immunofluorescence. EUROIMMUN provides a large range of
transfected cell test substrates, for example, for the detection of antibodies against aquaporin-4
(AQP-4), glutamate receptors (type NMDA), AMPA receptors 1 and 2 (GluR1/GluR2), contactin-
associated protein 2 (CASPR2), leucine-rich glioma-inactivated protein 1 (LGI1) and GABA
receptors B1 (GABARB1). Use of these substrates in combination with neuronal tissue, e.g.
cerebellum and hippocampus, enables comprehensive diagnostics for neurological diseases.

Stability: Stability is guaranteed for 18 months after the date of manufacture if stored properly.

Measurement range: The dilution starting point for this measurement system is 1:100. Samples
can be further diluted by a factor of 10 so that the dilution series is 1:100, 1:1000, 1:10000 etc.
There is no upper limit to the measurement range.

Intra-assay reproducibility: Ten determinations for each of two characterized samples were
incubated in parallel. In quantitative evaluation of results, the deviation amounted to no more than
± 1 fluorescence intensity level for all samples. The intensity of the specific fluorescence as a
numeric value is called fluorescence intensity level by EUROIMMUN. These values can reach from
“0“ (no specific fluorescence) to “5“ (extremely strong specific fluorescence).
Inter-assay reproducibility: Two characterised samples were incubated in duplicate on at least 2
different days in 5 test runs. In quantitative evaluation of results, the deviation amounted to no
more than ± 1 fluorescence intensity level for all samples.
Inter-lot reproducibility: Two characterised samples were incubated with slides from three
different lots. In quantitative evaluation of results, the deviation amounted to no more than ± 1
fluorescence intensity level for all samples.
Cross reactivity: There is no data known to EUROIMMUN in which cross reactivities are
described. Neurone-specific cell nuclear antibodies (ANNA) cannot be reliably differentiated from
other cell nuclear antibodies (ANA) using sections of brain alone.
Interference: Hemolytic, lipemic and icteric samples had no influence on analysis results.
Reference range: titer 1: < 10
titer 1: < 100 (anti-myelin, anti-neuroendothelium, anti-neurofilaments )

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Specificity and sensitivity:

Substrate Ig Reference Specificity Sensitivity

class (number and origin of samples)
Panel of healthy blood donors
Nerves (Monkey): (n = 35, origin: Germany)
IgG 94 % 100 %
Anti-Myelin IIFT: Cerebellum (Monkey),Cerebrum (Monkey)
(n = 12, origin: Germany)
Panel of healthy blood donors
Nerves (Monkey): IgAGM, (n = 200, origin: Germany)
100 % 100 %
Anti-MAG IgG Anti-MAG Immunoblot (n = 6, origin: Europe)
monoclonal antibody ab: anti-MAG
Cerebellum reference centers (n = 34, origin: Europe),
(Monkey): IgAGM Anti-Neuronal Westernblot 100 % 100 %
Anti-Yo, -Hu, -Ri (n = 11, origin: Germany)
Cerebellum Anti-GAD RadioImmunoAssay
IgAGM 96 %* 97 %*
(Monkey) GAD (n = 89, origin: Germany)
Intestine (Monkey): reference centers (n = 34, origin: Europe),
Anti-non-medullated IgAGM Anti-Neuronal Westernblot 100 % 100 %
nerves (n = 7, origin: Germany)
Panel of healthy blood donors
Cerebrum
IgAGM, (n = 200, origin: Germany)
(Monkey): Anti- 100 % 83 %
IgG Anti-MAG Immunoblot (n = 6, origin: Europe),
MAG
monoclonal antibody ab: anti-MAG
Panel of healthy blood donors
Cerebrum
IgAGM, (n = 200, origin: Germany)
(Monkey): 100 % 100 %
IgG Anti-Neuronal Westernblot
anti-Hu, anti-Ri)
(n = 6, origin: Germany)
Panel of healthy blood donors
Cerebrum
(n = 34, origin: Germany)
(Monkey): IgG 97 % 100 %
IIFT: Nerves (Monkey)
Anti-myelin
(n = 12, origin: Germany)
Panel of healthy blood donors
Spinal cord
IgAGM, (n = 200, origin: Germany)
(Monkey): 100 % 67 %
IgG Anti-MAG-Immunoblot
Anti-MAG
(n = 6, origin: Europe)
Panel of healthy blood donors
Spinal cord
(n = 34, origin: Germany)
(Monkey): IgG 97 % 100 %
IIFT: Nerves (Monkey)
Anti-myelin
(n = 12, origin: Germany)
Panel of healthy blood donors
Spinal cord
IgAGM, (n = 200, origin: Germany)
(Monkey): 100 % 100 %
IgG Anti-Neuronal Westernblot
Anti-Hu, anti-Ri
(n = 6, origin: Germany)
Panel of healthy blood donors
Spinal cord IgAGM, (n = 200, origin: Germany)
100 % 100 %
(Monkey): Anti-Yo IgG Anti-Neuronal Westernblot (n = 2, origin: Germany)
reference centers (n = 5, origin: Germany)
Pancreas (Monkey): reference centers
IgG 100 %* 100 %*
ICA (n = 65, origin: Germany)
* Incubation time during the first incubation step: 18 hrs

