Test Id : EPS2

To determine the necessity of laboratory testing for patients with suspected autoimmune encephalitis, epilepsy or dementia, see the Antibody Prevalence in Epilepsy and Encephalopathy (APE2) scorecard.

If the immunofluorescence assay (IFA) patterns suggest collapsin response-mediator protein-5 (CRMP-5)-IgG, then the CRMP-5-IgG IFA titer and CRMP-5-IgG Western blot will be performed at an additional charge.

If the IFA pattern suggests adenylate kinase 5 protein (AK5) antibody, then the AK5 IFA titer and AK5 cell binding assay (CBA) will be performed at an additional charge.

If the IFA patterns suggest amphiphysin antibody, then the amphiphysin immunoblot and amphiphysin IFA titer will be performed at an additional charge.

If the IFA pattern suggests antiglial nuclear antibody (AGNA)-1, then the AGNA-1 immunoblot and AGNA-1 IFA titer will be performed at an additional charge.

If the IFA pattern suggests antineuronal nuclear antibody type 1 (ANNA-1), then the ANNA-1 IFA titer, ANNA-1 immunoblot, and ANNA-2 immunoblot will be performed at an additional charge.

If the IFA pattern suggests ANNA-2 antibody, then the ANNA-2 IFA titer, ANNA-2 immunoblot, ANNA-1 immunoblot will be performed at an additional charge.

If the client requests or if the IFA pattern suggests ANNA-3 antibody, then the ANNA-3 IFA titer will be performed at an additional charge.

If the IFA pattern suggests Purkinje cytoplasmic antibody type 2 (PCA-2), then the PCA-2 IFA titer and MAP1B immunoblot will be performed at an additional charge.

If the IFA pattern suggests PCA-Tr antibody, then the PCA-Tr immunoblot and PCA-Tr IFA titer will be performed at an additional charge.

If the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-receptor antibody CBA result is positive, then the AMPA-receptor antibody IFA titer will be performed at an additional charge.

If the gamma-aminobutyric acid B (GABA-B) receptor antibody CBA result is positive, then the GABA-B-receptor antibody IFA titer assay will be performed at an additional charge.

If the N-methyl-D-aspartate (NMDA) receptor antibody CBA result is positive, then the NMDA-receptor antibody IFA titer assay will be performed at an additional charge.

If the dipeptidyl-peptidase-like protein-6 (DPPX) antibody CBA result is positive, then the DPPX IFA titer will be performed at an additional charge.

If the Kelch-like protein 11 (KLHL11) CBA result is reactive, then the KLHL11 antibody IFA titer will be performed at an additional charge.

If the IFA pattern suggests metabotropic glutamate receptor 1 (mGluR1) antibody, then the mGluR1 antibody CBA and mGluR1 IFA titer will be performed at an additional charge.

If the IFA pattern suggests metabotropic glutamate receptor 5 (mGluR5) antibody, then the mGluR5 antibody CBA and mGluR5 IFA titer will be performed at an additional charge.

If the IFA pattern suggests neurochondrin antibody, then the neurochondrin antibody CBA and neurochondrin IFA titer will be performed at an additional charge.

If the IFA pattern suggests tripartite motif-containing protein 46 (TRIM46) antibody, then TRIM46 antibody CBA and TRIM46 IFA titer will be performed at an additional charge.

If the IFA pattern suggests phosphodiesterase 10A (PDE10A) antibody, then PDE10A antibody IFA titer will be performed at an additional charge.

For more information see:

-Autoimmune/Paraneoplastic Epilepsy Evaluation Algorithm-Serum

-Epilepsy: Unexplained Refractory and/or Familial Testing Algorithm

Multiple neurological phenotype-specific autoimmune/paraneoplastic evaluations are available. For more information as well as phenotype-specific testing options, see Autoimmune Neurology Test Ordering Guide.

When more than one evaluation is ordered on the same order number, the duplicate test will be canceled.

For a list of antibodies performed with each evaluation, see Autoimmune Neurology Antibody Matrix.

This test should not be requested for patients who have recently received radioisotopes, therapeutically or diagnostically, because of potential assay interference. The specific waiting period before specimen collection will depend on the isotope administered, the dose given, and the clearance rate in the individual patient. Specimens will be screened for radioactivity prior to analysis. Radioactive specimens received in the laboratory will be held 1 week and assayed if sufficiently decayed or canceled if radioactivity remains.

