**4. Clinical features of epilepsy-associated autoimmune encephalitis**

Each of the currently known neuronal cell surface or synaptic autoantibody associates with a specific syndrome or limited set off symptoms (**Table 1**). NMDAR antibody-associated encephalitis is a recently described disorder in which infrequent seizures are associated with the presence of autoantibodies directed against the extracellular domain of the NR1 subunit of the NMDAR. This disorder was first described as a clinical entity in 2005, in one in four young women who developed acute psychiatric symptoms, seizures, memory deficit, in association with the presence of an ovarian teratoma. In a study of 100 patients, it was shown that although the majority are young women (mean age 23 years), the disorder could occur in men and in children. This fact has allowed the number of pediatric cases to grow steadily and appears to represent approximately 40% of all cases [27, 37, 38].

#### **4.1. NMDA receptor**

Encephalitis associated with antibodies against GABAB1 receptor is generally presented as limbic encephalitis, as well as drug-refractory seizures. In a series of 15 patients, the mean age of presentation was 62 years (range 24–75) and both sexes were similarly affected. About half of the patients had an associated tumor, either a small cell lung carcinoma or a neuroendocrine lung tumor. These patients usually have antibodies to various non-neuronal proteins of

**Figure 1.** (A) Structure of AMPA receptor subunits. The transmembrane topology is shown, along with the flip/flop alternatively spliced exon, and the two ligand-binding domains (S1 and S2). Glycosylation sites are shown as trees in the N-terminal region; this region is associated with immune response. (B) Flow cytometry demonstrates the presence of T lymphocytes of the CD8+ class with greater activation, as well as B lymphocytes; here it can be known that the immune process has extravasated to the cerebral parenchyma, (C) and (D) the tissue based assay. Mouse brain tissue sections, such as hippocampus are stained with the patient's serum or CSF by indirect immunoperoxidase technique. (C) Shows CSF immunoreaction at the hippocampus level of the cytoplasmic and a neuronal surface in D (anti-human IgG-Px, Abcamab97225). (E) TBA in F, shown a reaction at neuronal surface level that colocalizes with GAD65 / 67 (Alexafluor 546, Invitrogen Molecular probes). (G) Immunoblot, CSF recognizes 100 and 50 Kd proteins(Anti-human IgG-Px, Abcam- ab97225).

In the knockout mice to the GABAB1 receptor, a variety of neurological and behavioral alterations are found, including spontaneous seizures, increased anxiety, hyperactivity, hyperalge-

In contrast, patients present with limbic encephalitis in conjunction with antibodies to the AMPAR were not present with seizures as frequently: only 3/10 had seizures as presenting

sia and memory impairment, suggesting a dysfunction of the limbic system [31, 32].

uncertain significance, which suggests a susceptibility to autoimmunity [30].

feature with one other patient having seizures after a relapse [33].

18 Seizures

Symptoms of anti-NMDA receptor encephalitis develop and resolve in a multi-stage process; most patients experience a prodromal similar to a viral picture, which is followed by a pattern


**Epilepsyassociated** 

**Anti-LGI1**

**Anti-CASPR2**

**LGI1 >**

**CASPR2** 

**NMDAR**

**GAD**

**GABABR**

**AMPA**

**vs.**

**GluR1/2**

**vs.**

**GABABR**

**vs.**

**GAD-65**

**(VGKC-complex)**

**vs.**

**Subunit NR1**

**vs.**

**vs.**

**CASPR2**

**vs.**

**LGI1 >**

**CASPR2**

**LGI1**

**(***Channels Kv+***)**

**Treatment and** 

Good response to

Good response to

Good response to

Slow response to

Refractory to

Good response to

Good response to

immunotherapy

with recurrence

immunotherapy

treatment with

AED's and

immunotherapy

Immunotherapy

with recurrence

immunotherapy

immunotherapy

immunotherapy

**prognosis**

**References**

[49, 54–56]

[31]

[31, 63]

[31, 55, 58, 65, 67]

NMDAR, N-methyl-d-aspartate receptor; LGI1, leucine-rich glioma-inactivated 1; CASPR2, contactin-associated protein-like 2; AMPAR, amino-3-hydroxy-5-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; GABA a/B R, gamma-aminobutyric acid A/B receptor; mGluR1/2, metabotropic glutamate receptor type 1/2; LE, limbic

, faciobrachial dystonic seizures; GTC, generalized tonic-clonic.