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Clinical specificity and sensitivity:

Antibodies Ig Sample characterisation Prevalence
n=
substrate class Clinical patient cohort Positive %
NMO 32 25 78%
Longitudinal extensive transverse myelitis 12 8 67%
Recurrent opticus neuritis 5 1 20%
Recurrent opticus neuritis/
2 2 100 %
Anti-AQP-4 IgG Non-extensive transverse myelitis
Transfected cells Total 51 36 71%
Multiple sclerosis 66 0 0%
Other neurological diseases 23 0 0%
Healthy blood donors 100 0 0%
Total 189 0 0%
Anti-NMDAR encephalitis 39 39 100 %
Anti-NMDA
IgG Other encephalitides 31 0 0%
Transfected cells
Healthy blood donors 100 0 0%
Anti-NMDAR encephalitis 39 39 100 %
Anti-NMDA
IgG Other encephalitides 31 7 23 %
Rat hippocampus
Healthy blood donors 100 0 0%
Anti-NMDAR encephalitis 39 0 100 %
Anti-NMDA
IgG Other encephalitides 31 7 23 %
Rat cerebellum
Healthy blood donors 100 0 0%
Anti-CASPR2 Limbische Enzephalitis 9 9 100 %
IgG
Transfected cells Healthy blood donors 150 1 0,7 %
Anti-GABARB1 Limbische Enzephalitis 5 5 100 %
IgG
Transfected cells Healthy blood donors 150 0 0%