Provide the following information:

-Relevant clinical information

-Ordering healthcare professional's name, phone number, mailing address, and email address

Patient Preparation: For optimal antibody detection, collect specimen before starting immunosuppressant medication or intravenous immunoglobulin (IVIg) treatment.

Supplies: Sarstedt Aliquot Tube, 5 mL (T914)

Collection Container/Tube:

Preferred: Red top

Acceptable: Serum gel

Submission Container/Tube: Plastic vial

Specimen Volume: 4 mL Serum

Collection Instructions: Centrifuge and aliquot serum into a plastic vial.

• Autoimmune/Paraneoplastic Epilepsy Evaluation Algorithm-Serum
• Epilepsy: Unexplained Refractory and/or Familial Testing Algorithm

If not ordering electronically, complete, print, and send a Neurology Specialty Testing Client Test Request (T732) with the specimen.

Serum: 2.5 mL

Investigating new onset cryptogenic epilepsy with incomplete seizure control and duration of less than 2 years, using serum specimens

Investigating new onset cryptogenic epilepsy plus 1 or more of the following accompaniments:

-Psychiatric accompaniments (psychosis, hallucinations)

-Movement disorder (myoclonus, tremor, dyskinesias)

-Headache

-Cognitive impairment/encephalopathy

-Autoimmune stigmata (personal history or family history or signs of diabetes mellitus, thyroid disorder, vitiligo, premature graying of hair, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, idiopathic adrenocortical insufficiency), or multiple sclerosis

-History of cancer

-Smoking history (20 or more pack-years) or other cancer risk factors

-Investigating seizures occurring within the context of a subacute multifocal neurological disorder without obvious cause, especially in a patient with a past or family history of cancer

-A rising autoantibody titer in a previously seropositive patient suggests cancer recurrence

To determine the necessity of laboratory testing for patients with suspected autoimmune encephalitis, epilepsy or dementia, see the Antibody Prevalence in Epilepsy and Encephalopathy (APE2) scorecard.

If the immunofluorescence assay (IFA) patterns suggest collapsin response-mediator protein-5 (CRMP-5)-IgG, then the CRMP-5-IgG IFA titer and CRMP-5-IgG Western blot will be performed at an additional charge.

If the IFA pattern suggests adenylate kinase 5 protein (AK5) antibody, then the AK5 IFA titer and AK5 cell binding assay (CBA) will be performed at an additional charge.

If the IFA patterns suggest amphiphysin antibody, then the amphiphysin immunoblot and amphiphysin IFA titer will be performed at an additional charge.

If the IFA pattern suggests antiglial nuclear antibody (AGNA)-1, then the AGNA-1 immunoblot and AGNA-1 IFA titer will be performed at an additional charge.

If the IFA pattern suggests antineuronal nuclear antibody type 1 (ANNA-1), then the ANNA-1 IFA titer, ANNA-1 immunoblot, and ANNA-2 immunoblot will be performed at an additional charge.

If the IFA pattern suggests ANNA-2 antibody, then the ANNA-2 IFA titer, ANNA-2 immunoblot, ANNA-1 immunoblot will be performed at an additional charge.

If the client requests or if the IFA pattern suggests ANNA-3 antibody, then the ANNA-3 IFA titer will be performed at an additional charge.

If the IFA pattern suggests Purkinje cytoplasmic antibody type 2 (PCA-2), then the PCA-2 IFA titer and MAP1B immunoblot will be performed at an additional charge.

If the IFA pattern suggests PCA-Tr antibody, then the PCA-Tr immunoblot and PCA-Tr IFA titer will be performed at an additional charge.

If the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-receptor antibody CBA result is positive, then the AMPA-receptor antibody IFA titer will be performed at an additional charge.

If the gamma-aminobutyric acid B (GABA-B) receptor antibody CBA result is positive, then the GABA-B-receptor antibody IFA titer assay will be performed at an additional charge.

If the N-methyl-D-aspartate (NMDA) receptor antibody CBA result is positive, then the NMDA-receptor antibody IFA titer assay will be performed at an additional charge.

If the dipeptidyl-peptidase-like protein-6 (DPPX) antibody CBA result is positive, then the DPPX IFA titer will be performed at an additional charge.