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encephalitis; SPS, stiff-person syndrome; CPS, complex partial seizure; EEG, electroencephalogram; FBDS

Neuronal cell surface autoantibodies, associated epilepsy, and the clinical symptoms.

**Table 1.**

[59–61]

[31, 41, 51, 57,

[34, 57, 61, 66]

62, 64]

**antibody**


**Epilepsyassociated** 

**Anti-LGI1**

**Anti-CASPR2**

**LGI1 >**

**CASPR2** 

**NMDAR**

**GAD**

**GABABR**

**AMPA**

20 Seizures

**vs.**

**GluR1/2**

**vs.**

**GABABR**

**vs.**

**GAD-65**

**(VGKC-complex)**

**vs.**

**Subunit NR1**

**vs.**

**vs.**

**CASPR2**

**vs.**

**LGI1 >**

**CASPR2**

**LGI1**

**(***Channels Kv+***)**

**Gender/Age of** 

M > F

M > F

M > F

F > M

F > M

M > F

F > M >40 years

>40 years

70% childhood

Viral pathway:

Diabetes mellitus

LE

LE

It is associated

It is associated

with the presence

of SCLC, thymus

and breast cancer

tumors

with the presence

of SCLC

type 1, Stiffperson syndrome,

fever, headache and

fatigue of infectious

etiology, delirium

cerebellar ataxia,

non-paraneoplastic

LE

and disorientation.

>20 years

>50 years

**involvement**

**Clinical** 

Hyponatremia.

Morvan

TLE

Hyponatremia and

Synchronus dystonic

arm posturing and

grimacing facial

ipsilateral associated

10–20 days of evolution: orofacial

Severe cognitive

impairment

dyskinesias,

choreoatetotic

movements,

nystagmus,

decreased

consciousness and

dysautonomia

**Psychiatric** 

Confusion and

Cognitive

Sub-acute amnesia,

Personality changes,

Depression and

Cognitive

Confusion,

amnesia,

disorientation and

impairment,

behavioral

disorders such

psychosis

as psychosis and

hallucinations

hallucinations (visual

anxiety.

and auditory),

difficulty speaking

confusion, sleep

disorders, psychosis,

anxiety, personality

changes and

depression

impairment,

memory loss and

hallucinations

behavior and

REM sleep

disorders

**Seizure** 

GTC

GTC

FBDS

GTC

CPS.

CPS

GTC CPS

GTC

SE

CTsG

CPS

SE refractory to

treatment

GTC

CPS

**activity**

**Electrographic** 

Slow focal or

Slow focal or

Slow focal or

Focal or diffuse

Slow focal or

Slow focal or

Focal activity

generalized

activity

generalized

activity

delta/theta activity

and delta brush

activity

generalized activity

generalized

activity

generalized

activity

**activity (EEG)**

**comorbidity**

with paraneoplasias

(thymomas and lung

cancer (SCLC)).

syndrome

Complication

with

Cognitive

impairment.