Clinical significance
The inflammatory autoimmune disease neuromyelitis optica (NMO, opticospinal
encephalomyelitis, Delvic’s syndrome) is a rare form (around 1%) of the group of acquired
demyelinating diseases of the central nervous system (CNS) with degradation of the insulating
sheath of at least one optical nerve (neuritis nervi optici) and at the same time or a few months
later the spinal cord (myelitis) [1, 2, 3, 4, 5, 6, 7]. NMO was first described in 1870 by Clifford Albut.
In 1894 it was systemised and categorised as a neuro-optic-myelitic syndrome by Eugène Devic
and his student Fernand Gault [4].
Highly specific serum autoantibody markers are found very frequently in NMO, while they are not
detected in multiple sclerosis (MS) patients or in healthy subjects. Antibodies known as NMO-IgG
from their initial description cause a characteristic colouring of the Virchow-Robin’s space along the
small arterioles in the grey and white matter in immunofluorescence (IIFT) on CNS tissue [8, 9].
The protein aquaporin-4 (AQP4) was later identified as the target antigen [10]. AQP4 is a water-
channel protein which is involved in the regulation of water and electrolyte balance in the CNS. It is
expressed in astrocytes, predominantly in the region of the glial endfeet [1, 7, 11, 12]. Interestingly,
these water channels occur with particularly high frequency in sections of the CNS that are
typically attacked in NMO, namely the optic nerves and the spinal cord. Autoantibodies against
AQP4 are produced in peripheral plasma cells and, after binding to their target antigen in the CNS,
cause activation of complement with local inflammatory demyelination and necrosis. The disease
pattern is neuritis nervi optici and local myelitis over 3 or more spinal column segments with
localisation predominantly at or in the blood-brain barrier region [9, 10, 12].
NMO was previously considered a localised form of MS. With today’s knowledge it is known to be
a separate disease with respect to its pathogenesis. In contrast to MS, which is still considered as
a predominantly T-cell-mediated disease, it appears that humoral mechanisms are responsible for
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the development of NMO [14]. Indications for this include, in addition to the detection of
autoantibodies, histopathological features (perivascular antibody and complement deposition) and
the favourable response to therapy strategies based on removal of immunoglobulin from serum
and reduction of B lymphocytes (plasmapheresis, monoclonal anti-CD20 antibody Rituximab) [15,
16]. The disease symptoms of NMO are, on the one hand, acute visual disorders up to blindness
(amaurosis) in one or both eyes which develop within hours or days, and on the other hand,
symptoms of paraplegic syndrome, sometimes with advancing severity, sensitivity disorders,
muscle weakness or paralysis of the extremities and loss of control of intestines and bladder,
which develop acutely or within 1 to 14 days. Histologically, demyelinating lesions (similar to MS)
are found, which can recover well. However, damage often remains because of tissue destruction
(necrosis) [2, 4, 7].
In contrast to MS, NMO is much more frequent in Asians and dark-skinned people than in whites.
Therefore, in Asian and African countries the prevalence of the disease far exceeds that of classic
MS, while in Europe and North America it is very rare [3]. An “Asian” and a “Western” form of NMO
are differentiated on the basis of distinct genetic features [1, 3]. The average age of onset is 35
years (5-55 years), whereby there is no difference in the disease course between adults and
children [17]. Women are more frequently affected than men (1.5:1 to 9:1 depending on the
author). Without adequate therapy half of patients become blind in one or both eyes or cannot walk
without supports within 5 years [1]. Prognosis depends on the number and severity of flare-ups
during the first two years. The 5-year survival rate is given as 70%, with the cause of death usually
being neuropathic breathing insufficiency.
Diagnosis is made mostly clinically with anamnesis, neurological and neurophysiological tests
(EMG, NLG, evoked potential), MRT of the CNS and lumbar puncture [12]. In CSF there is
sometimes strong pleocytosis with polymorphonuclear and mononuclear cells; oligoclonal IgG
bands are only detectable in 15-30% of cases (in MS in 90%) [3].
The investigation of autoantibodies against AQP4 in the diagnostic laboratory secures a diagnosis
of NMO [12, 17, 18]. Moreover, it could be shown that these antibodies are also detected in a
subgroup of patients with isolated longitudinal transverse myelitis over 3 or more segments (LETM)
and in patients with isolated recurring opticus neuritis (ON) [11, 19]. Because of the repeatedly
demonstrated high specificity of NMO-IgG and AQP4 antibodies for NMO it is assumed that
seropositive LETM and ON cases are an incomplete form of NMO, with consequences for
prognosis and therapy [11, 19]. Determination of AQP4 autoantibodies is possible with
radioimmunoprecipitation assay (RIPA). However, the low sensitivity of RIPA of 56% carries the
risk of false-negative results [20]. Indirect immunofluorescence, in particular IIFT Mosaics, offers a
simple and state-of-the-art method to detect autoantibodies with high specificity and sensitivity,
while avoiding the use of radioactive materials [21, 22]. In the EUROIMMUN Neurology Mosaic
antibodies against AQP4 are detected with a sensitivity of 80% and a specificity of 100% using a
cell line which has been molecular biologically modified to produce large quantities of AQP4 [23].
With the tissue substrates cerebellum, cerebrum and optic nerve, the specific autoantibodies can
be identified additionally in the same test run on the basis of their characteristic pattern (earlier
description: NMO-IgG). At the same time any possible further autoantibodies in the patient serum
are also detected (e.g. ANA, paraneoplastic antibodies, anti-MAG antibodies, etc). Even
reactivities against neuronal and glial antigens of as yet unknown specificity are detected. NMO-
IgG may be associated with other autoantibodies, such as those against the cell nucleus (ANA),
SS-A (Ro) or thyroperoxidase (TPO). This suggests that NMO can occur together with autoimmune
diseases such as systemic lupus erythematosus (SLE), Sjögren's syndrome, etc [1, 24].
The Neurology Mosaic 17 provides, for the first time, an easy standardised method to detect
antibodies against AQP4 (synonym: NMO-IgG) in any laboratory that knows how to perform IFT.
The major significance of the antibody detection is that antibodies against AQP4 (NMO-IgG) allow
serological differentiation of prognostically poor NMO from classic MS, which can influence therapy
decisions [9, 10, 11, 19]. While MS is treated with immune-modulating substances such as beta-
interferon (IFN- ) and glatiramer acetate, NMO requires the use of immunosuppressive drugs such
as azathioprine, cyclophosphamide and the monocloncal anti-CD20 antibody Rituximab which is
directed against B-cells [6, 15, 16, 20].