If the Kelch-like protein 11 (KLHL11) CBA result is reactive, then the KLHL11 antibody IFA titer will be performed at an additional charge.

If the IFA pattern suggests metabotropic glutamate receptor 1 (mGluR1) antibody, then the mGluR1 antibody CBA and mGluR1 IFA titer will be performed at an additional charge.

If the IFA pattern suggests metabotropic glutamate receptor 5 (mGluR5) antibody, then the mGluR5 antibody CBA and mGluR5 IFA titer will be performed at an additional charge.

If the IFA pattern suggests neurochondrin antibody, then the neurochondrin antibody CBA and neurochondrin IFA titer will be performed at an additional charge.

If the IFA pattern suggests tripartite motif-containing protein 46 (TRIM46) antibody, then TRIM46 antibody CBA and TRIM46 IFA titer will be performed at an additional charge.

If the IFA pattern suggests phosphodiesterase 10A (PDE10A) antibody, then PDE10A antibody IFA titer will be performed at an additional charge.

For more information see:

-Autoimmune/Paraneoplastic Epilepsy Evaluation Algorithm-Serum

-Epilepsy: Unexplained Refractory and/or Familial Testing Algorithm

Antiepileptic drugs (AED) are the mainstay of treatment for epilepsy, but seizures continue in one-third of patients despite appropriate AED therapeutic trials. The etiology of epilepsy often remains unclear. Seizures are a common symptom in autoimmune neurological disorders, including limbic encephalitis and multifocal paraneoplastic disorders. Seizures may be the exclusive manifestation of an autoimmune encephalopathy without evidence of limbic encephalitis.

Autoimmune epilepsy is increasingly recognized in the spectrum of neurological disorders characterized by detection of neural autoantibodies in serum or spinal fluid (CSF) and responsiveness to immunotherapy. The advent of more sensitive and specific serological detection methods is increasingly revealing previously underappreciated autoimmune epilepsies. Neural autoantibodies specific for intracellular and plasma membrane antigens aid the diagnosis of autoimmune epilepsy, but no single antibody is specific for this diagnosis.

Autoantibody specificities most informative for autoimmune epilepsies include leucine-rich glioma inactivated protein-1 (LGI1), glutamic acid decarboxylase-65 (GAD65), N-methyl-D-aspartate receptor (NMDA-R), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-R), and gamma-aminobutyric acid type B receptor (GABA-B-R) antibodies.

Autoantibodies recognizing onconeural proteins shared by neurons, glia, or muscle (eg, antineuronal nuclear antibody, type 1 [ANNA 1]; collapsin response-mediator protein-5 neuronal [CRMP-5-IgG]; N-type calcium channel antibody) also serve as markers of paraneoplastic or idiopathic autoimmune epilepsies. A specific neoplasm is often predictable by the individual patient's autoantibody profile.

Suspicion for autoimmune epilepsy on clinical grounds justifies comprehensive evaluation of CSF and serum for neural autoantibodies. Selective testing for individual autoantibodies is not advised because each is individually rare, and a timely diagnosis is critical. Collectively, the antibodies tested for in the autoimmune epilepsy evaluations represent a broad spectrum of treatable disorders, some of which are associated with occult cancer. Testing of CSF for autoantibodies is particularly helpful when serum testing is negative, although, in some circumstances, testing both serum and CSF simultaneously is pertinent. Testing of CSF is recommended for some antibodies (eg, NMDA-R antibody and glial fibrillary acidic protein [GFAP]-IgG) because CSF testing is both more sensitive and specific. In contrast, serum testing for LGI1 antibody is more sensitive than CSF testing. Failure to detect a neural antibody does not exclude the diagnosis of autoimmune epilepsy when other clinical clues exist. A trial of immunotherapy is justifiable in those cases.

