Characterization of two monoclonal antibodies to human epidermal keratohyalin: reactivity with filaggrin and related proteins.
Characterization of two monoclonal antibodies to human epidermal keratohyalin: reactivity with filaggrin and related proteins.
复制标题
两种针对人表皮透明角质蛋白的单克隆抗体的表征：与聚丝蛋白和相关蛋白的反应性。
DOI：
10.1111/1523-1747.ep12466185
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Resing,KA
中科院分区：
文献类型：
--
作者：
Dale,BA;Gown,AM;Fleckman,P;Kimball,JR;Resing,KA
用部分纯化的人聚丝蛋白诱导两种单克隆抗体（ARH1和AKH2），并通过正常和异常皮肤活检、免疫印迹技术和抗原纯化进行免疫组化鉴定。这两种抗体与与角膜透明素分布一致的颗粒细胞层反应强烈，在正常皮肤活检中与角质层反应更广泛。在培养的人角质形成细胞中，反应仅限于融合培养中扁平的、分化良好的细胞中的免疫荧光颗粒，在这种情况下，我们之前已经证明了角质透明素。在免疫印迹上，AKH1与聚丝蛋白（37 kD）和聚丝蛋白（400 kD）反应，而AKH2主要染色150和300 kD的条带。在用于免疫的阳离子蛋白片段中鉴定出AKH2抗原，并通过凝胶渗透和高效液相色谱纯化。这些蛋白质的氨基酸组成与聚丝蛋白仅略有不同。除了寻常性鱼鳞病外，这两种抗体的免疫组化染色模式在角化遗传疾病中非常相似，反映了角透明素的存在和分布。在寻常型鱼鳞病中，AKH1染色较弱，与形态学一致，生化上缺乏聚丝蛋白/聚丝蛋白；然而，AKH2染色呈阳性，尽管比正常情况弱，这表明即使角膜透明素缺失或异常，也存在AKH2抗原。抗体AKH1和AKH2可作为组织和培养细胞角化的分化标记物。抗体AKH1可专门用于检测组织、培养的角质形成细胞和提取物中的聚丝蛋白/聚丝蛋白。
Two monoclonal antibodies (ARH1 and AKH2) were elicited with partially purified human filaggrin and characterized by immunohistochemistry on normal and abnormal skin biopsies, immunoblotting techniques, and antigen purification. Both antibodies react strongly with the granular cell layer consistent with the distribution of keratohyalin and show a more diffuse reaction with the stratum corneum in normal skin biopsies. Reaction in cultured human keratinocytes is limited to immunofluorescent granules in flattened, well-differentiated cells in confluent cultures, in which we have previously demonstrated keratohyalin. On immunoblots AKH1 reacts with filaggrin (37 kD) and profilaggrin (400 kD), while AKH2 primarily stains bands of 150 and 300 kD. The AKH2 antigens were identified in the cationic protein fraction used for immunization and were purified by gel permeation and high-performance liquid chromatography. Amino acid composition of these proteins differs only slightly from filaggrin. Immunohistochemical staining patterns of the two antibodies are very similar in the genetic disorders of keratinization tested, except for ichthyosis vulgaris, and reflect the presence and distribution of keratohyalin. In ichthyosis vulgaris, AKH1 staining is weak, consistent with the morphology and with biochemical absence of profilaggrin/filaggrin; however, AKH2 staining is positive, although weaker than normal, suggesting the presence of the AKH2 antigens even when keratohyalin is absent or abnormal. Antibodies AKH1 and AKH2 may be useful as differentiation markers for keratinization in tissues and for cells in culture. Antibody AKH1 can be used specifically for detection of profilaggrin/filaggrin in tissues, cultured keratinocytes, and extracts.