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Antibodies against myelin are present in multiple sclerosis (MS) and other neurological diseases
[25, 26, 27]. The diagnostic value of antibodies in serum is controversial, as high titers in healthy
persons can also be detected [28, 29, 30].
Anti-MAG autoantibodies (MAG = myelin associated glycoprotein) could be determined in half of
all patients with IgM gammopathic associated neuropathy [31, 32].
In Guillain-Barré syndrome autoantibodies against MAG can also sometimes be detected. The
disease is characterized by multifocal inflammation accompanied by cell infiltration into the myelin
sheath of peripheral nerves and spinal ganglia. Clinical symptoms include sensibility and mobility
dysfunction beginning with a decrease in reflexes in the legs. In the course of the disease
symptoms from paralysis to tetraplegia and respiratory depression develop [33].
Autoantibodies against Hu, Ri, Yo:
Two thirds of the patients with PND = paraneoplastic neurologic disorders show autoantibodies
against onconeuronal antigens in serum or in spinal fluid. These test results are often the first sign
of an underlying tumor. It not only proves the paraneoplastic etiology, but also alleviates the search
for tumors due to the association with definite tumor types [34, 35, 36, 37, 38]. Immunomodulatory
therapy and prognosis are tumor and syndrome dependent [38, 49, 50].
Hu-antibodies can indicate the first signs of an underlying tumor. Antibodies against Hu should be
examined in patients with nonspecific neuropathies, in particular with sensory neuropathies and
encephalitis with emphasis on the brainstem, cerebellum and the limbic system (in reference to
differential diagnostics see also: autoantibodies against onconeural antigens) [20, 42, 43]. The
most common tumors associated with Hu antibodies are: small cell lung cancer (SCLC),
neuroblastoma, prostate carcinoma [34, 44, 45].
Autoantibodies against onconeural Ri proteins (NOVA-1 and NOVA-2) have been described in
opsoclonus myoclonus syndrome in connection with a gynecological tumor, predominantly with
mamma carcinomas [46, 47]. Anti-Ri antibodies can indicate the first signs of an underlying tumor
(see autoantibodies against onconeural antigens) [48]. The most common tumors associated with
Ri antibodies: small cell lung cancer (SCLC) and mamma carcinoma [49, 50].
Anti-Ri antibodies in connection with a paraneoplastic syndrome can be observed in patients that
have a developing fast growing brainstem tumor. For breast and lung cancer must also be
examined [46].
The rare autoantibodies against Yo show cytoplasmic immune reactions, primarily against
cerebellar Purkinje cells and exhibit a symptomatic (paraneoplastic) cerebellar syndrome [39].
Normally the antibodies are associated with particular tumors, most often with ovary, mamma and
uterus carcinoma; they have also been found in prostate carcinomas or adeno-carcinomas of the
esophagus as well as in Hodgkin’s lymphoma with antibodies against Purkinje cells, that
degenerate after a successful lymphoma treatment [51]. In many cases cerebellar syndrome
clinically precedes the tumor, the determined antibodies against Yo indicates cause for further
examination [52].
Autoantibodies against islet cells react with the endocrine layer of the pancreas tissue,
autoantibodies against acinus cells with the exocrine layer of the pancreas. Autoantibodies against
glutamate decarboxylase (GAD) can react with the endocrine layer of the pancreas tissue in high
titers [53, 54, 55, 56]. The determination of GAD antibodies plays an important diagnostic role
amongst others in Stiff Man syndrome, a disease with progradient muscle rigidity and secondary
effects to almost all extremities as well as with progressive encephalomyelitis with rigidity and
myoclonus (PERM) [57, 58].
The determination of autoantibodies against antigens of islet cells serves on the one hand to
securely diagnose type I diabetes mellitus, and on the other hand to reveal preclinical autoimmune
reactions in persons at risk [59, 60, 61, 62, 63, 64].