Reflex Information:

*Methodology abbreviations:

Cell-binding assay (CBA)

Enzyme-linked immunosorbent assay (ELISA)

Immunoblot (IB)

Immunofluorescence assay (IFA)

Radioimmunoassay (RIA)

Western Blot (WB)

Antibodies specific for neuronal, glial, or muscle proteins are valuable serological markers of autoimmune epilepsy and of a patient's immune response to cancer. These autoantibodies are not found in healthy subjects and are usually accompanied by subacute neurological symptoms and signs. It is not uncommon for more than 1 of the following autoantibodies to be detected in patients with autoimmune dementia:

-Plasma membrane antibodies (N-methyl-D-aspartate [NMDA] receptor; 2-amino-3-[5-methyl-3-oxo-1,2-oxazol-4-yl] propanoic acid [AMPA] receptor; gamma-amino butyric acid [GABA-B] receptor); these autoantibodies are all potential effectors of dysfunction

-Antineuronal nuclear antibody, type 1 (ANNA-1) or type 3 (ANNA-3)

-Neuronal or muscle cytoplasmic antibodies (amphiphysin, Purkinje cell antibody-type 2 [PCA-2], collapsin response-mediator protein-5 neuronal [CRMP-5-IgG], or glutamic acid decarboxylase [GAD65] antibody)

Negative results do not exclude autoimmune epilepsy or cancer.

Intravenous immunoglobulin treatment prior to the serum collection may cause a false-positive result.

1. Smith KM, Britton JW, Thakolwiboon S, et al. Seizure characteristics and outcomes in patients with neurological conditions related to high-risk paraneoplastic antibodies. Epilepsia. 2023;64(9):2385-2398. doi:10.1111/epi.17695

2. Garrido Sanabria ER, Zahid A, Britton J, et al. CASPR2-IgG-associated autoimmune seizures. Epilepsia. 2022;63(3):709-722. doi:10.1111/epi.17164

3. Smith KM, Zalewski NL, Budhram A, et al. Musicogenic epilepsy: Expanding the spectrum of glutamic acid decarboxylase 65 neurological autoimmunity. Epilepsia. 2021;62(5):e76-e81. doi:10.1111/epi.16888

4. Steriade C, Britton J, Dale RC, et al. Acute symptomatic seizures secondary to autoimmune encephalitis and autoimmune-associated epilepsy: Conceptual definitions. Epilepsia. 2020;61(7):1341-1351. doi:10.1111/epi.16571

5. Dubey D, Singh J, Britton JW, et al. Predictive models in the diagnosis and treatment of autoimmune epilepsy. Epilepsia. 2017;58(7):1181-1189. doi:10.1111/epi.13797

Cell-Binding Assay:

Patient specimen is applied to a composite slide containing transfected and nontransfected EU90 cells. After incubation and washing, fluorescein-conjugated goat-antihuman IgG is applied to detect the presence of patient IgG binding.(Klein CJ, Lennon VA, Aston PA, et al. Insights from LGI1 and CASPR2 potassium channel complex autoantibody subtyping. JAMA Neurol 2013;70[2]:229-234; package insert: IIFT: Neurology Mosaics, Instructions for the indirect immunofluorescence test. EUROIMMUN; FA_112d-1_A_UK_C13, 02/25/2019)

Indirect Immunofluorescence Assay:

The patient's specimen is tested by a standardized immunofluorescence assay that uses a composite frozen section of mouse cerebellum, kidney, and gut tissues. After incubation with the specimen and washing, fluorescein-conjugated goat-antihuman IgG is applied. Neuron-specific autoantibodies are identified by their characteristic fluorescence staining patterns. Specimens that are scored positive for any neuronal nuclear or cytoplasmic autoantibody are titrated. 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;4[5]:e385. Published 2017 Jul 18. doi:10.1212/NXI.0000000000000385)

Radioimmunoassay:

(125)I-labeled recombinant human antigens or labeled receptors are incubated with patient specimen. After incubation, anti-human IgG is added to form an immunoprecipitate. The amount of (125)I-labeled antigen in the immunoprecipitate is measured using a gamma-counter. The amount of gamma emission in the precipitate is proportional to the amount of antigen-specific IgG in the specimen. Results are reported as units of precipitated antigen (nmol) per liter of patient specimen.(Griesmann GE, Kryzer TJ, Lennon VA. Autoantibody profiles of myasthenia gravis and Lambert-Eaton myasthenic syndrome. In: Rose NR, Hamilton RG, eds. Manual of Clinical and Laboratory Immunology. 6th ed. ASM Press; 2002:1005-1012; 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[12]:1161-1166; Jones AL, Flanagan EP, Pittock SJ, et al. Responses to and outcomes of treatment of autoimmune cerebellar ataxia in adults. JAMA Neurol. 2015;72[11]:1304-1312. doi:10.1001/jamaneurol.2015.2378)

