ABSTRACT
OBJECTIVE: To compare performance of contemporary aquaporin-4-immunoglobulin (Ig) G assays in clinical service. METHODS: Sera from neurologic patients (4 groups) and controls were tested initially by service ELISA (recombinant human aquaporin-4, M1 isoform) and then by cell-based fluorescence assays: fixed (CBA, M1-aquaporin-4, observer-scored) and live (fluorescence-activated cell sorting [FACS], M1 and M23 aquaporin-4 isoforms). Group 1: all Mayo Clinic patients tested from January to May 2012; group 2: consecutive aquaporin-4-IgG-positive patients from September 2011 (Mayo and non-Mayo); group 3: suspected ELISA false-negatives from 2011 to 2013 (physician-reported, high likelihood of neuromyelitis optica spectrum disorders [NMOSDs] clinically); group 4: suspected ELISA false-positives (physician-reported, not NMOSD clinically). RESULTS: Group 1 (n = 388): M1-FACS assay performed optimally (areas under the curves: M1 = 0.64; M23 = 0.57 [p = 0.02]). Group 2 (n = 30): NMOSD clinical diagnosis was confirmed by: M23-FACS, 24; M1-FACS, 23; M1-CBA, 20; and M1-ELISA, 18. Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2. Group 3 (n = 31, suspected M1-ELISA false-negatives): results were positive for 5 sera: M1-FACS, 5; M23-FACS, 3; and M1-CBA, 2. Group 4 (n = 41, suspected M1-ELISA false-positives): all negative except 1 (positive only by M1-CBA). M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells. CONCLUSION: Aquaporin-4-transfected CBAs, particularly M1-FACS, perform optimally in aiding NMOSD serologic diagnosis. High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.
ABSTRACT
Anti-IgA may cause anaphylactic transfusion reactions in IgA-deficient individuals. Testing for IgG anti-IgA is useful to identify persons at risk. This report describes an immunoassay for anti-IgA that uses polyclonal IgA coupled to fluorescent microspheres as an immunosorbent. Anti-IgA is detected by phycoerythrin-labeled anti-IgG. The assay is calibrated in arbitrary units by use of a serum that contains anti-IgA. Dose-response studies with sera that contain anti-IgA showed positive responses at dilutions up to 32-fold greater than the dilution used to test patients' samples. Inhibition studies with purified IgA and IgA-deficient serum showed no inhibition with IgA-deficient serum and complete inhibition with soluble IgA. Clinical tests performed in more than 90 assays had a CV of 13.6 percent for measurements of an internal positive control. The fluorescent immunoassay method is rapid, reproducible, and sensitive to low concentrations of IgG anti-IgA.