It is associated

with the presence

Myasthenia

of a thymoma or

gravis

Not associated

with neoplasias

SCLC

**manifestations**

**antibody**

**Table 1.** Neuronal cell surface autoantibodies, associated epilepsy, and the clinical symptoms.

encephalitis; SPS, stiff-person syndrome; CPS, complex partial seizure; EEG, electroencephalogram; FBDS

5-methyl-4-isoxazolepropionic acid receptor; GABA a/B R, gamma-aminobutyric acid A/B receptor; mGluR1/2, metabotropic glutamate receptor type 1/2; LE, limbic

, faciobrachial dystonic seizures; GTC, generalized tonic-clonic.

of memory alterations, behavior, cognition, developing psychotic pictures, convulsions, dyskinesia (orofacial, trunk, and limb), and autonomic respiratory instability. Most adults are initially seen by psychiatric services and may be confused with acute psychotic disturbance or drug abuse. Most children are taken to medical care due to changes in mood, behavior and/ or personality, seizures, or language impairment [38–40]. Autonomic instability is a common manifestation in adults. Some patients develop severe cardiac arrhythmias that require the use of pacemakers. Signs of more frequent autonomic dysfunction in children include urinary incontinence and sleep disturbances [38]. Nuclear magnetic resonance (MRI) findings in these patients may be hyperintensities in FLAIR or T2 sequences in the cerebral cortex, cerebellar or temporal medial lobes, as well as in the corpus callosum and brainstem. In some cases, a transient increase in contrast, of the cerebral cortex, cerebellum, basal ganglia and meninges is observed.

symptoms. The most common disorder effects were in the middle-aged women. Most patients present with a sub-acute appearance of confusion, disorientation and memory loss, and seizures may also be part of the clinical describe. About 70% of patients have an underlying tumor in the lung, breast, or thymus. AMPAR is the predominant receptor subtype in the hippocampus, and it has been found that these antibodies in patients caused a decrease in the pre- and postsynaptic GluR1/2 receptor groups in cultures of rat hippocampal neurons. Since the receptor levels have been more affected at synapses than along dendrites, the findings suggested a mechanism by which patients' antibodies disrupted the receptor traffic, moving them from synaptic sites to extra-cellular sites and intracellular pool. These effects are similar to neuronal plasticity models that decrease synaptic strength, also called long-term depression.

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Glutamic acid decarboxylase (GAD) is a cytoplasmic enzyme that catalyzes the conversion of l-glutamic acid to gamma-aminobutyric acid (GABA), considered the main inhibitory neurotransmitter of the central nervous system. GAD is expressed primarily in GABAergic neurons and in pancreatic β cells, and has two isoforms with different molecular weight; GAD65 and GAD67. GAD antibodies act as a marker of the underlying autoimmune disease, although it is not known how antibodies against an intracellular enzyme can directly initiate pathological events; however, it is known that anti-GAD Abs inhibit the activity of GAD, and the synthesis of GABA antibodies to GAD is associated with several autoimmune disorders, including limbic encephalitis [44, 45], type 1 diabetes mellitus [46]. Stiff Person Syndrome (SPS) [47], and cerebellar ataxia [48], as well as overlapping syndromes. Recent work highlighting the response of these patients to immunotherapy and association with forms of epilepsy related to localization suggest that antibodies may also be present with specific cell surface. This is supported by a functional study of magnetic resonance spectroscopy in patients with TLE and elevated levels of serum GAD antibodies that demonstrated significantly lower GABA levels within their cortex compared to paired control patients [44]. On the other hand, in one study with 138 patients over 18 years old, investigated with recent onset epilepsy, were prospectively studied to determine the clinical and radiological characteristics of LE, and response to treatment. Fifty-three adult patients fulfilled the criteria for LE; nine had high-titer GAD antibodies and ten had voltage-controlled potassium channel (VGKC) antibodies. Patients with GAD antibodies were younger's (range, 17–66 years) and had seizures only, whereas polymorphic limbic features were more frequent in the VGKC positive group. Patients with anti-GAD antibodies had more frequently oligoclonal bands of cerebrospinal fluid and intrathecal secretion of the specific antibody. Which after monthly, patients were treated with intravenous methylprednisolone pulses, however GAD antibodies remained elevated in 6/6 patients, however VGKC antibodies normalized in 6/9 patients (p < 0.03). Despite the more intense anticonvulsant treatment in the group with anti-GAD antibodies (p < 0.01), none of these patients were seizure free, unlike all patients with VGKC antibodies (p < 0.001). High-titer GAD antibodies define a form of non-paraneoplasic LE. It is a chronic non-persistent disorder, and should be included in the differential

The effects of the antibodies were shown to be reversible [42, 45].

diagnosis of patients with LE and mediotemporal encephalitis [49].

