| We made the retrospective studies about the serum samples of 37 patients routinely diagnosed as having bullous pemphigoid ( BP) to obtain the incidence of the epidermolysis bullosa acquisita (EBA) a-mong this group of the people and tested 4 saliva samples with BP to define whether there are anti - basement membrane zone ( BMZ) antibodies in the saliva or not.METHODSI. Materials1. Sera: The serum samples of 37 patients (20 men and 17 women, aged range 20 -85 and median 62. 1) diagnosed as having BP by the clinical features, pathology, and direct immunofluorescence ( DIF) at the Dermatology Department, the No. 1 Hospital attached to China Medical University between 1995 and 2003 and 10 normal volunteers served as controls.2. Saliva: The 4 patients ( 2 men and 2 women, aged range 54 -85 and median 69. 3) with BP and 4 normal saliva samples as controls.3. Skin specimens of normal individuals; The specimens are normal skin from mammoplasty or plastic surgery.n. Methods1. Indirect Immunofluorescence (IIF)(1) Standard IIFNormal skin was served as substrate, serum or saliva samples as the first antibody, and fluorescein isothiacyanate ( FITC ) - conjugated goat anti - human IgG as the second antibody, finally observed by fluorescence microscope.(2) Salt - split skin IIFFresh normal skin was incubated for 72 hours at 4 ℃ in 1.0 mol/ 1 NaCl solution replaced every 24 hours, and then mounted in OCT compound, and others were done as standard IIF.2. Western Immunoblot(IB)(1) Separation of dermis and epidermisFresh normal skin specimens from surgery with subcutaneous fatty tissue removed at most were cut the strip at about 0.5 mm thick. The dermis and epidermis are separated by being incubated in 1 mol/1 NaCl solution containing 1 mmol/1 PMSF and 2 mmol/1 EDTA at 4t for 72 hours, replaced every 24 hours, and then epidermis was separated from the dermis with forceps.(2) Protein ExtractsThe epidermis was added to the extraction buffer of 65 mmol/1 Tris - HC1 pH 6. 8 containing 2% SDS, 5% 2 - ME, 10 mmol/1 EDTA, 2 mmol/1 PMSF, and pepstatin A, antipain, leupeptin, Chymo-statin at 0. Olmg/ml, respectively) ,and every 25 cm2 epidermis was added to 1 ml extraction buffer, homogenized with a homogenizer, then ultrasonicated for 30 seconds (A=60, C = 0. 5 ) , and centri-fuged at 15000 r/m for 30 minutes at 4℃. Then supernatant was collected as the epidermal extracts kept at - 201!.The dermis was added to the extraction buffer of 31. 2 mmol/1 Tris - HC1 pH 6. 8 containing 8 mol/1 urea, 2% SDS, 5% 2 - ME,10 mmol/1 EDTA, 10 mmol/1 PMSF) , and every 15 cm2 dermis was added to 1 ml extraction buffer., vortexed for 1 hour at room temperature, then kept for 16 hours at 4℃ overnight, and centrifuged at 15000 r/m for 1 hour at 4℃. 'Then supernatant was collected as the dermal extracts kept at - 20℃.(3)SDS-PAGEThe dermis and epidermal protein extracts were electrophoresed on 6 % polyacrylamide slab gel for about 2 hours at steady voltage. (4) TransmembraneThe proteins on the gel were transferred to nitrocellulose paper through semi - drying proteins transferring method.(5) ImmunoblotThe nitrocellulose papers at 2 mm in width were placed to reaction vessel incubated with: (i) 5 % dried milk in 50 mmol/1 Tris -HCl pH 7.4 for 2 hours at room temperature; (ii) 1: 100 dilution of each serum or 1: 2 dilution of each saliva in 50mmol/l Tris - HCl pH 7.4 containing 0. 5 moVl NaCl, 5 % dried milk and 0. 3% Tween 20 for 1 hour at 37℃; (iii) after washed by 10mmol/1 Tris - HCl pH 7. 4 containing 0.5 mol/1 NaCl and 0.3 % Tween 20 for 10 minutes x 4,1:2000 dilution of horseradish peroxidase - conjugated goat anti -human IgG in 50mmol/l Tris - HCI pH 7. 4 containing 0. 5 mol/1 NaCl, 5 % dried milk and 0. 3% Tween 20 for 1 hour at 37℃ ; (iv) washed again, and developed with DAB until showing brown in strips.RESULTSI. Results of Sera samples tested2 sera of 37 patients with BP were proved having EBA, 5.4 % of all the patients examined with BP in their ser...
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