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Juvenile diabetes mellitus (type I) manifests itself predominantly before the age of 35 years; in
some cases already in infancy [65]. In particular first-degree relatives of diabetes mellitus patients
are at risk (risk rate 2 to 7 %), also women, during pregnancy can develop a diabetic metabolism
imbalance [66].
What is of particular importance is that in 90% of all cases one or several diabetes mellitus
associated autoantibodies can already be detected in serum before the time of clinical
manifestation [59, 60]. The number of autoantibodies against islet cells is proportional to the risk of
developing type I diabetes [67]. Early detection enables identification of persons at high risk of
acquiring the disease [34, 67]. Prevention of disease outbreak can in some cases be achieved by
implementing appropriate intervention such as regulating glucose levels at low levels or with
immunosuppressive treatments [66].
Emphasis is to be put on the observation that paradoxically the antibody titer levels decrease as
the disease progresses [66].
The clinical symptom of the latent syndrome is characterized by microangiopathies (diabetic
arteriosclerosis). Additional complications such as polyendocrinopathies, neuropathies,
retinopathies, diabetic glomerulosclerosis and gangrene can occur. These secondary illnesses are
responsible for life expectancy reduction. [68].

Anti-Glutamate receptor (type NMDA)

Antibodies against glutamate receptor (type NMDA) are specific markers for anti-glutamate
receptor (type NMDA) encephalitis, an inflammatory encephalopathic autoimmune disease which
was first described in 2007 and is currently still widely underdiagnosed [69].
NMDA receptors belong to the ionotropic glutamate receptors and were named according to their
ability to be activated by the synthetic amino acid N-methyl-D-aspartate (NMDA) [70]. They are
localised in the post-synaptic membrane and form cation canals with major significance for
synaptic transmission and plasticity [71, 72]. The receptors consist of two subunits, NR1 and NR2.
Their activity is regulated by the binding of ligands, such as the neurotransmitter glutamate. In the
serum and cerebrospinal fluid (CSF) of patients with anti-glutamate receptor (type NMDA)
encephalitis are autoantibodies directed against an extracellular epitope of the NR1 subunit. These
can be determined by immunohistochemical detection procedures or recombinant assays [37].
The occurrence of these specific antibodies and the possibility of immunotherapeutic intervention
suggest an immune-mediated pathogenesis for anti-glutamate receptor (type NMDA) encephalitis
[69, 73, 74, 75, 76]. In cell culture experiments on hippocampal neurons it could be demonstrated
that the binding of the antibodies induced a reversible reduction in glutamate receptors (type
NMDA) on the neuronal cell surface [73].
Furthermore, a pharmacological blockade of the receptors with NMDA antagonists causes clinical
symptoms similar to those of anti-glutamate receptor (type NMDA) encephalitis, in particular
psychosis [77, 78].
For anti-glutamate receptor (type NMDA) encephalitis a virtually stereotypical clinical course
occurring in phases is typical. In 100% of affected persons a flu-like prodromal phase (subfebrile
temperature, headache, fatigue) is followed by a psychotic stage with severe behavioural and
personality changes, delusions, disturbed thoughts and hallucinations. Because of these features a
large proportion of patients end up in psychiatric therapy, and in many cases a drug-induced
psychosis is initially diagnosed. In the following phase consciousness disorders, hypoventilation,
epileptic attacks, autonomous instability and dyskinesia develop. Due to the severity of this disease
(coma, status epilepticus, etc.) affected individuals must often be treated in intensive stations for
long periods of time [69, 73, 75, 76, 79, 80, 81, 82, 83, 84].
