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AQP4 autoantibody assay performance in clinical laboratory service.

Fryer JP, Lennon VA, Pittock SJ, Jenkins SM, Fallier-Becker P, Clardy SL, Horta E, Jedynak EA, Lucchinetti CF, Shuster EA, Weinshenker BG, Wingerchuk DM, McKeon A - Neurol Neuroimmunol Neuroinflamm (2014)

Bottom Line: Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2.M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells.High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

View Article: PubMed Central - PubMed

Affiliation: Departments of Laboratory Medicine and Pathology (J.P.F., V.A.L., S.J.P., E.H., E.A.J., A.M.), Neurology (V.A.L., S.J.P., S.L.C., C.F.L., B.G.W., A.M.), Immunology (V.A.L.), and Health Sciences Research (S.M.J.), College of Medicine, Mayo Clinic, Rochester, MN; Institute of Pathology and Neuropathology (P. F.-B.), University of Tubingen, Germany; Department of Neurology (E.A.S.), College of Medicine, Mayo Clinic, Jacksonville, FL; and Department of Neurology (D.M.W.), College of Medicine, Mayo Clinic, Scottsdale, AZ.

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.

No MeSH data available.


Related in: MedlinePlus

M1 and M23 AQP4 isoforms compared by freeze-fracture electron microscopic and Western blot analyses(A) Plasma membranes of HEK293 cells expressing recombinant M1-AQP4 or M23-AQP4 viewed by freeze-fracture electron microscopy. (B) Western blot analysis of the proportion of recombinant AQP4 expressed in higher-order arrays or as tetramers in HEK293 cells transfected with plasmids encoding M23 alone (lane 1), M1 alone (lane 5), or different ratios of each (lanes 2–4). Intramembranous particles in M1-AQP4 cells are predominantly singlet (A, left). Compare the large lattices of orthogonal array-like assemblies in M23-AQP4 cells (A, right). M1-AQP4 coexpression inhibits high-order array formation by M23-AQP4 (B). The y-axis indicates molecular weight (kDa) of AQP4 structures. Solubilized proteins, separated by Blue Native gel electrophoresis and transferred to PDF membrane, were probed with monoclonal AQP4-specific IgG. AQP4 immunoreactivity in largest-sized arrays (lane 1) diminishes with increasing M1:M23 ratio, and the proportion in tetrameric form increases (lane 5). AQP4 = aquaporin-4; Ig = immunoglobulin; HEK = human embryonic kidney.
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Figure 2: M1 and M23 AQP4 isoforms compared by freeze-fracture electron microscopic and Western blot analyses(A) Plasma membranes of HEK293 cells expressing recombinant M1-AQP4 or M23-AQP4 viewed by freeze-fracture electron microscopy. (B) Western blot analysis of the proportion of recombinant AQP4 expressed in higher-order arrays or as tetramers in HEK293 cells transfected with plasmids encoding M23 alone (lane 1), M1 alone (lane 5), or different ratios of each (lanes 2–4). Intramembranous particles in M1-AQP4 cells are predominantly singlet (A, left). Compare the large lattices of orthogonal array-like assemblies in M23-AQP4 cells (A, right). M1-AQP4 coexpression inhibits high-order array formation by M23-AQP4 (B). The y-axis indicates molecular weight (kDa) of AQP4 structures. Solubilized proteins, separated by Blue Native gel electrophoresis and transferred to PDF membrane, were probed with monoclonal AQP4-specific IgG. AQP4 immunoreactivity in largest-sized arrays (lane 1) diminishes with increasing M1:M23 ratio, and the proportion in tetrameric form increases (lane 5). AQP4 = aquaporin-4; Ig = immunoglobulin; HEK = human embryonic kidney.

Mentions: To analyze differences in the antigenic substrates used in live CBAs (i.e., HEK293 cells transfected with M1 or M23 AQP4 isoform), we compared plasma membrane preparations by freeze-fracture electron microscopy, native gel electrophoresis/Western blot, and flow cytometry. Freeze-fracture images revealed singlet particles in plasma membranes of M1-AQP4 cells and large orthogonal array-like assemblies in M23-AQP4 cells (figure 2A). Analysis of plasma membrane proteins solubilized from cells transfected with M1-AQP4 or M23-AQP4 alone, or with both AQP4 isoforms (varying ratios), by Western blot using AQP4-specific monoclonal antibody confirmed that the proportion of AQP4 in higher-order assemblies was directly proportional to the transfection ratio of M23:M1 cDNA (figure 2B).


