Detection Methods for Autoantibodies in Suspected Autoimmune Encephalitis

Abstract

This review provides an overview on different antibody test methods that can be applied in cases of suspected paraneoplastic neurological syndromes (PNS) and anti-neuronal autoimmune encephalitis (AIE) in order to explain their diagnostic value, describe potential pitfalls and limitations, and discuss novel approaches aimed at discovering further autoantibodies. Onconeuronal antibodies are well-established biomarkers for PNS and may serve as specific tumor markers. The recommended procedure to detect onconeuronal antibodies is a combination of indirect immunohistochemistry on fixed rodent cerebellum and confirmation of the specificity by line assays. Simplification of this approach by only using line assays with recombinant proteins bears the risk to miss antibody-positive samples. Anti-neuronal surface antibodies are sensitive and specific biomarkers for AIE. Their identification requires the use of test methods that allow the recognition of conformation dependent epitopes. These commonly include cell-based assays and tissue based assays with unfixed rodent brain tissue. Tissue based assays can detect most of the currently known neuronal surface antibodies and thus enable broad screening of biological samples. A complementary testing on live neuronal cell cultures may confirm that the antibody recognizes a surface epitope. In patients with peripheral neuropathy, the screening may be expanded to teased nerve fibers to identify antibodies against the node of Ranvier. This method helps to identify a novel subgroup of peripheral autoimmune neuropathies, resulting in improved immunotherapy of these patients. Tissue based assays are useful to discover additional autoantibody targets that play a role in diverse autoimmune neurological syndromes. Antibody screening assays represent promising avenues of research to improve the diagnostic yield of current assays for antibody-associated autoimmune encephalitis.

Keywords: anti-neuronal antibodies; autoimmune encephalitis; cell-based assay; onconeuronal antibodies; paraneoplastic neurological syndrome; test methods; tissue-based assay.

Figure 1

Staining pattern of antibodies targeting intracellular antigens. Indirect immunohistochemistry (avidin-biotin peroxidase method) on rat cerebellum shows a specific staining pattern of intracellular antibodies: (A) Anti-Hu-antibodies label the cytoplasm and nuclei of Purkinje and granule cells. (B) Anti-Yo antibodies show labeling of the cytoplasm of Purkinje cells (arrows) and stellate and basket cells in the molecular layer. (C) Anti-Ri-antibodies show the same staining pattern like Hu-antibodies in the cerebellum (differentiation is possible by staining enteric neurons of the gut that are positive with anti-Hu but negative with anti-Ri-antibodies). (D) Anti-Tr/DNER antibodies strongly label the Purkinj cell somata and dendrites (arrows). (E) Anti-amphiphysin antibodies show an intensive synaptic staining pattern in the molecular layer of the cerebellum. (F) Anti-CV2-antibodies mark a subgroup of oligodendrocytes in the cerebellar cortex and white matter (arrows). (G) Anti-Ma1/2-antibodies show a dot-like staining pattern in large neurons of the brainstem (arrows). (H) Anti-GAD65-antibodies display a dot-like staining of the base of Purkinje cells and a rosette-like staining pattern in the granular layer of the cerebellar cortex (I) Anti-SOX1-antibodies stain the nuclei of Bergmann glia in the cerebellar cortex (arrows). (J) Serum of a healthy control remains negative. Magnification: (A–J): x400.

Figure 2

Screening of autoantibodies on teased nerve fibers in patients with peripheral neuropathies. Rat sciatic nerve fibers were immunostained with a polyclonal rabbit anti-CASPR2 antibody (red) and serum from a patient with (A) anti-CASPR2 antibodies (green), (B) anti-contactin1 antibodies (green), and (C) anti-neurofascin155/186 antibodies (green). CASPR2 labels the juxtaparanodal region of the node of Ranvier, contactin1 the paranodal and neurofascin155/186 the paranodal and nodal region. CNTN1, contactin1; NF155/186, neurofascin155/186; Scale bar = 10 μm.

Figure 3

Comparison of reactivity of different antibodies against cell surface antigens on different primary neuronal and glioneuronal cell cultures. (A) The serum of a patient with anti-IgLON5 antibodies shows an intensive labeling of live nonpermeabilized rat hippocampal neurons, (B) rat dorsal root ganglion cells (DRGs), and (C) dissociated rat retinal cell culture. In contrast, (D) the serum of a patient with anti-GABA(B)R antibodies labels hippocampal neurons but not (E) DRGs. (F) The retinal cell culture is strongly GABA(B)R positive. (G–I) A serum of a patient with anti-contactin1 antibodies labels all three types of cell cultures. A serum of a patient with (J) anti-aquaporin4 antibodies is negative on hippocampal neurons and (K) DRGs, but (L) labels the end feet membranes of GFAP-positive Müller cells (red: rabbit polyclonal anti-AQP4 antibody; green: serum of a patient with AQP4 antibodies; blue: mouse monoclonal anti-GFAP antibody). (M–O) A healthy control is negative. HCN, hippocampal neurons; DRG, dorsal root ganglion cells; retinal culture, dissociated rat retinal cell culture; CNTN1, contactin1; AQP4, aquaporin-4; CO, healthy control; Scale bar = 10 μm.