that this has become so serious

 

Neuronal antibodies

The autoimmune disorder of the nervous system linked to an underlying tumour has been known since the 18th century. This is due to an attempt by the immune system to subjugate the growth of the tumour expressing neuronal antigens. The unfortunate consequence is a cross reaction by the immune components against the nervous tissue resulting in rapid onset of a variety of neurological deficits known as Paraneoplastic Neurological Syndrome. The resulting disability is considered irreversible because the nervous system lacks the capacity to regenerate following the immune onslaught. This phenomenon is rare, occurring at an approximate frequency of less than 1% and often accompanied by the presence of specific high-titre autoantibodies in both the cerebrospinal fluid and blood. These antibodies are non-pathogenic and are very useful early diagnostic markers of the brain disease and also, in some cases, underlying malignancy thus facilitating faster diagnosis and earlier treatment with better prognosis. 

Summary of paraneoplastic neurological antibodies

Antibody  MW (kDa) Staining pattern PND  Associated tumours
Recoverin 23, 65  Retinal photoreceptor  Retinopathy SCLC
Yo (PCA-1) 34, 52, 62  Purkinje cell cytoplasm & axons PCD Ovary, breast
Ma (Ma1) 37, 40  Neuronal nuclei PCD, BE  Various cancer
Ta (Ma2) 41.5  Neuronal nuclei, perikaryon PCD, LE Testicular cancer
Hu (ANNA1) 34-40  Nuclei of both central and peripheral neurones PCD, PEM, SN SCLC
Ri (ANNA2) 55, 80  Nuclei of central neurones OM, PCD, BE Breast, SCLC, gynaecological 
GAD  65, 67  Islet cells & grey matter SPS Breast, colon, SCLC
CV2/CRMP5  66  Oligodendrocytes cytoplasm PEM, SN SCLC, thymoma
Amphiphysin 128  Central presynaptic terminals  SPS, SN Breast cancer, SCLC 
mGluR1 ~140  Purkinje cell cytoplasm, climbing fibre PCD Hodgkin’s lymphoma
ANNA-3 170 Purkinje cell cytoplasm & nucleus + glomerular podocytes  PCD, PEM, SN SCLC
PCA-2 280 Purkinje cell cytoplasm and other neurones PEM, PCD, LEMS SCLC
AGNA ??  Nuclei of Bergmann glia of cerebellar Purkinje layer and glial in white matter  PND SCLC
Tr ?? Purkinje cell cytoplasm with “dots” in molecular layer PCD Hodgkin’s lymphoma


KEY: PND = paraneoplastic neurological disorder, PCD = paraneoplastic cerebellar degeneration, PEM = paraneoplastic encephalomyelitis, SN = sensory neuropathy, OM= opsoclonus/myclonus, BE = brainstem encephalomyelitis, LE = limbic encephalomyelitis, LEMS = Lambert-Eaton myasthenic syndrome, SPS = Stiff person syndrome, SCLC= small cell lung carcinoma. ?? = No common band has been identified by Western blot analysis

For the detection of paraneoplastic neurological antibodies, it is necessary to be familiar with cerebellar histology (see below).

Cerebellar architecture

The cerebellum consists of the white and the grey matter. The latter is subdivided into Molecular, Purkinje cell and Granular layer (contains densely packed granular cells|). The Purkinje cells can easily be identified due their large size (click on image). These are located on the border of granular and molecular layer.

The method of choice for detection of paraneoplastic antibodies is to screen the patient's serum on cerebellum and any positive reaction producing identifiable pattern is further confirmed by an alternative method such as line blot which consists of painted recombinant proteins.

Detection method for paraneoplastic antibodies

Antibodies produced in specific autoimmune diseases can be screened on an appropriate tissue using indirect immunofluorescence techniques.

PRINCIPLE: Autoantibodies, which attach to the appropriate antigen during an initial incubation with substrate tissue, can be detected by an immunoglobulin reagent (IgG, IgA or IgM), which has been conjugated with a fluorochrome for visualization under UV light following excitation.

ROUTINE SUBSTRATE SLIDES (commercially available) 

Primate cerebellum can be used to detect the following antibodies:- GAD, PCA (Yo), ANNA1 (Hu), ANNA2 (Ri), Ma/Ta, amphiphysin, CV2/CRMP5, AGNA, and Tr.

METHOD:

  • Diluted serum is incubated with tissue section in a moist chamber at room temperature.
  • Then unbound antibodies are removed by washing the slides in PBS.
  • Sections are incubated with diluted anti-human immunoglobulins (monkey adsorbed)-FITC at room temperature.
  • The FITC conjugate is washed off as above.
  • The sections are mounted under a glass cover slip using buffered glycerol +DABCO.
  • The slides are ready for viewing under the fluorescence microscope.
  • Pattern indicative of neuro-autoantibodies must be confirmed by other means (Western blot etc) and results disseminated to the appropriate physician as soon as possible.

VK, Buffalo, NY, USA "I very much appreciate your contributions in the area of autoantibody detection specifically in the field of paraneoplastic antibody detection.  We enjoy visiting your web site which is full of information....... "