What is the significance of neuronal antibody testing in cases suspected of autoimmune encephalitis?

Autoimmune encephalitis is a rapidly progressive and potentially debilitating neurological disorder characterized by inflammation of the brain. It often involves antibodies targeting neuronal cell-surface or synaptic proteins. Neuronal antibody testing is pivotal for making a definite diagnosis of autoimmune encephalitis however, it must be interpreted within the appropriate clinical context.1-3 For a comprehensive outline of diagnostic criteria and clinical management, please refer to the Mellen Center Approach for Diagnosis and Management of Autoimmune Encephalitis.

How is neuronal antibody testing performed?

Various testing methods are used, each method has its advantages and limitations, so a combination of 2 techniques (screening followed by confirmatory testing) is typically employed to maximize diagnostic yield.

  1. Tissue-Based Indirect Immunofluorescence (TIIF): This is often the first screening step. TIIF involves incubating patient serum or cerebrospinal fluid (CSF) on fresh-frozen or formalin-fixed composite sections of rodent brain tissue (e.g., cortex, hippocampus, cerebellum) and non-neuronal tissues. Antibodies bound to tissue antigens are visualized using fluorescent-labeled anti-human secondary antibodies.1-2,4
  2. Western Blot and Line Blot: These methods are commonly used as confirmatory tests following a positive TIIF result. They employ recombinant antigens immobilized on blot strips, allowing for the screening of multiple well-characterized neuronal autoantibodies in a single assay. While these assays provide valuable antigen-specific confirmation, their results should be interpreted with caution when used in isolation.1,4
  3. Enzyme-Linked Immunosorbent Assay (ELISA): Plays a role in detection of glutamic acid decarboxylase 65 (GAD65) antibody. However, commercial ELISA kits may produce false-positive results, particularly at low antibody titers.1
  1. Radioimmunoprecipitation Assay (RIPA): RIPA is particularly useful for quantifying low-concentration antibodies, such as in CSF samples where even very low titers of antibody carry clinical significance. There is a risk of false positives due to nonspecific immunoglobulins’ binding.1-2
  1. Cell-Based Assays (CBA): Cell-based assays are currently the gold standard for detecting antibodies against neuronal surface antigens. These assays use transfected cells expressing the target antigen in its native, conformationally intact state on the cell surface. CBAs have a very low false-positive rate (approximately 0.2%), making them a reliable choice for clinical diagnostics.2,5.
    • Fixed CBAs use transfected cells that are fixed and incubated with patient samples. These assays are convenient for storage and widely available in clinical laboratories. However, fixation can alter the antigen conformation, reducing sensitivity and potentially impacting test performance.1,6
    • Live CBAs preserve the antigen’s native structure, resulting in higher sensitivity and specificity. However, the assays are technically complex and limited to specialized or academic centers.6

Should neuronal antibodies be tested in serum or CSF?

A critical consideration for accurate detection of neuronal autoantibodies is to obtain both CSF and serum sample for analysis. Each specimen provides complementary diagnostic value: CSF generally offers higher specificity and a lower false-positive rate, though it may fail to detect certain antibodies, such as those targeting leucine-rich, glioma inactivated 1 protein (LGI-1).7 Conversely, serum testing typically demonstrates greater sensitivity due to higher antibody titers but is more prone to false-positive results, particularly with antibodies such as contactin-associated protein-like 2 (CASPR-2). In contrast, N-methyl-D-aspartate receptor (NMDAR), glial fibrillary acidic protein (GFAP), antibodies are more sensitively detected in CSF.8-9 Ideally, neuronal antibody testing should be performed prior to initiating therapies such as plasma exchange, intravenous immunoglobulin (IVIg) or intravenous methylprednisolone (IVMP) to avoid potential impact on test results.10

Is there any significance of neuronal antibody titers?

Neuronal antibody titers have been demonstrated to be important for specificity of diagnosis/ clinical relevance in a few scenarios. For instance, GAD65 serum titers exceeding 20 nmol/L using RIPA, and any detectable level in CSF, are considered to have clinical relevance in neurological syndromes.11 Similarly, CASPR-2 cell-based assay, titers of 1:100 have higher positive predictive values than values of 1:10.8,12 Overall, low antibody titers should be interpreted with caution.

Is there a role for serial titers or repeated neuronal antibody testing in monitoring of autoimmune encephalitis?

Serial measurements of neuronal antibody titers in serum or CSF have not been validated as reliable markers for monitoring disease activity, treatment effect or prognosticating outcomes, but studies are limited to date.13

Repeated neuronal antibody testing has limited utility following initial negative results.14 However, if there is a significant change in the patient's clinical status, such as new or worsening neurological symptoms, initial testing was performed after starting immunotherapy, or if a specific antibody that was not included in the initial panel is highly suspected, repeat testing may be justified.6,14

What are the preferred neuronal antibody testing panels?

