Serum Anti-AQP4 Antibody Detection Based On HEK293 Cells Stably Express M23-AQP4

Objective: To detect serum anti-AQP4 antibody in patients with demyelinating diseases like Optical neuromyelitis, Multiple sclerosis and patients with non-demyelinating diseases as control using cells based assay based on recombinant HEK293 cells stably express M23-AQP4. To explore an easy-to-use assay for serum anti-AQP4 antibody detection in clinic by storing the M23-AQP4 stably expressed HEK293 cells and repetitive freeze-thawing serum under different temperature conditions.Methods: pEGFP-N1-M23-AQP4 plasmid transfect HEK293 cells using calcium phosphate transfection reagents and screen the M23-AQP4 stable expression using screening medium containing G418. Then select monoclonal cells and identify M23-AQP4 expression and distribution. We detected serum anti-AQP4 antibody of samples from 72 patients with demyelinating diseases including NMO, MS and other demyelinating diseases(Optic neuritis, Myelitis, Guillain-Barré syndrome, Demyelinating encephalopathy) using CBA based on the selected M23-AQP4 stably expressed monoclonal cells. Serum samples from 30 patients with non-demyelinating diseases were also detected as control. Anti-AQP4 antibody serum positive samples and an equal number of randomly selected anti-AQP4 antibody negative ones were detceted using CBA based on HEK293 cells stably express M23-AQP4, which were fixed with 4% paraformaldehyde and stored at room temperature, 4℃ and-20℃ for 4 weeks to check the stability of M23-AQP4. Anti-AQP4 antibody serum positive specimens and an equal number of randomly selected anti-AQP4 antibody negative ones were stored at room temperature, 4℃ and-20℃ for 1 week after 3 freeze/thaw cycles to check the stability of serum anti-AQP4 antibody using CBA based on fresh M23-AQP4 stably expressed HEK293 cells.Results: M23-AQP4 stably expressed HEK293 were successfully constructed in this study and we confirmed that M23-AQP4 were mainly expressed in the cell’s membrane. 6/7(85.7%)patients with NMO, 1/24(4.2%)patients with MS and 1/41(2.4%)patients with other demyelinating disease were serum positive for anti-AQP4 antibody and all non-demyelinating diseases were serum negative. The sensitivity were 85.7% for NMO and the specificity were 95.8%(23/24)when MS were treated as control, 96.9%(63/65)when demyelinating disease without NMO as control, or 100% when non-demyelinating diseases as control. No discrepancies were found between initial results and results from repeat testing based on M23-AQP4 stably expressed HEK293 cells stored for four weeks under different temperature conditions or using serum specimens that went through 3 freeze/thaw cycles and stored below 4℃. Compared with the initial results the titers of serum anti-AQP4 antibody declined significantly when specimens stored at room temperature(p<0.01).Conclusions: We successfully established M23-AQP4 stably expressed HEK293 cells. Anti-AQP4 antibody extensively existed in serum of patients with NMO, anti-AQP4 antibody may be treated as reliable biomarker of NMO. The stability of M23-AQP4 and serum anti-AQP4 antibody were quite well, the detection method used in this study may be useful and easy-to-use for diagnosis of NMO and distinguish NMO from other demyelinating diseases in clinic.