Development of a live cell-based assay for the detection of anti-NMDA receptor antibodies in patients with autoimmune encephalitis

Abstract

N-methyl-D-aspartate receptor (NMDAR) is a glutamate receptor and an ion channel protein expressed on the surface of neuronal cells. NMDAR is composed of three different subunits, the NR1, NR2 and NR3, which are encoded by the splicing of different genes, including one GluN1 gene, four GluN2 genes and two GluN3 genes. The NR1 protein is the essential molecule in forming a functional ion channel. Anti-NMDAR encephalitis is an autoimmune disease correlated to the central nervous system (CNS), with a slightly female predominance. This disease was found to be strongly related to the antibodies in the serum or cerebrospinal fluid (CSF) against the NMDAR on the neurons. The binding of these antibodies to the NMDAR decrease the number of NMDAR on the cell surface, and hence leading to the abnormality in neuronal functions, such as synaptic plasticity and neurodegradation in the CNS. Laboratory diagnosis of Anti-NMDAR encephalitis could be done by detecting the presence of anti-NMDAR in patient serum or CSF using indirect immunofluorescence assay (IIFA) or cell-based flow cytometry. In this study, a cell line stably transfected with NMDAR gene, GluN1, and empty vector, as control cell line, were firstly established. Using these stably transfected cell lines, a live cell-based IIFA by examination of culture slides and live cell-based flow cytometry tests were set up and evaluated for detection of anti-NMDAR in patient serum. In this study, a human embryonic kidney cell line, HEK-293, was transfected separately with a green fluorescence protein (GFP) tagged NMDAR gene plasmid and an empty GFP vector. Transfected cells were selectively maintained in a selection medium with G418 at a concentration of 800-1000 μg/ml. Before setting up the cell-based assays, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blotting analysis were done to confirm the presentation of the GluN1 gene and the expression of NR1 protein respectively in the NMDAR gene transfected cells.

A cohort of, patient serum samples were collected from the Clinical Immunology Laboratory of the Queen Mary Hospital for testing and evaluating the live cell-based assays. Six positive, seven weakly positive and seven negative patient sera were selected after testing for the presence of anti-NMDAR antibody by a commercial fixed cell-based IIFA. Results between the commercial fixed and in-house developed live cell-based assays were compared. Although positive membranous and cytoplasmic staining patterns of the anti-NMDAR could be found in transfected cells when treated with some positive patient samples, the fluorescence signal on live cell-based culture slide was still too weak. There was no significant different in the overall fluorescent signal between positive and negative serum samples when treated with NMDAR gene transfected cells. Moreover, the overall interpretations were not agreed with that of the fixed cell-based assay. On the other hand, the number of anti-NMDAR antibody bound positive staining cells was found higher in anti-NMDAR positive samples treated cells than that of negative samples treated cells, when testing by live cell-based flow cytometry analysis. Further analysis of the stained cells after flow cytometric analysis by wet mounting confirmed the detection of anti-NMDAR in the testing patient samples with observation of membranous and cytoplasmic staining in the NMDAR gene transfected cells but not in the empty vector transfected cells.

In summary, our study has successfully established a stably transfected HEK 293 cell line with the expression of NMDAR which was confirmed by RT-PCR and Western blot analysis. The stable clone of cells was used as a live cell-based substrate for the development of an IFA and Flow cytometric analysis for the detection of anti-NMDAR antibodies. When looking into the fluorescence staining patterns, Positive staining of the cell membrane and cytoplasm could be observed when cells were treated with anti-NMDAR antibody positive or weakly positive patient serum. However, the expression level of NMADR in transfected cells was too low to provide a sensitive assay. Due to the low expression of NMDAR, results of both live cell-based IFA and flow cytometric analysis were not concur with that of the commercial fixed cell-based assay. Further studies and works should be done to enhance the expression of NMDAR in transfected cells and to optimize the assay condition and protocols, improve the sensitivity and specificity of the live cell-based assays with a larger sample size.