Two groups of bullous pemphigoid antigens are identified by affinity-purified antibodies.
Two groups of bullous pemphigoid antigens are identified by affinity-purified antibodies.
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通过亲和纯化的抗体鉴定两组大疱性类天疱疮抗原。
DOI：
10.1111/1523-1747.ep12876194
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Zone,JJ
中科院分区：
文献类型：
--
作者：
Meyer,LJ;Taylor,TB;Kadunce,DP;Zone,JJ
大疱性类天疱疮抗原最初被描述为从人表皮中提取的 240 kD 蛋白质，但随后的报告描述了患者血清可与分子量为 240、200、180、97 和 77 kD 的表皮蛋白质发生反应。我们评估了 10 名临床典型大疱性类天疱疮患者的血清所鉴定的类天疱疮抗原的异质性。我们使用间接免疫荧光和蛋白质印迹法对由 1 M 盐或 20 mM EDTA 分离的表皮制备的表皮提取物进行表征，以表征粗血清和亲和纯化抗体的反应性。使用正常人表皮或印迹到作为免疫吸收剂的硝酸纤维素上的蛋白质条带进行抗体的亲和纯化。在盐水和 EDTA 分离的表皮上通过间接免疫荧光测定的抗基底膜抗体滴度是相同的。尽管如此，从 EDTA 分离的表皮制备的提取物的蛋白质印迹显示，与盐水分离的皮肤相比，240-kD 抗原的含量更高。大多数粗制 BP 血清可在蛋白质印迹上识别表皮提取物中的多种抗原，包括 240、200、160 和 100 kD 的条带。不同的血清以明显不同的强度与这些抗原反应，分为两大类，即带有针对240-200-kD抗原的抗体的血清和带有针对160-100-kD成分的抗体的血清。当表皮用作大疱性类天疱疮抗体亲和纯化的底物时，洗脱的抗体与蛋白质印迹上的多个条带发生反应，证明了抗基底膜区抗体与多种蛋白质的反应性。发现从免疫印迹的几个单独条带中洗脱的抗体在间接免疫荧光上与基底膜发生反应。当这些硝化纤维纯化的抗体重新应用于蛋白质印迹时，它们在两组（240-200 kD 抗原和 160-100 kD 抗原）内发生交叉反应。我们的结论是，当使用 EDTA 分割皮肤提取时，类天疱疮抗原得到最好的证明，并且不同的类天疱疮血清可能含有针对两组不同的基底膜区抗原的抗体。
The bullous pemphigoid antigen was originally described as a 240-kD protein extracted from human epidermis, but a subsequent report has described patients' sera which react with epidermal proteins of molecular masses 240, 200, 180, 97, and 77 kD. We have evaluated the heterogeneity of the pemphigoid antigens identified by the sera of 10 patients with clinically typical bullous pemphigoid. We used indirect immunofluorescence and Western immunoblots of epidermal extracts prepared from epidermis separated by either 1 M salt or 20 mM EDTA to characterize the reactivity of both crude sera and affinity-purified antibodies. Affinity purification of antibodies was performed with either normal human epidermis or protein bands blotted onto nitrocellulose as immunoabsorbents. The anti-basement membrane antibody titers determined by indirect immunofluorescence on the saline- and EDTA- separated epidermis were identical. Despite this, Western blots of extracts prepared from EDTA-separated epidermis demonstrated greater amounts of the 240-kD antigen than saline split skin. Multiple antigens were recognized in epidermal extracts on Western blots by most crude BP sera, including bands at 240, 200, 160, and 100 kD. Different sera reacted with these antigens with a markedly different intensity, falling into two major groups, those bearing antibodies to the 240- 200-kD antigens and those with antibodies to the 160-1 00-kD components. When epidermis was used as a substrate for affinity purification of bullous pemphigoid anti- bodies, the eluted antibodies reacted with multiple bands on Western blots, demonstrating the reactivity of anti-basement membrane zone antibodies with multiple proteins. Antibodies eluted from several individual bands of immunoblots were found to react with the basement membrane on indirect immunofluorescence. When these nitrocellulose-purified antibodies were reapplied to Western blots, they cross-reacted within two groups, the 240-200 kD anigens and the 160-100 kD antigens. We conclude that perphigoid antigens are best demonstrated when EDTA-split skin is used for extraction and that different pemphigoid sera may contain antibodies to two separate groups of basement membrane zone antigens.