Amelia Cecchin¹, Alexander Troelnikov^1,2, Tatjana Banovic^1,3

¹ SA Pathology, Adelaide, Australia; 

² Flinders University, Adelaide, Australia;

³ Pathology Queensland, Royal Brisbane Hospital, Australia

 

Presently, anti- N-methyl-D-aspartate receptor (NMDAR) antibodies are commonly detected with indirect immunofluorescence (IIF) of NMDAR transfected, formalin-fixed human embryonic kidney cells (HEK 293). However, positive results rely on subjective identification of typical staining patterns, which can be prone to ambiguity. We sought to investigate the feasibility of a modified¹ commercial IIF method employing an optimised, dual-fluorescence IIF strategy to disambiguate borderline results. 

 

A commercial (EUROIMMUN®) assay was altered with additional incubation step of a mouse anti- NMDAR1 monoclonal antibody, detected by Cyanine-3 conjugated anti-mouse IgG, and subsequently human sera detected by Alexa Fluor 488 conjugated anti-human IgG. The method was tested on 34 patient samples, including 20 atypical or indeterminate, 6 paired serum and CSF and 2 positive and negative controls.

 

We observed an average HEK293 transfection rate of 25%. Dual-fluorescence pattern confirmed non-specific staining in 18/20 (90%) of atypical/indeterminate cases, whilst in 2 cases, co-localised cytoplasmic staining, suggestive of intra-cytoplasmic antigen binding, was observed and correlated with clinical features. 2/7 (29%) previously determined positive results were deemed negative by dual IIF. 

 

Modifying existing commercial anti-NMDAR antibody assay is feasible and provides unambiguous determination of indeterminant or weak positive results.

 

¹ Chiu N-C, et al. Optimisation of an Anti-NMDA Receptor Autoantibody Diagnostic Bioassay. Front. Neurol. 2018; 9:661. 

 

The work in this study was performed and based on an original concept/hypothesis by the authors, however the method was adapted from reference 1.