Introduction

Autoantibodies against glutamic acid de-carboxylase (GAD) are the most prevalent markers in patients with stiff-person syn-drome (SPS) and also appear in a propor-tion of patients with type I diabetes. Up to now, they are determined by indirect immunofluorescence assay (IFA) using cryosections of primate pancreas and cerebellum, by radio immunoassay or by bridge ELISA. Here we show the evalua-tion of a novel recombinant cell-based IFA based on HEK293 expressing full-length GAD65 for the specific detection of SPS-associated antibodies.

Methods

The coding sequence for GAD65 was in-serted into the eukaryotic expression vec-tor pTriEx-1. Acetone-fixed HEK293 ex-pressing the recombinant or an unrelated control protein were used as substrates in IFA (RC-IFA) in parallel to cryosections of rat and primate cerebellum. IgG binding was analyzed in sera from 44 patients with SPS, 30 with progressive encephalomyeli-tis with rigidity and myoclonus (PERM), 7 with paroxysmal or truncal dystonia/dys-

kinesia (DD), 6 with hyperekplexia (HE), and 19 with other neurological disorders as well as from 50 healthy controls.

Results

Antibodies against GAD65 were found in 28 patients with SPS (64%), 20 with PERM (67%), 3 with dystonia/dyskinesia (43%), 2 with cerebellitis, and 2 with stiff-leg syndrome by RC-IFA but in none of the healthy controls. The titers ranged from 1:32 to 1:3,200 without any significant dif-ferences between the different disorders. All sera also produced a GAD65 pattern on rat and primate cerebellum (titer range: 1:10 to 1:3,200), although the evaluation using the sections was in some cases im-peded by unrelated staining.

Conclusion

RC-IFA is a valid tool for the determina-tion of anti-GAD65 antibodies in move-ment disorders. Microscopic interpreta-tion of RC-IFA results is generally easier compared to IFA using frozen tissue sec-tions, while titers are almost identical.

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A new recombinant cell-based IFA for the determination of autoantibodies against GAD in stiff-person syndrome

I.-M. Bloecker1, S. Mindorf1, W. Stoecker1, B. Balint2,3, H.-M. Meinck2,

L. Komorowski1, C. Probst1, and W. Schlumberger1

1 Institute for Experimental Immunology, affiliated to EUROIMMUN AG, Luebeck, Germany2 Department of Neurology, University of Heidelberg, Germany

3 Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, London, United Kingdom

GAD65 (HEK293) Control (HEK293) Cerebellum (rat) Hippocampus (rat) Cerebellum (primate)

An

ti-G

AD

65-r

c-IF

A [

tite

r]

Cohort

SPS PERM DD HE Other

0

1:10

1:32

1:100

1:320

1:1000

1:3200

IFA

, cer

ebel

lum

rat

[ti

ter]

Anti-GAD65-rc-IFA [titer]

0 1:10 1:32 1:100 1:320 1:1000 1:3200

0

1:10

1:32

1:100

1:320

1:1000

1:3200

IFA

, cer

ebel

lum

pri

mat

e [t

iter

]

0

1:10

1:32

1:100

1:320

1:1000

1:3200

Anti-GAD65-rc-IFA [titer]

0 1:10 1:32 1:100 1:320 1:1000 1:3200

Scientific presentation at the 12th Congress of the International Society of Neuroimmunology (ISNI), Mainz, Germany, November 2014