Canis ISSN: 2398-2942

Pemphigus antibodies

Contributor(s): Michael Day, Helen Milner

Overview

Sampling

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Tests

Methodologies

  • Application of direct immunohistochemistry (immunofluorescence or immunoperoxidase Immunohistochemistry (IHC) ) for detection of tissue-bound autoantibody follows the standard protocols for this test.
  • In general, tissue will be screened with species-specific antisera specific for IgG, IgM, IgA and complement factor C3 where these reagents are available.
  • Several methods are reported for detection of serum autoantibody.
  • In the indirect immunofluorescence (or immunoperoxidase) technique Immunofluorescent antibody tests , a section of normal substrate tissue containing squamous epithelium is used. This may be normal skin, but tissues such as lip, tongue or esophagus may provide greater sensitivity. The substrate tissue need not come from the species of animal that is being tested for the presence of serum autoantibody.
    The autoantibody has affinity to protein antigen(s) that are well conserved between species.
  • The substrate tissue is generally snap-frozen and cryostat sections are used.
  • Appropriately diluted serum from the patient is overlaid onto the substrate tissue, and during a period of incubation any serum autoantibody will bind to the appropriate antigen within the tissue.
  • Binding of autoantibody is then detected by subsequent incubation with a fluorochrome or enzyme-labeled secondary antibody.
  • For the pemphigoid disorders, the sensitivity of indirect detection of autoantibody can be increased by using frozen sections of salt-split skin. In these, there is separation of the epidermis from dermis through the basement membrane zone. In bullous pemphigoid, the autoantibody will bind selectively to the epidermal side of the split.
  • Antigenic extracts from cultured keratinocytes can also be used in western blotting with patient serum. Using this technique, a range of autoantibodies can be detected. For example, autoantibodies in canine bullous pemphigoid are directed against 180kd and 230kd epitopes equivalent to the target antigens in human bullous pemphigoid, and autoantibodies in canine pemphigus foliaceus bind a 160kd antigen equivalent to desmoglein-1.
  • Recently, synthetic peptides derived from the sequence of dominant antigens, or recombinant antigens, have become available for use in ELISA Enzyme linked immunosorbent assay (ELISA). These are used to coat ELISA plates that are then incubated with patient serum, and subsequently an enzyme-linked secondary antibody. The use of a panel of four peptides derived from the human collagen XVII sequence, and of recombinant segments of desmoglein 1 and 3, are reported. The application of these methods has permitted subtypes of the subepidermal blistering diseases to be defined.

Availability

  • Direct immunohistochemistry for detection of lesional autoantibody can generally be arranged by most histopathology laboratories, after initial screening of samples for compatible histopathological changes.
  • Detection of serum autoantibodies by indirect methods is rarely available outside research laboratories.

Validity

Sensitivity

  • Direct immunohistochemistry is a relatively insensitive technique.
  • The sensitivity may increase with the use of frozen tissue, and particular antisera may be better suited to this application.
  • In some reported studies, only 25% of skin biopsies have been positive by immunohistochemistry (on fixed tissue) in patients where there is clear clinical and histopathological suggestion of autoimmune skin disease.
  • Indirect immunohistochemistry may also be relatively insensitive. Even in the presence of positive direct immunohistochemistry, not all patient sera will have sufficient levels of circulating autoantibody to give a positive result by indirect methods.
  • Similarly, whilst ELISA with specific recombinant proteins is a sensitive technique, serum antibodies may not necessarily be present in sufficient quantity to give a positive result.

Specificity

  • Direct immunohistochemistry is interpreted subjectively, and care must be taken to distinguish specific staining from non-specific percolation of serum immunoglobulins into a spongiotic epidermis.
  • It has been reported that epidermis from areas such as planum nasal and footpad may give false positive results in direct immunohistochemistry. The specificity of indirect methods will be greater, and ELISA using recombinant proteins should be a highly specific technique.

Result Data

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Further Reading

Publications

Refereed papers

  • Recent references from VetMed Resource and PubMed.
  • Perez J et al (2002) Comparison of three monoclonal and there polyclonal antibodies in the immunohistochemical diagnosis of canine autoimmune skin diseases. Vet Dermatol 13, 231-6.
  • Olivry T, Dunston S M, Schachter M et al (2001) A spontaneous canine model of mucous membrane (cicatricial) pemphigoid, an autoimmune blistering disease affecting mucosae and mucocutaneous junctions. J Autoimmunity 16, 411-421.
  • Olivry T, Alhaidari Z & Ghohestani R F (2000) Anti-plakin and desmoglein autoantibodies in a dog with pemphigus vulgaris. Vet Pathol 37, 496-499.
  • Olivry T, Chan L S, Xu L et al(1999) Novel feline autoimmune blistering disease resembling bullous pemphigoid in humans: IgG autoantibodies target the NC16A ectodomain of type XVII collagen (BP180/BPAG2). Vet Pathol 36, 328-335.
  • Iwasaki T, Shimizu M, Obata H et al (1997) Detection of canine pemphigus foliaceus autoantigen by immunoblotting. Vet Immunol Immunopathol 59, 1-10.
  • Iwasaki T, Olivry T, Lapiere J C et al (1995) Canine bullous pemphigoid (BP): identification of the 180kd canine BP antigen by circulating autoantibodies. Vet Pathol 32, 387-393.
  • Iwasaki T, Shimizu M, Obata H et al (1996) Effect of substrate on indirect immunofluorescence test for canine pemphigus foliaceus. Vet Pathol 33, 332-336.
  • Day M J, Hanlon L & Powell L M (1993) Immune-mediated skin disease in the dog and cat. J Comp Pathol 109, 395-407.

Other sources of information

  • Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing, London.


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