About half of patients show irregularities in cerebral MRT. The EEG is pathologically altered in over
90% of persons with the disease. Investigation of CSF reveals mild lymphocytic pleocytosis in 90%
of cases, intrathecal protein increase in 33% and oligoclonal bands in 25% [69, 73]. In the majority
of mostly young female patients ovarian tumours (teratoma) are found, which amongst other things
contain nerve cells. These cases involve a paraneoplastic syndrome (PNS) in anti-glutamate
receptor (type NMDA) encephalitis [69, 73, 76, 80, 84, 85, 86, 22]. The probability of an associated
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tumour disease is, on average, around 60%, although this is dependent on age and gender. Anti-
glutamate receptor (type NMDA) encephalitis is increasingly diagnosed not just in young women,
but also in older patients, in women without teratoma, in men (some with teratoma of the testis)
and in children [73, 81, 85].
Prognosis for patients is improved with appropriate immunomodulatory therapy, and, in PNS,
tumour detection and resection as early as possible. In around 75% of cases a substantial
regression of symptoms can be achieved [73, 84]. However, 25% of patients die or suffer from
severe neurological deficits. Survivors have memory loss for the duration of the illness, and there is
a risk of relapses of the encephalitis syndrome, the latter in particular when the tumour is removed
too late or not at all or if no tumour could be found [73, 80].
Diagnosis of anti-glutamate receptor (type NMDA) encephalitis is based on a combination of the
characteristic clinical picture, with supporting results from brain MRT, EEG and CSF analysis if
necessary, and the detection of anti-glutamate receptor (type NMDA) antibodies in serum/CSF.
Infectious encephalitides (especially HSV) and other autoimmune aetiologies (limbic encephalitis
with autoantibodies against Hu, Ma2, CV2 and amphiphysin) must be excluded by differential
diagnostics. In general, antibodies against glutamate receptors (type NMDA) should be determined
in all patients with encephalitis where no pathogen has been detected and in suspected cases of
limbic encephalitis. When a positive serological result is obtained a comprehensive teratoma
investigation should be undertaken [82, 86, 22, 87].
Indirect immunofluorescence (IIFT) is a simple and modern method that enables highly sensitive,
monospecific detection of anti-glutamate receptor (type NMDA) antibodies by means of a
recombinant cell line transfected with an expression construct for the receptor subunit NR1 [88]. In
addition, anti-glutamate receptor (type NMDA) positive sera yield a characteristic, although less
specific colouring of the neuropils of the molecular layer of the hippocampus as well as the
granular layer of the cerebellum (neuropil: network of neurons and glial cell appendages). If the
monospecific detection of antibodies against glutamate receptors of type NMDA is negative, a
neuropil fluorescence can also indicate the presence of other autoantibodies associated with limbic
encephalitis (e.g. ant-VGKC antibodies, anti-AMPA receptor antibodies) [86, 87, 88, 89, 90, 91, 92,
93]. Possible further autoantibodies that are relevant for differential diagnostics can be determined
using EUROIMMUN Neurology Mosaics (IIFT) as supplementary tests (e.g. ANA, paraneoplastic
antibodies, anti-MAG antibodies, etc.) [22, 87, 24]. Even reactivities against neuronal and glial
antigens of as yet unknown specificity are detected.
Alongside to serum analysis, parallel investigation of CSF is of great significance, since in most
patients intrathecal synthesis of anti-glutamate receptor (type NMDA) antibodies is in the
foreground [73]. If immunomodulatory therapy has already been started, the antibody titer can be
significantly decreased and may therefore no longer be detectable. Clinical improvement
accompanies a reduction in antibody titer [73].

