|Values are valid only on day of printing.|
Indirect Immunofluorescence Assay (IFA):
The patient's sample is tested by a standardized IFA that uses a composite frozen section of mouse cerebellum, kidney, and gut tissues. After incubation with sample and washing, fluorescein-conjugated goat antihuman IgG is applied. Neuron-specific autoantibodies are identified by their characteristic fluorescence staining patterns. Autoantibody of antineuronal nuclear antibody-type 1 (ANNA-1) specificity is pan-neuronal in reactivity; it binds to the cytoplasm and nucleus (sparing nucleolus) of central and peripheral nervous system neurons. Autoantibody of ANNA-2 specificity has a similar binding pattern but reactivity is restricted to neurons of the central nervous system; neurons of the peripheral nervous system are nonreactive. Autoantibody of Purkinje cell cytoplasmic antibody (PCA)-1 specificity bind distinctively to endoplasmic reticulum in Purkinje cells, molecular neurons, and other large neurons in the central and peripheral nervous system. Samples that are scored positive for any neuronal nuclear or cytoplasmic autoantibody are titrated to an endpoint on mouse Purkinje cells or peripheral neurons. Interference by coexisting non-neuron-specific autoantibodies can usually be eliminated by serologic absorption. Occasionally Western blot analysis is required.(Yu Z, Kryzer TJ, Griesmann GE, Kim KK, et al: CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol 2001;49:146-154)
Western blot testing is indicated in the infrequent situation that interference by coexisting nonneuronal-specific autoantibodies precludes IFA interpretation. It is not cost-effective for routine serologic screening. However, results obtained by Western blot analyses in Mayo Clinic's Neuroimmunology Laboratory have shown 100% concordance with IFA results for PCA-1, and superior sensitivity of the IFA for detection of ANNA-1 and ANNA-2.
An aqueous extract of adult rat cerebellar proteins is used as the source of neuronal antigens on the paraneoplastic autoantibody Western blot. Western blot is performed on denatured and reduced proteins separated by electrophoresis on 10% polyacrylamide gel.(Vernino S, Lennon VA: New Purkinje cell antibody [PCA-2]: marker of lung cancer-related neurological autoimmunity. Ann Neurol 2000 March;47:297-305). Full-length recombinant human CRMP-5 antigen is used to confirm CRMP-50IgG.(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). Denatured full-length recombinant human amphiphysin protein is used to confirm amphiphysin antibody.
Radioimmunoassay (RIA) :
(125)I-labeled recombinant human GAD65 and nonimmune human serum are incubated with the patient's diluted cerebrospinal fluid. Antihuman IgG and IgM are then added to form an immunoprecipitate. After washing the precipitated immune complexes, specific antibodies are detected by counting gamma-emission from the pellet's bound I-GAD65.(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)
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)
ANNA-1, ANNA-2, ANNA-3, AGNA-1, PCA-1, PCA-2, PCA-Tr, Amphiphysin, CRMP-5-IgG, NMDIC, AMPIC, GABIC: Monday through Friday; 12 p.m. and 5 p.m.
Paraneoplastic autoantibody Western blot confirmation, CRMP-5-IgG Western blot, amphiphysin Western blot: Monday through Friday; 8 a.m.
NMO/AQP4-IgG CBA, NMDCC, AMPCC, GABCC: Monday through Friday; 4 a.m.
GAD65, VGKC-complex antibody: Monday through Friday; 2 a.m.