**4.4. GAD**

Movement disorders are common and can be misinterpreted as a convulsive activity, the most common being dyskinesia, usually orofacial, choreoatetoid limb movements, rigidity, opisthotonos, or a combination of these. In most patients, EEG shows a slow generalized activity, disorganized without ictal discharges. These findings may overlap with ictal discharges in the EEG [41]. Niehusmann and colleagues [42] reported presence of NMDAR-antibodies in women (age range, 15–45 years), which had extra-temporal epilepsy, a reduction in level of consciousness and altered speech, as well as nystagmus, dyskinesia, dystonia, and hypoventilation. Clinical improvements in seizure frequency were seen in treatment of three patients treated with an immunomodulator such as corticosteroids and IVIg.

#### **4.2. GABAb**

GABA receptors are essential to inhibition. The presence of autoantibodies against these receptors has been associated with seizures and changes in memory and behavior. In a study with 15 patients with GABABR and LE antibodies (median age of 62 years, range 24–75 years), the clinical features where the presence of seizures, confusion and altering memory. Seizures were the predominant characteristic in 87%, and were mainly onset of temporal lobe with secondary generalization. 13% of patients presented epileptic status. CSF findings showed lymphocytic pleocytosis (n = 4) and MRI showed an increased signal, typical of LE. Clinical improvement was observed in 40% of patients who received IT alone and 20% who had IT, and 46% of patients were taken for a surgery to remove tumors. On the other hand, in a series of 15 patients, the mean age of presentation was 62 years (range 24–75) and both sexes were equally affected. About half of the patients had an associated tumor, either a small cell lung carcinoma or a neuroendocrine lung tumor. These patients often have additional antibodies to glutamic acid decarboxylase (anti-GAD) and several non-neuronal proteins of uncertain significance, suggesting a susceptibility to autoimmunity [32, 43].

#### **4.3. AMPA receptor**

Antibodies to the AMPAR have recently been described in patients with limbic encephalitis (LE). The AMPAR antibodies are the least frequent of these antibodies, however, also these patients develop a limbic dysfunction that may be associated with significant psychiatric symptoms. The most common disorder effects were in the middle-aged women. Most patients present with a sub-acute appearance of confusion, disorientation and memory loss, and seizures may also be part of the clinical describe. About 70% of patients have an underlying tumor in the lung, breast, or thymus. AMPAR is the predominant receptor subtype in the hippocampus, and it has been found that these antibodies in patients caused a decrease in the pre- and postsynaptic GluR1/2 receptor groups in cultures of rat hippocampal neurons. Since the receptor levels have been more affected at synapses than along dendrites, the findings suggested a mechanism by which patients' antibodies disrupted the receptor traffic, moving them from synaptic sites to extra-cellular sites and intracellular pool. These effects are similar to neuronal plasticity models that decrease synaptic strength, also called long-term depression. The effects of the antibodies were shown to be reversible [42, 45].

#### **4.4. GAD**

of memory alterations, behavior, cognition, developing psychotic pictures, convulsions, dyskinesia (orofacial, trunk, and limb), and autonomic respiratory instability. Most adults are initially seen by psychiatric services and may be confused with acute psychotic disturbance or drug abuse. Most children are taken to medical care due to changes in mood, behavior and/ or personality, seizures, or language impairment [38–40]. Autonomic instability is a common manifestation in adults. Some patients develop severe cardiac arrhythmias that require the use of pacemakers. Signs of more frequent autonomic dysfunction in children include urinary incontinence and sleep disturbances [38]. Nuclear magnetic resonance (MRI) findings in these patients may be hyperintensities in FLAIR or T2 sequences in the cerebral cortex, cerebellar or temporal medial lobes, as well as in the corpus callosum and brainstem. In some cases, a transient increase in contrast, of the cerebral cortex, cerebellum, basal ganglia and meninges

