|Values are valid only on day of printing.|
Indirect Immunofluorescence Assay (IFA):
Before testing, patient's serum is preabsorbed with liver powder to remove nonorgan-specific autoantibodies. After applying to a composite substrate of frozen mouse tissues (brain, kidney, and gut) and washing, fluorescein-conjugated goat antihuman IgG is applied to detect the distribution and pattern of patient IgG binding.(Pittock SJ, Kryzer TJ, Lennon VA: Paraneoplastic antibodies coexist and predict cancer, not neurological syndrome. Ann Neurol 2004;56:715-719)
Goat antihuman IgG and IgM is used as precipitant in all assays. Cation channel protein antigens are solubilized from neuronal or muscle membrane, in nonionic detergent, and complexed with a selective high-affinity ligand labeled with (125)I. (125)I-labelled recombinant human glutamic acid decarboxylase-65 (GAD65) antigen is used to confirm GAD65 autoantibody (when suspected from immunofluorescent staining pattern).(Griesmann GE, Kryzer TJ, Lennon VA: Autoantibody profiles of myasthenia gravis and Lambert-Eaton myasthenic syndrome. In Manual of Clinical and Laboratory Immunology. Sixth edition. Edited by NR Rose, RG Hamilton, et al: Washington, DC, ASM Press, 2002, pp 1005-1012; Walikonis JE, Lennon VA: Radioimmunoassay for glutamic acid decarboxylase [GAD65] autoantibodies as a diagnostic aid for stiff-man syndrome and a correlate of susceptibility to type 1 diabetes mellitus. Mayo Clin Proc 1998;73:1161-1166)
Western Blot (WB):
WB is performed when IFA screening for antineuronal nuclear antibody-type 1 (ANNA-1) is not interpretable due to interfering autoantibodies. A mixture of neuronal antigens extracted aqueously from adult rat cerebellum is denatured, reduced, and separated by electrophoresis on 10% polyacrylamide gel. Full-length recombinant human CRMP-5 antigen is used to confirm CRMP-5-IgG.(Yu Z, Kryzer TJ, Griesmann GE, et al: CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol 2001;49:145-154)
Cell-Binding Assay (CBA):
Patient serum is applied to a composite slide containing transfected and nontransfected HEK-293 cells. After incubation and washing, fluorescein-conjugated goat antihuman IgG is applied to detect the presence of patient IgG binding.(Package insert: EUROIMMUN AG. Stocker W. et al: Differenzierte Autoantikorper-Diagnostik mit BIOCHIP-Mosaiken. U Conrad, K. [Hrsg] Autoantikorper. Pabst-Verlag  78-99)
NMO-IgG Fluorescence-Activated Cell Sorting Assay (FACS)
Human embryonic kidney cells (HEK 293) are transfected transiently with a plasmid (pIRES2- Aequorea coerulescens green fluorescent protein [AcGFP]) encoding both green fluorescent protein (AcGFP) and AQP4-M1. After 36 hours, a mixed population of cells (transfected expressing AQP4 on the surface and AcGFP in the cytoplasm and nontransfected lacking AQP4 and AcGFP) are lifted and resuspended in live cell-binding buffer. Patient serum is then added to cells at a 1 in 5 screening dilution. After incubation and washing, the cells are resuspended in secondary antibody (AlexaFluor 647-conjugated goat antihuman IgG; 1:2000 in LCBB), held on ice, washed, fixed with 4% paraformaldehyde, and analyzed by flow cytometry (BD FACSCanto; Becton, Dickinson and Co). Two populations are gated on the basis of AcGFP expression: positive (high AQP4 expression) and negative (low or no AQP4 expression). The median Alexafluor 647 fluorescence intensity (MFI) for the AcGFP-positive population indicates relative abundance of human IgG potentially bound to AQP4 surface epitopes; MFI for the GFP-negative population indicated non-specifically-bound IgG. The IgG binding index is calculated as the ratio of the average MFI for duplicate aliquots of each cell population (MFI GFP positive/MFI GFP negative). We established conservative cutoff IgG binding index values of 2.00 for M1-FACS.
If FACS assay is positive at screening dilution, FACS Titer Assay is performed at an additional charge. The patient serum is titrated to endpoint. The dilution where the IgG binding index is greater than or equal to 2, is considered the endpoint dilution. If a patient is positive at a 1:5 dilution, but negative at 1:10 dilution, the endpoint will be reported as 5.
ANN1S, ANN2S, ANN3S, AGN1S, PCABP, PCAB2, PCATR, AMPHS, CRMS, AMPIS, GABIS, NMDIS:
Monday through Friday; 11:30 a.m. and 8:00 p.m.; Saturday and Sunday 8:00 a.m.
AMPCS, GABCS, NMDCS: Monday through Friday; 6 a.m.
Paraneoplastic autoantibody Western blot confirmation, CRMP-5-IgG Western blot, Amphiphysin Western blot: Monday, Wednesday, Friday; 8 a.m.
ARBI: Monday through Saturday; 5 p.m.
CCPQ, CCN, GANG, VGKC: Monday through Friday 11:00 a.m. and 6:00 p.m.; Saturday 6:00 a.m.; Sunday 6:00 a.m.
GD65S: Monday to Friday; 6:00 a.m. and 4:00 p.m.