In recent years, tests such as CIIFA, radioimmunoprecipitation assay (RIPA), FIPA and ELISA have been developed for the detection of the presence of specific AQP4 antibodies. The cell-based assay (CBA), which utilizes AQP4 expressed on the cell surface in a naturally folded state as an antigen, showed advantages in sensitivity and specificity over NMO-IgG using mouse brain tissue or ELISA using synthesized recombinant peptide or protein [15
]. Subsequently, in-house CBAs based on different detection principles such as indirect immunofluorescence [20
], flow cytometry [23
], or cytotoxicity [25
] were developed. All of these methods utilize HEK cell lines transfected with full-length recombinant human AQP4 for antigenic preparations, thus providing the naturally folded AQP4 protein as an antigenic target.
Recently, EUROIMMUN AG has introduced the commercially available CIIFA [16
]. In our comparison set between the in-house CIIFA and the commercially available CIIFA, we had several discordant results due to an inconclusive fluorescence pattern observed in the in-house CIIFA, which was derived primarily from the poor quality of cellular substrate preparation. In contrast, by using the commercially available kit we completely avoided inconclusive results in 147 samples, including 46 comparison samples and 101 consecutively enrolled patient samples. Nevertheless, the preparation of transfected cells as an antigenic source in the CBA might have caused the variation of results. Test kits manufactured under standardized conditions may improve the consistency of in-house test preparation. In our prospective cohort in which we evaluated the clinical usefulness of CIIFA, AQP4 antibodies were present in 100% (6/6) of patients with NMO and 23% (8/35) of high-risk NMO such as LETM and BON/RON patients, but was virtually absent in patients with MS and other inflammatory and non-inflammatory neurological diseases with the exception of 1 patient with an autoimmune disease and 2 CIS patients. Considering the differences in diagnostic criteria, the study design (i.e., whether patients and sera were acquired consecutively on a clinical basis rather than selected from a pool of known cases and controls), and the test method, direct comparisons of diagnostic values of AQP4 antibody tests reported in other studies may not be applicable. However, the sensitivity of the CIIFA used in this study was superior compared to those of other studies, such as 91% sensitivity reported by Takahashi et al. [18
] and 73% sensitivity reported by McKeon et al. [26
]. More importantly, the specificity of CIIFA for detection of AQP4 antibodies was excellent (94%), thus the clinical relevance of this test in the discrimination of NMO from MS and other demyelinating diseases was significant.
NMOSDs have been reported in patients with systemic connective-tissue diseases such as systemic lupus erythematosus (SLE) or Sjögren's syndrome [27
]; of note, autoantibody markers of SLE or Sjögren's syndrome are found in almost 40% of patients with NMO and high-risk NMO [14
]. However, AQP4 antibodies are not present in patients with systemic connective-tissue diseases in the absence of CNS involvement or with CNS involvement other than NMO [29
]. In our study, 4 Sjögren's syndrome patients were diagnosed with NMO and LETM (1 patient) and high-risk NMO (3 patients), and 3 of these patients had AQP4 antibodies. Min et al. reported that 75% of Sjögren's syndrome patients with recurrent brain lesions had AQP4 antibodies and met the criteria for NMOSDs such as NMO in Korean patients with Sjögren's syndrome [30
FIPA has been developed by using the principle of an immunoprecipitation assay for routine use and quantitative measurement of AQP4 antibodies [20
]. Detection of AQP4 antibodies using FIPA was reported to have comparable sensitivity and specificity and correlate highly with the results of CBAs [15
]. The concordance between FIPA and CIIFA performed at our hospital was 86-100%, depending on the diagnosis. The AQP4 antibody level detected by FIPA and titers detected by CIIFA derived from the dilution factor of the patient sera were proportional and had a tendency to correlate well with each other. Since the in vivo
pathogenic role of AQP4 antibodies (which are predominantly of the IgG1 subclass and activate complement after binding to extracellular epitopes) is well described [20
], the quantitative measurement of AQP4 antibodies may provide insight into the clinical course and treatment response of AQP4 antibody-related diseases. Serial measurements of the AQP4 antibody level by FIPA to monitor the treatment response or relapse during the clinical course have been reported [18
]. Takahashi et al. [18
] observed that the AQP4 antibody titer was related to spinal cord lesion length and Jarius et al. [31
] noted that antibody levels were higher if serum samples were obtained during a relapse and before commencement of immunosuppression. However, in spite of these potential applications, the establishment of in-house FIPA is quite problematic since there are several steps that can cause variability during the test procedure. In particular, the preparation of antigenic material in each batch of test includes multiple procedures such as maintenance of HEK cell lines, preparation of the transfecting vector and DNA, transfection, and cell lysate processing. Moreover, establishment of a cut-off point is arbitrary in each laboratory, thus the transferability of quantitative data is limited, and there is no standardized control material to validate the quantitative value generated from each test. In this respect, CIIFA, a CBA using indirect immunofluorescence principles has several advantages over FIPA. First, the antigenic material prepared on slides can be manufactured on a large scale and stored for a relatively long duration of time. Second, the test procedure is conventional IIFA, which is widely performed in clinical laboratories. Third, the interpretation of fluorescence intensity is a standardized concept among clinical pathologists.
In this study, we demonstrated that the commercially available CIIFA was well correlated with FIPA for the detection and quantitation of AQP4 antibodies, and exhibited a high sensitivity and excellent specificity for the diagnosis of NMO and high-risk NMO diseases. Nevertheless, the usefulness of titration of CIIFA for the prediction of the extent of spinal cord lesions and monitoring of disease progression or treatment response needs to be actively investigated in a prospective study on a larger scale.