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. EUROIMMUN AG · D-23560 Luebeck (Germany) · Seekamp 31 · Tel +49 451 58550 · Fax 5855591 · E-mail [email protected] A new recombinant cell-based IFA for the determination of autoantibodies against GAD in stiff-person syndrome I.-M. Bloecker 1 , S. Mindorf 1 , W. Stoecker 1 , B. Balint 2,3 , H.-M. Meinck 2 , L. Komorowski 1 , C. Probst 1 , and W. Schlumberger 1 1 Institute for Experimental Immunology, affiliated to EUROIMMUN AG, Luebeck, Germany 2 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) Anti-GAD65-rc-IFA [titer] Cohort SPS PERM DD HE Other 0 1:10 1:32 1:100 1:320 1:1000 1:3200 IFA, cerebellum rat [titer] 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, cerebellum primate [titer] 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