Mayo Clinic laboratory testing is utilized at our center; however, other centers may use different laboratories, such as Associated Regional and University Pathologists (ARUP) and Quest Diagnostics. Therefore, it is important to remain cognizant of the specific testing methodologies employed by each laboratory. Commonly used diagnostic panels at our center include:

  1. Autoimmune/Paraneoplastic Encephalopathy Evaluation – Serum/CSF (ENS2/ENC2): This comprehensive panel includes majority of currently recognized neuronal antibodies implicated in autoimmune and paraneoplastic encephalopathies. In contrast, the paraneoplastic autoantibody panel alone includes a limited number of neuronal antibodies and therefore has reduced breadth.
  2. Autoimmune/Paraneoplastic Movement Disorder Evaluation – Serum/CSF (MDS2/MDC2): This panel is used to evaluate patients with suspected autoimmune or paraneoplastic movement disorders, including dyskinesias, myoclonus, chorea, parkinsonism, and ataxia. Unique antibodies included in this panel are:
    • P/Q-type voltage-gated calcium channels, GTPase Regulator Associated with Focal Adhesion Kinase-1(GRAF1), Inositol 1,4,5-trisphosphate receptor type 1 (ITPR1), Kelch-like protein 11 (KLHL11), Adaptor Protein 3 beta2 (AP3B2), and Septin 5 and 7.
  3. Stiff-Person Spectrum Disorders Evaluation – Serum/CSF (SPPS/SPPCI): This targeted panel is designed for the evaluation of stiff-person syndrome and its spectrum disorders, including progressive encephalomyelitis with rigidity and myoclonus (PERM). Key antibodies included are:
    • GAD65, glycine receptor, dipeptidyl-peptidase-like protein, and amphiphysin.
  4. Autoimmune/Paraneoplastic Myelopathy Evaluation – Serum/CSF (MAS1/MAC1): This panel supports the diagnosis of autoimmune or paraneoplastic myelopathies, including transverse myelitis and longitudinally extensive spinal cord lesions. It includes antibodies to myelin oligodendrocyte glycoprotein (MOG) and aquaporin-4 (AQP4).
  5. Autoimmune/Paraneoplastic Epilepsy Evaluation – Serum/CSF (EPS2/EPCS): Recommended for patients with new-onset cryptogenic epilepsy of less than two years duration, especially those with poor seizure control.
  6. Autoimmune/Paraneoplastic Dementia Evaluation – CSF (DMS2/DMC2): Designed for patients with rapidly progressive cognitive decline or atypical dementia presentations.

There is considerable overlap among the available antibody panels, particularly in the detection of key neuronal autoantibodies. However, some panels are more targeted and tailored to specific clinical syndromes such as the stiff person spectrum disorder panel.14-16 Refer to the hyperlinks included for serum and CSF comprehensive autoimmune encephalopathy panels performed at Mayo Clinic, ARUP, and Quest laboratory. These panels are updated frequently.

Which neuronal antibodies need to be ordered separately?

  1. Ma2 antibody: Ma2 is an onconeural antibody associated with paraneoplastic neurologic syndromes (PNS), most commonly in patients with testicular germ cell tumors, though other malignancies may also be involved. Clinical presentations often include limbic, brainstem, and/or hypothalamic encephalitis, manifesting as memory loss, behavioral changes, sleep disturbances, and oculomotor dysfunction.17

Ma2 Antibody by ELISA, Serum - Mayo Clinic Laboratories | Neurology Catalog

  1. GABA-A receptor antibody: GABA-A receptor encephalitis should be considered in patients presenting with encephalopathy and seizures, particularly when seizures are refractory to antiepileptic medications. Neuroimaging in these patients typically reveals multifocal, large T2 signal abnormalities in both temporal and extra-temporal cerebral regions, usually without contrast enhancement.18 Currently, GABA-A receptor antibody is available only for research testing via Mayo clinic lab.

What are the caveats in the interpretation of neuronal antibody testing?

Several caveats in the interpretation of neuronal antibody testing may contribute to misdiagnosis of autoimmune encephalitis.19

  1. Low titer positive serum GAD65 antibody
  2. Voltage-gated potassium channel complex antibody in setting of negative for LGI-1 & CASPR-2
  3. Immunoblot or line blot antibody positivity in isolation
  4. Low titer positive serum CASPR-2 antibody
  5. Antibody detection in non-certified laboratories.20

Table 2: Summary of recommendations for neuronal antibody testing

  • Appropriate testing should be based on diagnostic criteria for autoimmune encephalitis or paraneoplastic syndrome
  • Paired serum and CSF samples should be ordered (ideally prior to initiating immunotherapy)
  • Avoid overlapping diagnostic panels with similar antibodies (e.g. ordering both ENS2 and EPS2 panels)
  • Avoid ordering “miscellaneous send out tests” as they may not be visible to all providers
  • Avoid ordering same diagnostic panels from different laboratories
  • Avoid repeat neuronal antibody testing unless:
  • Significant change in clinical presentation
  • Specific antibody not included in initial testing
  • Isolated antibody testing is reserved for certain conditions *
  • Low antibody titers should be interpreted with caution

* Anti- Ma-2 associated encephalitis, KLHL11 antibody-associated paraneoplastic neurologic syndrome and GABA-A receptor encephalitis. At our center, isolated GAD65 testing is performed using ELISA; however, when ordered as part of the stiff person spectrum disorder panel, it is performed using radioimmunoprecepitation assay.

References:

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  8. Bien CG, Mirzadjanova Z, Baumgartner C, et al. Anti-contactin-associated protein-2 encephalitis: relevance of antibody titres, presentation and outcome. Eur J Neurol. 2017;241:175-186. doi:10.1111/ene.13180
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  10. Grüter T, Möhn N, Schwabe K, et al. Effects of IVIg treatment on autoantibody testing in neurological patients: marked reduction in sensitivity but reliable specificity. J Neurol. 2020;2673:715-720. doi:10.1007/s00415-019-09614-4
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  16. Mayo Clinic Laboratories. The evolution of autoimmune neurology. Mayo Clinic Laboratories. https://news.mayocliniclabs.com/autoimmune-neurology-evolution/
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