Method Description

Indirect Immunofluorescence Assay:

The patient's specimen is tested by a standardized indirect immunofluorescence assay (IFA) that uses a composite frozen section of mouse cerebellum, kidney, and gut tissues. After incubation with specimen and washing, fluorescein-conjugated goat-antihuman IgG is applied. Neuron-specific autoantibodies are identified by their characteristic fluorescence staining patterns.. Samples that are scored positive for any neuronal nuclear or cytoplasmic autoantibody are titrated to an endpoint. Interference by coexisting non-neuron-specific autoantibodies can usually be eliminated by serologic absorption.(Honorat JA, Komorowski L, Josephs KA, et al: IgLON5 antibody: neurological accompaniments and outcomes in 20 patients. Neurol Neuroimmunol Neuroinflamm 2017 Jul 18;4(5):e385. doi: 10.1212/NXI.0000000000000385

Radioimmunoassay:

Duplicate aliquots of patient specimen are incubated with I(125)-labeled antigen. Immune complexes, formed by adding secondary (goat) antihuman immunoglobulin, are pelleted by centrifugation and washed. Gamma emission from the washed pellet is counted, and mean counts per minute (cpm) are compared with results yielded by high positive and negative control sera. Specimen yielding cpm higher than the background cpm yielded by normal human specimen are retested to confirm positivity and titrated as necessary to obtain a value in the linear range of the assay. The antigen binding capacity (nmol per liter) is calculated from the cpm precipitated at a dilution yielding a linear range value.(Vernino S, Kryzer TJ, Lennon AV: Chapter 114: Autoimmune autonomic neuropathy and neuromuscular hyperexcitability disorders. In Manual of Clinical and Laboratory Immunology. Sixth edition. Edited by NR Rose, RG Hamilton, B Detrick. ASM Press,  2002, pp 1013-1017; Jones AL, Flanagan EP, Pittock SJ, et al: Responses to and Outcomes of Treatment of Autoimmune Cerebellar Ataxia in Adults. JAMA Neurol 2015 Nov;72[11]:1304-1312 doi: 10.1001/jamaneurol.2015.2378)

Live-cell Assay:

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; Kang S, Oh JH, Song SK, et al: Both binding and blocking antibodies correlate with disease severity in myasthenia gravis. Neurol Sci 2015; 36:1167-1171)

Enzyme-Linked Immunosorbent Assay:

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-linked immunosorbent assay in myasthenia gravis, thymoma, and recipients of D-penicillamine or allogeneic bone marrow. Mayo Clin Proc 1988;63:474-481; McKeon A, Lennon V, LaChance DH, et al: Striational antibodies in a paraneoplastic context  Muscle Nerve. 2013 Apr;47(4):585-587)

Western Blot:

Neuronal antigens extracted aqueously from adult rat cerebellum, full-length recombinant human collapsin response-mediator protein-5 (CRMP-5), or full-length recombinant human amphiphysin protein is denatured, reduced, and separated by electrophoresis on 10% polyacrylamide gel. IgG is detected autoradiographically by enhanced chemiluminescence. (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[2]:146-154; Dubey D, Jitprapaikulsan J, Bi H, et al: Amphiphysin-IgG autoimmune neuropathy: A recognizable clinicopathologic syndrome. Neurology 2019 Oct 17 pii: 10.1212/WNL.0000000000008472. doi: 10.1212/WNL.0000000000008472)

Immunoblot:

All steps are performed at room temperature (18-28°C) utilizing the EUROBlot One instrument. Diluted patient serum (1:12.5) is added to test strips (strips containing recombinant antigen manufactured and purified using biochemical methods) in individual channels and incubated for 30 minutes. Positive serums will bind to the purified recombinant antigen and negative serums will not bind. Strips are washed to remove unbound serum antibodies and then incubated with anti-human IgG antibodies (Alkaline phosphatase-labelled) and incubated for 30 minutes. The strips are again washed to remove unbound anti-human IgG antibodies and Nitroblue tetrazolium chloride/5-Bromo-4-chloro-3-indolylphosphate (NBT/BCIP) substrate is added. Alkaline phosphatase enzyme converts the soluble substrate into a colored insoluble product on the membrane to produces a black band. Strips are digitized via picture capture on the EUROBlot One instrument and evaluated with the EUROLineScan software.(O'Connor K, Waters P, Komorowski L, et al: GABAA receptor autoimmunity: A multicenter experience. Neurol Neuroimmunol Neuroinflamm 2019 Apr 4;6[3]:e552 doi: 10.1212/NXI.0000000000000552)

Cell-Binding Assay:

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: IIFT: Neurology Mosaics, Instructions for the indirect immunofluorescence test. EUROIMMUN, Lubeck, Germany, FA_112d-1_A_UK_C13, 02/2019)