Human placental amnion is a novel substrate for detecting autoantibodies in autoimmune bullous diseases by immunoblotting.
BACKGROUND
Identification of antigens by immunoblotting techniques, using epidermal and dermal extracts, is regarded as essential for making a definitive diagnosis in autoimmune bullous diseases (AIBDs). These procedures involve epidermal-dermal separation for subsequent protein extraction, which may result in partial loss of some antigenic polypeptides and changes in the conformational epitopes targeted by autoantibodies in AIBDs. It may therefore be necessary to use different substrates for consistent results. Objectives To evaluate the usefulness of human placental amnion extract as a substrate for immunoblotting in the diagnosis of AIBDs.


METHODS
We checked the structural components of the desmosomes and basement membrane zone (BMZ) of amnion by electron microscopy. Using immunofluorescence and immunoblotting techniques, we tested the amnion immunoreactivity with antibodies to desmosomal and BMZ proteins, and with sera from 76 patients with AIBDs including pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid (BP), pemphigoid gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, paraneoplastic pemphigus and mucous membrane pemphigoid.


RESULTS
The desmosomes and BMZ of the amnion tissue were ultrastructurally similar to those in skin. Antigen mapping confirmed that amnion contains all the proteins that were recognized by a panel of monoclonal and polyclonal antibodies. Immunoblotting showed that the antibodies clearly detected bands corresponding to desmogleins 1 and 3, desmocollins 1 and 2, desmoplakins 1 and 2, three subunits (alpha3, beta3 and gamma2) of laminin 5, BP antigens 1 and 2, the 97-kDa LAD antigen and type VII collagen. In addition, most of the patient sera (82%) reacted exclusively with their respective antigens.


CONCLUSIONS
Harvesting proteins from amnion does not require epidermal-dermal separation, and a sufficient yield of desmosomal and hemidesmosomal proteins can be obtained. Therefore, amnion may be a more reliable source of substrate than skin samples for immunoblot analysis of AIBDs.
Oyama
N
N
Department of Immunofluorescence, St John's Institute of Dermatology, Guy's, King's and St Thomas' School of Medicine, St Thomas' Hospital, London SE1 7EH, UK. norider@wine.plala.or.jp
Bhogal
B S
BS
Carrington
P
P
Gratian
M J
MJ
Black
M M
MM
D000650:Amnion / Q000276:immunology* / Q000648:ultrastructure; D001323:Autoantibodies / Q000032:analysis*; D001327:Autoimmune Diseases / Q000175:diagnosis*; D016022:Case-Control Studies; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008854:Microscopy, Electron; D011247:Pregnancy; D012872:Skin Diseases, Vesiculobullous / Q000276:immunology*
D001323:Autoantibodies