In a recent case-control study of 836 human serum samples, including 332 AQP4-IgG–positive and 504 negative samples, the novel immunodot assay showed a 99.4% sensitivity and a 99.2% specificity.
Recently published in JAMA Neurology, findings from a case-control study demonstrated that the sensitivity and specificity of a novel immunodot assay for detecting immunoglobulin G autoantibodies for aquaporin 4 (AQP4-IgG) in patients with neuromyelitis optica spectrum disorder (NMOSD) were comparable with gold standard cell-based assays (CBAs). The findings suggest that the immunodot assay may be a reliable alternative for detecting AQP4-IgG, especially for community medical institutions with relatively scarce equipment and limited economic resources, since it is more time- and cost-efficient than CBA.1
In the validation analysis involving 436 cases of patients with NMOSD, less than 1% were false positive (n = 2) and none reported as false negative. The CBA identified 332 AQP4-IgG-positive samples and 504 negative samples (200 [40%] in controls and 304 [60%] in patients with other diseases). In that sample, less than 1% of the positive cases (n =2) were false negative and less than 1% negative cases (n = 4) were false positive. Overall, the immunodot’s sensitivity was 99.4% (95% CI, 97.8%-99.9%), and specificity was 99.2% (95% CI, 98.0%-99.8%).
Senior author Wei Qiu, MD, PhD, chief physician, department of neurology, The Third Affiliated Hospital of Sun Yat-sen University, in Guangzhou, China, and colleagues wrote, “In our study, the presence of AQP4-IgG was rigorously confirmed using 2 methods: an in-house, live cell–based assay and a commercial CBA. However, for participants who tested negative using the immunodot test or CBA test, it did not mean they did not have NMOSD. It just meant that AQP4 antibodies were not detected. There are variations in the specific details of CBA procedures across different laboratories. The CBA used in this study involved only 2 of the many types available. Therefore, we cannot extrapolate our results to all methods of CBA.”1
In this multicenter case-control study, researchers validated an enzyme immunodot assay for simple and fast AQP4-IgG detection in NMOSD cases between May 2020 and February 2023 from 4 medical centers (3 China; 1 Korea). The study included patients who were AQP4-IgG-positive with NMOSD, patients with other immune-related diseases, and healthy controls. Those not included were patients who did not agree to participate or if their serum sample had turbidity since the serum AQP4 antibodies were measured with immunodot assay. The main outcome was the performance of the immunodot assay compared with the gold standard CBA for AQP4-IgG detection. Authors also examined generalizability with cross-validation in Korea and at a second site in China, assessing the validation of patients with other immune-related diseases, and following up with the original cohort.
There was a total of 836 serum samples gathered with 400 included in the diagnostic study and 436 used in the validation sets. In a head-to-head diagnostic study involving 200 patients with NMOSD with AQP4-IgG (mean age, 43.1 [SD, 13.5] years; 188 [94%] women) and 200 healthy controls, the immunodot assay showed an antibody detection performance comparable with that of the gold standard (κ = 98.0%). The validation sets included 47 patients NMOSD and 26 with other autoimmune diseases from Korea. For the same sets, there were 31 patients with NMOSD at a second site in China, 275 patients with other diseases, and 57 patients with NMOSD at follow-up.
All told, some of the follow-up data were collected retrospectively, meaning that some of the gathered information may have been incomplete and inadequate. The authors also noted that they did not cover all CBA methods available, which may result in referral bias and overrepresentation of immunodot assay generalizability. In addition, the immunodot assay represents a semiquantitative format and therefore, could not measure antibody concentrations with complete certainty. Authors acknowledged the need for more cooperation with international research centers for extensive and comprehensive methodologic validation in larger studies. Additionally, researchers did not investigate the immunodot assay in patients who were AQP4 antibody–negative with NMOSD and concluded that immunodot assay kits are needed to help enhance reagent stability in order to avoid restricted use.
“We found that the immunodot assay provides a practical option for AQP4-IgG detection without specialized laboratories. Consequently, this test has significant implications for cost-effective testing in low-income countries. However, improving a single method cannot thoroughly increase the positive detection rate. The clinical judgment and selection of patients for testing by physicians could significantly influence the positive detection rate,” Qiu et al noted.1 “If physicians can make more accurate judgments regarding the population that should be tested and the appropriate timing, it may help improve the positive detection rate. In addition, standardized protocols for sample collection and testing procedures are crucial for accurate results. We hope that authoritative physicians and scientists can contribute to a better understanding of NMOSD and facilitate more straightforward clinical diagnosis, ultimately benefiting more patients with NMOSD.”