Movement disorders are common and can be misinterpreted as a convulsive activity, the most common being dyskinesia, usually orofacial, choreoatetoid limb movements, rigidity, opisthotonos, or a combination of these. In most patients, EEG shows a slow generalized activity, disorganized without ictal discharges. These findings may overlap with ictal discharges in the EEG [41]. Niehusmann and colleagues [42] reported presence of NMDAR-antibodies in women (age range, 15–45 years), which had extra-temporal epilepsy, a reduction in level of consciousness and altered speech, as well as nystagmus, dyskinesia, dystonia, and hypoventilation. Clinical improvements in seizure frequency were seen in treatment of three patients

GABA receptors are essential to inhibition. The presence of autoantibodies against these receptors has been associated with seizures and changes in memory and behavior. In a study with 15 patients with GABABR and LE antibodies (median age of 62 years, range 24–75 years), the clinical features where the presence of seizures, confusion and altering memory. Seizures were the predominant characteristic in 87%, and were mainly onset of temporal lobe with secondary generalization. 13% of patients presented epileptic status. CSF findings showed lymphocytic pleocytosis (n = 4) and MRI showed an increased signal, typical of LE. Clinical improvement was observed in 40% of patients who received IT alone and 20% who had IT, and 46% of patients were taken for a surgery to remove tumors. On the other hand, in a series of 15 patients, the mean age of presentation was 62 years (range 24–75) and both sexes were equally affected. About half of the patients had an associated tumor, either a small cell lung carcinoma or a neuroendocrine lung tumor. These patients often have additional antibodies to glutamic acid decarboxylase (anti-GAD) and several non-neuronal proteins of uncertain

Antibodies to the AMPAR have recently been described in patients with limbic encephalitis (LE). The AMPAR antibodies are the least frequent of these antibodies, however, also these patients develop a limbic dysfunction that may be associated with significant psychiatric

treated with an immunomodulator such as corticosteroids and IVIg.

significance, suggesting a susceptibility to autoimmunity [32, 43].

is observed.

22 Seizures

**4.2. GABAb**

**4.3. AMPA receptor**

Glutamic acid decarboxylase (GAD) is a cytoplasmic enzyme that catalyzes the conversion of l-glutamic acid to gamma-aminobutyric acid (GABA), considered the main inhibitory neurotransmitter of the central nervous system. GAD is expressed primarily in GABAergic neurons and in pancreatic β cells, and has two isoforms with different molecular weight; GAD65 and GAD67. GAD antibodies act as a marker of the underlying autoimmune disease, although it is not known how antibodies against an intracellular enzyme can directly initiate pathological events; however, it is known that anti-GAD Abs inhibit the activity of GAD, and the synthesis of GABA antibodies to GAD is associated with several autoimmune disorders, including limbic encephalitis [44, 45], type 1 diabetes mellitus [46]. Stiff Person Syndrome (SPS) [47], and cerebellar ataxia [48], as well as overlapping syndromes. Recent work highlighting the response of these patients to immunotherapy and association with forms of epilepsy related to localization suggest that antibodies may also be present with specific cell surface. This is supported by a functional study of magnetic resonance spectroscopy in patients with TLE and elevated levels of serum GAD antibodies that demonstrated significantly lower GABA levels within their cortex compared to paired control patients [44]. On the other hand, in one study with 138 patients over 18 years old, investigated with recent onset epilepsy, were prospectively studied to determine the clinical and radiological characteristics of LE, and response to treatment. Fifty-three adult patients fulfilled the criteria for LE; nine had high-titer GAD antibodies and ten had voltage-controlled potassium channel (VGKC) antibodies. Patients with GAD antibodies were younger's (range, 17–66 years) and had seizures only, whereas polymorphic limbic features were more frequent in the VGKC positive group. Patients with anti-GAD antibodies had more frequently oligoclonal bands of cerebrospinal fluid and intrathecal secretion of the specific antibody. Which after monthly, patients were treated with intravenous methylprednisolone pulses, however GAD antibodies remained elevated in 6/6 patients, however VGKC antibodies normalized in 6/9 patients (p < 0.03). Despite the more intense anticonvulsant treatment in the group with anti-GAD antibodies (p < 0.01), none of these patients were seizure free, unlike all patients with VGKC antibodies (p < 0.001). High-titer GAD antibodies define a form of non-paraneoplasic LE. It is a chronic non-persistent disorder, and should be included in the differential diagnosis of patients with LE and mediotemporal encephalitis [49].