AQP4 autoantibody assay performance in clinical laboratory service.

Fryer JP, Lennon VA, Pittock SJ, Jenkins SM, Fallier-Becker P, Clardy SL, Horta E, Jedynak EA, Lucchinetti CF, Shuster EA, Weinshenker BG, Wingerchuk DM, McKeon A - Neurol Neuroimmunol Neuroinflamm (2014)

M1 and M23 AQP4 isoforms compared by freeze-fracture electron microscopic and Western blot analyses(A) Plasma membranes of HEK293 cells expressing recombinant M1-AQP4 or M23-AQP4 viewed by freeze-fracture electron microscopy. (B) Western blot analysis of the proportion of recombinant AQP4 expressed in higher-order arrays or as tetramers in HEK293 cells transfected with plasmids encoding M23 alone (lane 1), M1 alone (lane 5), or different ratios of each (lanes 2–4). Intramembranous particles in M1-AQP4 cells are predominantly singlet (A, left). Compare the large lattices of orthogonal array-like assemblies in M23-AQP4 cells (A, right). M1-AQP4 coexpression inhibits high-order array formation by M23-AQP4 (B). The y-axis indicates molecular weight (kDa) of AQP4 structures. Solubilized proteins, separated by Blue Native gel electrophoresis and transferred to PDF membrane, were probed with monoclonal AQP4-specific IgG. AQP4 immunoreactivity in largest-sized arrays (lane 1) diminishes with increasing M1:M23 ratio, and the proportion in tetrameric form increases (lane 5). AQP4 = aquaporin-4; Ig = immunoglobulin; HEK = human embryonic kidney.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: M1 and M23 AQP4 isoforms compared by freeze-fracture electron microscopic and Western blot analyses(A) Plasma membranes of HEK293 cells expressing recombinant M1-AQP4 or M23-AQP4 viewed by freeze-fracture electron microscopy. (B) Western blot analysis of the proportion of recombinant AQP4 expressed in higher-order arrays or as tetramers in HEK293 cells transfected with plasmids encoding M23 alone (lane 1), M1 alone (lane 5), or different ratios of each (lanes 2–4). Intramembranous particles in M1-AQP4 cells are predominantly singlet (A, left). Compare the large lattices of orthogonal array-like assemblies in M23-AQP4 cells (A, right). M1-AQP4 coexpression inhibits high-order array formation by M23-AQP4 (B). The y-axis indicates molecular weight (kDa) of AQP4 structures. Solubilized proteins, separated by Blue Native gel electrophoresis and transferred to PDF membrane, were probed with monoclonal AQP4-specific IgG. AQP4 immunoreactivity in largest-sized arrays (lane 1) diminishes with increasing M1:M23 ratio, and the proportion in tetrameric form increases (lane 5). AQP4 = aquaporin-4; Ig = immunoglobulin; HEK = human embryonic kidney.
Mentions: To analyze differences in the antigenic substrates used in live CBAs (i.e., HEK293 cells transfected with M1 or M23 AQP4 isoform), we compared plasma membrane preparations by freeze-fracture electron microscopy, native gel electrophoresis/Western blot, and flow cytometry. Freeze-fracture images revealed singlet particles in plasma membranes of M1-AQP4 cells and large orthogonal array-like assemblies in M23-AQP4 cells (figure 2A). Analysis of plasma membrane proteins solubilized from cells transfected with M1-AQP4 or M23-AQP4 alone, or with both AQP4 isoforms (varying ratios), by Western blot using AQP4-specific monoclonal antibody confirmed that the proportion of AQP4 in higher-order assemblies was directly proportional to the transfection ratio of M23:M1 cDNA (figure 2B).

Bottom Line: Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2.M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells.High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

View Article: PubMed Central - PubMed

Affiliation: Departments of Laboratory Medicine and Pathology (J.P.F., V.A.L., S.J.P., E.H., E.A.J., A.M.), Neurology (V.A.L., S.J.P., S.L.C., C.F.L., B.G.W., A.M.), Immunology (V.A.L.), and Health Sciences Research (S.M.J.), College of Medicine, Mayo Clinic, Rochester, MN; Institute of Pathology and Neuropathology (P. F.-B.), University of Tubingen, Germany; Department of Neurology (E.A.S.), College of Medicine, Mayo Clinic, Jacksonville, FL; and Department of Neurology (D.M.W.), College of Medicine, Mayo Clinic, Scottsdale, AZ.

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.

No MeSH data available.


Related in: MedlinePlus