Differentiating Anti-Lamina Lucida and Anti-Sublamina Densa Anti-BMZ Antibodies by Indirect Immunofluorescence on 1.0 M Sodium Chloride-Separated Skin

Abstract

Sixty-one bullous disease sera containing IgG anti-BMZ antibodies were examined by indirect immunofluorescence on intact skin and skin separated through the lamina lucida by incubation in 1.0 M NaCl. All sera produced an indistinguishable pattern of linear immunofluorescence on intact skin at dilutions of 1:10 or higher. On separated skin, antibodies bound to either the epidermal (epidermal pattern), dermal (dermal pattern), or epidermal and dermal (combined pattern) sides of the separation. The binding patterns were consistent on separated skin from several donors and titers of anti-basement membrane zone antibodies on separated skin were comparable to those on intact skin. Sera from 3 patients with herpes gestationis (HG), 36 patients with bullous pemphigoid (BP), and 1 patient with clinical and histologic features of epidermolysis bullosa acquisita (EBA) showed an epidermal pattern. Sera from 9 patients with BP showed a combined pattern and sera from 6 patients with EBA and 6 patients with clinical and histologic features of BP showed a dermal pattern. Indirect immunoelectron microscopy of selected sera showed antibodies producing the epidermal and combined patterns were anti-lamina lucida antibodies and those producing the dermal pattern were anti-sublamina densa antibodies. These results show indirect immunofluorescence on separated skin is a dependable method for differentiating bullous disease anti-lamina lucida and anti-sublamina densa antibodies and that differentiating between the antibodies is essential for accurate diagnosis in some patients. The results also suggest BP anti-lamina lucida antibodies may have more than one antigenic specificity.

Tissue form of type VII collagen from human skin and dermal fibroblasts in culture

“…Skin was subjected to artificial epidermolysis in 1 M NaCI, 0.05 M Tris/HCl, pH 7.4,20 mM EDTA, 10 mM phenylmethylsulfonyl fluoride, 20 mM Nethylmaleimide and 100 mM E-aminocaproic acid for 72 h at 4°C [15]. The epidermis was peeled off and the newly exposed dermal surface was extracted twice for 30 s with hot (95°C) buffer consisting of 0.125 M Tris/HCI, 8 M urea, 2% SDS, 0.1 M dithioerythrol, pH 6.8, and the same proteinase inhibitors as in the epidermolysis buffer [16].…”

“…This was achieved by incubation of the tissue with 1 M NaCl for 72 h at 4°C in the presence of proteinase inhibitors, a procedure which effects a separation in the basement membrane at the level of the lamina lucida [15]. The newly eposed dermal surface was most efficiently extracted with preheated buffers containing urea, SDS and a reducing agent.…”

Clinical features and diagnosis of epidermolysis bullosa acquisita

“…As in all pemphigoid diseases, antitype VII collagen antibodies bind along the BMZ on the monkey and rabbit esophagus. By indirect IF microscopy of human salt-split skin, antitype VII collagen antibodies label the floor of the artificial blister ( Figure 7) [138]. However, antibodies against laminin 332 and the p200 protein/ laminin γ1 also bind to the floor of the blister [3,127,139,140].…”

Subepidermal blistering disease in a 5-month-old Alaskan Malamute dog with concurrent megaesophagus

“…In human medicine (Gammon et al 1984), positive indirect immunofluorescence in salt-split skin test is classically used to differentiate BP from other subepidermal autoimmune bullous diseases. However, indirect immunofluorescence testing frequently fails to demonstrate circulating IgG antibodies and less than 20% of canine BP patients showed positive results in the past (Scott 1987 (Favrot et al 2002).…”

“…An alternative approach to the treatment of autoimmune skin disease was the use of non-steroidal immunosuppressive drugs, e.g. azathioprine, chlorambucil, gold salts, tetracycline/niacinamide, cyclophosphamide, and cyclosporine (Bennett et al 1980a;Gammon et al 1984;Ackerman 1985). In this case, we prescribed azathioprine and cyclosporine for an alternative treatment of BP in order to avoid the side effects of prednisolone.…”