#### **4.5. LGI**

Studies describing treatment of LGI1-antibody associated encephalopathy, LGI1 is a protein secreted by neurons that interact with pre- and postsynaptic receptors. LGI1 mutations have been associated with autosomal dominant temporal lobe epilepsy syndrome [50, 51]. Patients with antibodies against LGI1 develop alterations of memory, confusion, and seizures. The MRI results are typical of limbic encephalitis. Memory and cognitive deficits can be preceded by brief tonic seizures that can be confused for mimic myoclonic movements. Observational studies have provided evidence of a marked improvement with for high-dose steroids, IVIG and PLEX for patients with auto-antibodies to LGI1 and CASPR [37, 50–52]. In a retrospective study of 10 patients with high titers of VGKC-complex antibodies that had seizures and memory disorders, who received IVIG 2 g/kg/day, 100 mg prednisolone on alternate days and PLEX for 5 days, improvement was observed in frequency of seizures and cognition in six patients within 2 weeks to 12 months, correlating with reductions in antibody titers [37]. Earlier treatment, and possibly corticosteroids, appeared to provide greater benefits than before. This disorder had been included previously within the spectrum of antibodies against voltage-dependent potassium channels. Some patients develop hyponatremia and behavior or REM sleep disorders. Only 20% of cases are associated with a neoplasm, usually thymoma or small cell lung carcinoma.

In the cell brain adhesion test (CBA), cells (e.g., HEK293 cells) are transfected with the respective neural antigens (receptors, channels, etc.) and incubated with the CSF or serum of patients with an indirect immunofluorescence technique. Autoantibodies to the specifically expressed receptor result in the cell membrane marking cells, similar, primary cultures of hippocampal neurons can be used, with these methods autoantibodies are displayed on the surface of the

Autoimmune Epilepsy: New Development and Future Directions

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25

For the detection of classical intracellular and cytoplasmic antibodies, the immunoblot technique is used. Immunoblotting is the method that uses Abs to detect a specific protein from a mixture of several unrelated proteins separated by molecular weight. The diagnosis of antibodies with this technique involves several steps, including protein extraction of mice brain tissue followed by (30–50 μg) spitted proteins through electrophoresis, and transfer of a nitrocellulose membrane and overlapping of the primary antibody (the serum or CSF of the patient) and secondary on the membrane labeled with enzymes (**Figure 1(G)**) or fluorescent antibodies [54]. Recently, we introduced flow cytometry and have confirmed that in those patients with AE, a large presence of activated T and B cells is observed; in some cases, the CD8+ cells are dominated and in negative cases, there is no activation of lymphocytes (unpublished data); the results suggest that this tool could provide additional information on the patient's immune response (**Figure 1(B)**). On the basis of this data, the recommendation is to include both CSF and serum for citometric testing in patients with suspected autoimmune