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;49[2]:146-154; Dubey D, Jitprapaikulsan J, Bi H, et al. Amphiphysin-IgG autoimmune neuropathy: A recognizable clinicopathologic syndrome. Neurology. 2019;93[20]:e1873-e1880. doi:10.1212/WNL.0000000000008472)

Enzyme-Linked Immunosorbent Assay:

Ma2 antibodies are directed against their corresponding intracellular protein (PNMA2). The Ma2 autoantibody enzyme-linked immunosorbent assay (ELISA) is based on the principle of indirect ELISA that employs the ability of Ma2 autoantibodies to bind to antigenic protein (PNMA2) coated on the well surface of an ELISA plate. Detection of this bound antibody is accomplished using an antihuman secondary antibody that specifically recognizes the Fc region of the bound autoantibody. The secondary antibody is conjugated with alkaline phosphatase that enzymatically hydrolyzes a substrate molecule to produce an end product with a yellow color measurable at a wavelength of 405 nm. Controls and diluted samples are added in duplicate to coated plate wells and incubated for 2 hours, then washed extensively before adding the conjugated secondary antibody for an additional hour. After aspiration and washing, p-nitrophenyl phosphate substrate is added to each well and incubated for an additional hour before absorbance is measured at 405 nm.(Unpublished Mayo method)

Immunoblot for AGNA-1, amphiphysin, ANNA-1, ANNA-2, and PCA-Tr:

All steps are performed at room temperature (18 to 28 degrees C) utilizing the EUROBlot One instrument. Diluted patient sample is added to test strips (strips containing recombinant antigen manufactured and purified using biochemical methods) in individual channels and incubated for 30 minutes. Positive samples will bind to the purified recombinant antigen and negative samples will not bind. Strips are washed to remove unbound antibodies and then incubated with anti-human IgG antibodies (alkaline phosphatase-labelled) 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;6[3]:e552. doi:10.1212/NXI.0000000000000552)

Immunoblot for MAP1B:

Test strips are incubated with appropriately diluted sample, prepared to meet the assay's required end-dilution for each specimen type. Samples and controls are added directly to the strips, ensuring they are fully covered with dilution buffer, then mixed gently and incubated for 60 minutes at room temperature on a rocking platform. After incubation, the strips undergo a series of wash steps using diluted wash buffer, followed by a 30-minute incubation with a ready-to-use alkaline phosphatase-labeled IgG conjugate. The strips are washed again using the same procedure, then incubated with a ready-to-use substrate solution for 20 minutes at room temperature. The reaction is stopped by rinsing with distilled water through several wash cycles. Strips are then placed on absorbent material and allowed to dry fully, generally requiring at least two hours. Results are interpreted only after the strips have dried completely, and dried strips are stored protected from light.(Package insert: PCA-2 line assay. ravo Diagnostika GmbH; 05/2023)

Profile tests: Monday through Sunday; Reflex tests: Varies

• Authorized users can sign in to Test Prices for detailed fee information.
• Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
• Prospective clients should contact their account representative. For assistance, contact Customer Service.

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

86255 x 21

86341

0432U

83516

84182 AGNBS (if appropriate)

86256 AGNTS (if appropriate)

86255 AK5CS (if appropriate)

86256 AK5TS (if appropriate)

86256 AMPIS (if appropriate)

86256 APHTS (if appropriate)

84182 AMIBS (if appropriate)

84182 AN1BS (if appropriate)

86256 AN1TS (if appropriate)

84182 AN2BS (if appropriate)

86256 AN2TS (if appropriate)

86256 AN3TS (if appropriate)

86256 CRMTS (if appropriate)

84182 CRMWS (if appropriate)

86256 DPPTS (if appropriate)

86256 GABIS (if appropriate)

86256 K11TS (if appropriate)

84182 MP1BS (if appropriate)

86255 GL1CS (if appropriate)

86256 GL1TS (if appropriate)

86255 GL5CS (if appropriate)

86256 GL5TS (if appropriate)

86255 NCDCS (if appropriate)

86256 NCDTS (if appropriate)

86256 NMDIS (if appropriate)

86256 PC2TS (if appropriate)

84182 PCTBS (if appropriate)

86256 PCTTS (if appropriate)

86256 PDETS (if appropriate)

86255 T46CS (if appropriate)

86256 T46TS (if appropriate)