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Alternative Neurological
Name Antigen Function Related tumors
description syndrome
RNA Encephalomyelitis, SCLC,
Anti-Hu ANNA-1 Hu proteins
bonding neuropathy neuroblastoma
RNA Mamma
Anti-Ri ANNA-2 NOVA POMA
bonding carcinoma, SCLC
Neuropathy,
Anti-PP ANNA-3 170 kDa unknown SCLC
PKD, PLE
Ovarian, breast
DNA
Anti-Yo PCA-1 cdr2, cdr62 PKD and interine
bonding
carcinoma
Anti-
Vesicle Stiff person Mamma
amphi- Amphiphysin
endocytosis syndrome carcinoma, SCLC
physin
Neural
Anti-CV-2 Anti-CRMP5 CRMP5 Encephalitis SCLC, thymoma
development
PLE, Mamma
Anti-Ma Ma proteins unknown
rhombencephalitis carcinoma, various
Purkinje cell Encephalitis,
PCA -2 280 kDa unknown SCLC
antibody LEMS, neuropathy
Anti-
Recoverin Retina Retinopathy Lung carcinoma
recoverin
Anti- PLE,
Ma proteins unknown Testicle carcinoma
Ta/Ma2 rhombencephalitis
Muscle
Anti-titin Titin Myasthenia gravis Thymoma
filament
Anti-Tr PCA-Tr unknown unknown PKD Hodgkin´s disease

Abbreviations:

ANNA-1 Anti-neuronal nuclear autoantibodies, type 1

ANNA-2 Anti-neuronal nuclear autoantibodies, type 2
ANNA-3 Anti-neuronal nuclear autoantibodies, type 3
Anti-PP Autoantibodies against Purkinje cell nucleus and podocyte nucleus of kidney
glomeruli = ANNA-3
CRMP Collapsin response mediator protein
LEMS Lambert-Eaton myasthenic syndrome
PCA Purkinje cell antibodies
PKD paraneoplastic cerebellar degeneration
PLE paraneoplastic limbic encephalitis
POMA paraneoplastic opsoclonus myoclonus ataxia
SCLC small cell lung cancer/carcinoma

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 95.

BIOCHIP position on the fields:

This test instruction is valid for the following test kits (#### is a place holder for different test formats, e.g. 1005 = 10 slides with 5 fields):
Field
BIOCHIPs per field size
Order no. Description
(mm
1 2 3 4 5 6 )
Intestinal
FA 1111-####-1 Neurology Mosaic 1 Nerves, monkey Cerebellum, monkey 5x5
tissue, monkey

FA 1111-####-2 Neurology Mosaic 2 Nerves, monkey Cerebellum, monkey 5x5

Intestinal
FA 1111-####-8 Neurology Mosaic 8 Nerves, monkey Cerebellum, monkey Pancreas, monkey 5x5
tissue, monkey
Intestinal tissue,
FA 1111-####-14 Neurology Mosaic 14 Cerebellum, monkey 5x5
monkey
Intestinal
FA 1111-####-16 Neurology Mosaic 16 Cerebellum, monkey Nerves, monkey HEp-2 cells 5x5
tissue, monkey
Cerebrum, Nervus opticus,
FA 1111-####-17 Neurology Mosaic 17 Cerebellum, monkey EU 90 Aquaporin-4 5x5
monkey monkey**
IIFT: Glutamate Hippocampus Glutamate receptor
FA 111m-####-3 EU 90 Cerebellum , rat 5x5
Receptor Mosaic 3 rat (type NMDA)
Autoimmune Glutamate Leucine-rich Glutamate
IIFT: Glutamate Contactin- GABA
FA 112d-####-1 Encephalitis receptor (type glioma-inactivated receptor 5x5
Receptor Mosaic 4 associated protein 2 Receptor B1
Mosaic 1 AMPA1)** protein 1** (type AMPA2)**

** For the clinical evaluation the results obtained must be confirmed with a CE marked test system.