|Values are valid only on day of printing.|
Indirect Immunofluorescence Assay (IFA):
Before screening for neuronal nuclear and cytoplasmic autoantibodies, patient's serum is preabsorbed with liver tissue extract to remove nonorgan-specific autoantibodies. After application to a composite substrate of frozen mouse tissues (brain, kidney, and gut), washing, fluorescein-conjugated goat antihuman IgG is applied to detect the distribution and pattern of the patient's bound IgG.(Vernino S, Lennon VA: New Purkinje cell antibody [PCA 2]: marker of lung cancer related neurological autoimmunity. Ann Neurol 2000;47:297-305;Lennon VA: The case for a descriptive generic nomenclature: classification of immunostaining criteria for PCA-1, ANNA-1, and ANNA-2 autoantibodies. Neurology 1994;44:2412-2415; Chan KH, Vernino S, Lennon VA: ANNA-3 anti-neuronal nuclear antibody: marker of lung cancer-related autoimmunity. Ann Neurol 2001 September;50:301-311; Yu Z, Kryzer TJ, Griesmann GE, et al: CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol 2001 February;49:146-154)
Goat antihuman IgG and IgM is used as precipitant in all assays. Cation channel protein antigens are solubilized from neuronal or muscle membranes, in nonionic detergent and complexed with a selective high-affinity ligand that is labeled with (125)I. (125)I recombinant human GAD65 is used as antigen 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 December;73:1161-1166)
Acetylcholine receptor modulating antibodies (muscle AChR) are detected by incubating the patient's serum for 14 hours with viable, noninnervated, monolayer cultures of human muscle cells. Percent loss of surface AChR is then quantitated by probing with (125)I-alpha-bungarotoxin.(Howard FM Jr, Lennon VA, Finley J, et al: Clinical correlations of antibodies that bind, block, or modulate human acetylcholine receptors in myasthenia gravis. Ann NY Acad Sci 1987;505:526-538)
Enzyme Immunoassay (EIA):
A mixture of sarcomeric proteins extracted from innervated rat skeletal muscle is used as antigen to detect striational antibodies (IgG, IgM, and IgA).(Cikes N, Momoi MY, Williams CL, et al: Striational autoantibodies: quantitative detection by enzyme immunoassay in myasthenia gravis, thymoma, and recipients of D-penicillamine or allogeneic bone marrow. Mayo Clin Proc 1988;63:474-481)
Western Blot (WB):
WB is performed when IFA screening 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 (5% for PCA-2 and ANNA-3). Full-length recombinant human CRMP-5 antigen is used to confirm CRMP-5-IgG. Denatured full-length recombinant human amphiphysin protein is used to confirm amphiphysin antibody.(Yu Z, Kryzer TJ, Griesmann GE, et al: CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol 2001 February;49:145-154)
Cell-Binding Assay (CBA):
Patient serum is applied to a composite slide containing HEK293 cells: 1) transfected with AQP4 (M1 isoform), 2) nontransfected. After incubation and washing, fluorescein-conjugated goat IgG reactive with human IgG is applied to detect bound patient IgG.(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 647â€“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 nonspecifically-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.
ANNA-1, ANNA-2, ANNA-3, AGNA-1, PCA-1, PCA-2, PCA-Tr, Amphiphysin, CRMP-5-IgG, NMDIS, AMPIS, GABIS: Monday through Friday; 11:30 a.m. and 8 p.m.; Saturday and Sunday; 8 a.m.
Striational (striated muscle) antibodies: Monday through Friday; 4 a.m. and 3 p.m.; Saturday; 6 a.m.
P/Q-type calcium channel antibody, N-type calcium channel antibody, AChR ganglionic neuronal antibody, neuronal (V-G) K+ channel autoantibody: Monday through Friday; 11 a.m. and 6 p.m.; Saturday and Sunday; 6 a.m.
ACh receptor (muscle) binding antibody: Monday through Friday;11 a.m., 6 p.m., and 10 p.m.; Saturday; 6 a.m.; Sunday; 6 a.m. and 10 a.m.
Paraneoplastic autoantibody Western blot, CRMP-5-IgG Western blot, Amphiphysin Western blot: Monday, Wednesday, Friday; 8 a.m.
GAD65 antibody assay: Monday through Friday; 6 a.m. and 4 p.m.
ACh receptor (muscle) modulating antibodies: Monday through Thursday; 2 p.m., Saturday; 8 a.m.
NMO/AQP4-IgG CBA, NMDCS, AMPCS, GABCS: Monday through Friday; 6 a.m.