Test Catalog

Test ID: PAVAL    
Paraneoplastic, Autoantibody Evaluation, Serum

Method Description Describes how the test is performed and provides a method-specific reference

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



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: Autoimmune autonomic neuropathy and neuromuscular hyperexcitability disorders. In: Rose NR, Hamilton RG, Detrick B, eds. Manual of Clinical and Laboratory Immunology. 6th ed. ASM Press; 2002:1013-1017; 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)


Sorting Assay/Flow Cytometry:

This method uses flow cytometry to measure the loss of acetylcholine receptor modulating (AChR) molecules expressed on the surface of live cells expressing AchR on the cell surface. The cell line used is an immortalized human rhabdomyosacroma cell line that expresses endogenous muscle-type nicotinic AChR on its surface. Cells are plated in a 96-well plate and cultured 72 hours prior to the addition of patient serum for an additional 18 to 22 hours to enable internalization of AChR receptors (modulation). Modulation is then stopped by placing cells on ice. The amount of remaining AChRs on the cell surface is measured by flow cytometry. On ice, cells are incubated with a recombinant rat monoclonal antibody against alpha-subunit of the AChR followed by a secondary goat anti-rat IgG antibody conjugated with allophycocyanin (APC). The amount of AchR on the cell surface is proportional to the median fluorescence intensity (MFI) of APC. To calculate the amount of modulation (ie, % loss of AchR) the APC MFI is compared between cells treated with patient serum and cells treated with serum lacking AchR modulating antibodies. Background signal is established in each experiment utilizing cells stained with secondary antibody alone (no patient sera). The percent loss of AchR is calculated as 1-[(Patient MFI-Background MFI)/(Negative calibrator MFI - Background MFI)]*100%.(Unpublished Mayo method)


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 Nov 12;93(20):e1873-e1880. doi: 10.1212/WNL.0000000000008472)



All steps are performed at room temperature (18-28 degree C) utilizing the EUROBlot One instrument. Diluted patient serum (1:101) 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; FA_112d-1_A_UK_C13, 02/2019)

PDF Report Indicates whether the report includes an additional document with charts, images or other enriched information


Day(s) Performed Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.


Monday through Sunday



Monday through Friday



Monday, Wednesday, Saturday



Monday through Friday, Sunday



Monday, Thursday

Report Available The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

10 to 17 days

Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

28 days

Performing Laboratory Location Indicates the location of the laboratory that performs the test