Allen J, Schomberg K, Wojnarowska F
Department of Dermatology, Oxford Radcliffe Hospital, U.K.
Br J Dermatol. 1997 Dec;137(6):907-15.
Many of the techniques used in the diagnosis and characterization of the acquired blistering diseases of the skin and of their target antigens require the use of tissue that has been modified by heat, chemicals, or by proteolytic digestion. The effect these treatments may have on the blistering disease antigens is poorly understood and has seldom been taken into account in the interpretation of the results. Likewise, their effect on the immunodetection and expression of the ubiquitous proteins of the basement membrane zone and extracellular matrix has rarely been investigated. We have addressed this problem by probing tissue split after heat, chemical treatment and proteolytic digestion with an extensive panel of antibodies to the hemidesmosome-plasma membrane-anchoring filament complex, the extracellular matrix and the anchoring fibrils. The results showed that chemical modification by sodium chloride, calcium chloride and ethylenediaminetetraacetic acid, and incubation in 0.15 mol/L NaCl at 56 degrees C for 1 min did not adversely affect the expression of the alpha 6 beta 4 integrin, laminin-5, the LH39 antigen, laminin-1, collagen type IV or collagen type VII. The methods that involved proteolytic digestion had the greatest detrimental effect on these basement membrane components, with pepsin having a damaging effect on the greatest number of antigens, and the LH39 antigen being the most sensitive to proteolytic degradation. This project has highlighted the susceptibility of the basement membrane zone and extracellular matrix proteins to proteolytic hydrolysis. It has demonstrated a way of differentiating between specific proteins, and has shown its potential for the differential diagnosis of the autoimmune blistering disease antigens.
用于诊断和鉴定后天性皮肤水疱病及其靶抗原的许多技术，都需要使用经过加热、化学物质处理或蛋白水解消化处理过的组织。这些处理对水疱病抗原可能产生的影响，我们了解得很少，并且在结果解读中很少被考虑进去。同样，它们对基底膜区和细胞外基质中普遍存在的蛋白质的免疫检测和表达的影响，也很少被研究。我们通过用一组广泛的针对半桥粒-质膜-锚定细丝复合物、细胞外基质和锚定原纤维的抗体，对经过加热、化学处理和蛋白水解消化后的组织切片进行检测，解决了这个问题。结果表明，氯化钠、氯化钙和乙二胺四乙酸的化学修饰，以及在56℃下于0.15mol/L氯化钠中孵育1分钟，不会对α6β4整合素、层粘连蛋白-5、LH39抗原、层粘连蛋白-1、IV型胶原或VII型胶原的表达产生不利影响。涉及蛋白水解消化的方法对这些基底膜成分的损害最大，胃蛋白酶对最多数量的抗原具有破坏作用，而LH39抗原对蛋白水解降解最为敏感。这个项目突出了基底膜区和细胞外基质蛋白对蛋白水解的敏感性。它展示了一种区分特定蛋白质的方法，并显示了其在自身免疫性水疱病抗原鉴别诊断中的潜力。
