Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen

Abstract 

Background

Recently, a highly specific serum autoantibody was discovered in patients with neuromyelitis optica, called NMO-IgG, and aquaporin-4, the most abundant water channel in the CNS, was identified as the target antigen. Several assays for the detection of NMO-IgG/AQP4-Ab have been described. Tests based on recombinant human AQP4 have been repeatedly demonstrated to be more sensitive than the previous gold standard assay, i.e. immunohistochemistry (IHC) on mouse brain tissue. However, the sophisticated techniques applied restrict their availability to few laboratories worldwide.

Objective

To develop an easy-to-use, recombinant immunofluorescence assay (rIFA) suitable for standardized and high-throughput detection of NMO-IgG/AQP4-Ab.

Methods

HEK293 cells seeded on cover glasses were transfected with full-length recombinant human AQP4 at large scale. Cover glasses with the immobilized cells were cut into millimetre-sized fragments and transferred to microscopy slides. 151 serum samples from patients with NMO spectrum disorders (NMOSD) and controls were analysed both in the standard IHC assay and in the newly developed rIFA.

Results

25/32 (78.1%) patients with clinically definite NMO and 36/51 (70.6%) of total patients with NMOSD were positive for NMO-IgG/AQP4-Ab in the rIFA compared to 65.6% and 58.8%, respectively, in the IHC assay.

Conclusion

The recombinant IFA presented here provides laboratories familiar with indirect immunofluorescence microscopy with a highly sensitive and reproducible diagnostic tool for standardized detection of antibodies to AQP4. This new approach could make AQP4-Ab testing, which is of high clinical relevance, more widely available.

Abbreviations: AQP4, aquaporin-4, AQP4-Ab, aquaporin-4 antibody, HC, healthy controls, IHC, immunohistochemistry, LETM, longitudinally extensive transverse myelitis, MS, multiple sclerosis, NMO, neuromyelitis optica, NMO-IgG, neuromyelitis optica IgG, nLR, negative likelihood ratio, ON/NETM, optic neuritis with non-extensive transverse myelitis, OND, other neurological diseases, pLR, positive likelihood ratio, rIFA, recombinant immunofluorescence assay, rON, recurrent optic neuritis

Keywords: Neuromyelitis optica (Devic syndrome), NMO-IgG, Antibody to aquaporin-4, Diagnosis, Recombinant antigen, Immunofluorescence assay, Longitudinally extensive transverse myelitis, Optic neuritis, Multiple sclerosis