Recently, several reports that associate CNS disorders with autoantibodies are directed against cell surface proteins, which are likely to be pathogens. Many of these conditions have seizures as an early and prominent feature, which are commonly refractory to conventional drugs. In contrast, a good response with immunotherapy is often observed. The studies in patients coincide in clinical manifestations, but not in autoantibodies. For this reason, CSF is crucial in the identification of new antigens, including NMDAR, AMPAR, GABABR, GABAARr, mGluR5, DPPX and LGI1, and Caspr2. Serum negativity is more likely with a milder form of the disease, presenting with clinical pictures of psychosis but not requiring intensive care, particularly if the antibodies are generated predominantly in the brain, which makes it necessary to standardize the diagnostic methods in order to be safer and to offer a timely diagnosis. The effects of antibodies on children (the effects of antibodies on hippocampal synapses) are different from that of adults; this may explain some of the differences in clinical pictures between adults and children. For this reason, the selection of patients for the autoimmune evaluation requires a high level of suspicion in the initial consultation. Since there have been currently no universally agreement upon diagnostic criteria for autoimmune epilepsies, the clinical evidence, such as the high frequency of seizures, psychiatric co-morbidity and resistance to AEDs, are important indicators to decide it. More studies are needed to identify early

neuronal membrane [53].

encephalitis.

**6. Concluding remarks**

autoantibodies and to perform preventive treatments.

#### **5. Diagnostic approach**

The laboratory diagnosis of AD depends on the identification of the clinical symptoms of the patient, their association with each disease and their correspondence with the detection of AA. For this reason, laboratory tests are of great importance for the evaluation of patients when an AE is suspected. The results can confirm the diagnosis, estimate the severity of the disease, and are useful to follow up its evolution and establish a prognosis. The presence of autoantibodies (AA) alone in a patient does not mean the diagnosis of an AD, the associated signs and symptoms help to achieve the definitive diagnosis and are of crucial importance. Analysis of CSF plays a central part in all diagnostic criteria for encephalitis, including infectious encephalitis, relevant antibodies might be found only in the CSF, because the repertoire of antibodies in the CSF and serum can be different in the same patient (e.g., NMDA receptor in CSF and serum) [52]. By other way, serological tests to detect AA have demonstrated the presence of AA in healthy individuals and in known non-EA patients and approximately half of all autoimmune encephalitis series are Ab-negative cases, so AA is a confirmatory diagnostic test, for this reason the diagnostic tests must be combined. Three basic research techniques are used for this purpose must include: tissue-based assay (TBA), cell-based assay (CBA), and immune-precipitation (IP; in-house). In the TBA, rat or mouse brains are stained with CSF or serum of patients with an indirect immunohistochemistry or immunofluorescence technique or the combination of two fluorophores, one that identifies the autoantibody and the other to the antigen and use of confocal microscopy (**Figure 1(C)–(F)**).

In the cell brain adhesion test (CBA), cells (e.g., HEK293 cells) are transfected with the respective neural antigens (receptors, channels, etc.) and incubated with the CSF or serum of patients with an indirect immunofluorescence technique. Autoantibodies to the specifically expressed receptor result in the cell membrane marking cells, similar, primary cultures of hippocampal neurons can be used, with these methods autoantibodies are displayed on the surface of the neuronal membrane [53].

For the detection of classical intracellular and cytoplasmic antibodies, the immunoblot technique is used. Immunoblotting is the method that uses Abs to detect a specific protein from a mixture of several unrelated proteins separated by molecular weight. The diagnosis of antibodies with this technique involves several steps, including protein extraction of mice brain tissue followed by (30–50 μg) spitted proteins through electrophoresis, and transfer of a nitrocellulose membrane and overlapping of the primary antibody (the serum or CSF of the patient) and secondary on the membrane labeled with enzymes (**Figure 1(G)**) or fluorescent antibodies [54]. Recently, we introduced flow cytometry and have confirmed that in those patients with AE, a large presence of activated T and B cells is observed; in some cases, the CD8+ cells are dominated and in negative cases, there is no activation of lymphocytes (unpublished data); the results suggest that this tool could provide additional information on the patient's immune response (**Figure 1(B)**). On the basis of this data, the recommendation is to include both CSF and serum for citometric testing in patients with suspected autoimmune encephalitis.
