Usefulness of immunoblotting using purified laminin 5 in the diagnosis of anti-laminin 5 cicatricial pemphigoid
Usefulness of immunoblotting using purified laminin 5 in the diagnosis of anti-laminin 5 cicatricial pemphigoid
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DOI：
10.1016/s0923-1811(03)00158-0
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发表时间：
2003-11-01
影响因子：
4.6
通讯作者：
Hashimoto, T
中科院分区：
文献类型：
--
作者：
Hisamatsu, Y;Nishiyama, T;Hashimoto, T
背景：抗层粘连蛋白5瘢痕性类天疱疮（CP）是一种粘膜显性的皮下起泡性疾病，其特征是IgG抗基底膜带自身抗体，与1 M NaCl的皮侧结合。剥皮和免疫沉淀层粘连蛋白5。层粘连蛋白5是一种表皮特异性的细胞外基质，由α 3、β 3和γ 2亚基组成。最近的研究表明，抗层粘连蛋白5cp的自身抗体可以识别alpha3亚基的G结构域。目的：用纯化的层粘连蛋白5和重组α 3亚基蛋白进行免疫印迹检测抗层粘连蛋白5 CP的反应性。方法：首先用纯化的层粘连蛋白5免疫印迹法检测抗层粘连蛋白5cp的反应性。为了进一步研究α 3亚基G结构域的表位，我们制备了覆盖整个G结构域的G1-2、G1-3、G2-3、G3-5结构域的重组蛋白，并通过免疫印迹法检测了这些重组蛋白与抗层粘连蛋白5 CP血清的反应性。结果：用纯化的层粘连蛋白5进行免疫印迹，21份抗层粘连蛋白5 CP血清中有7份与alpha3亚基反应，8份与3亚基反应，1份与gamma2亚基反应。两种血清与α 3和β a3亚基均有反应，而7种血清无阳性反应。这一结果表明抗层粘连蛋白5cp血清的反应性更为异质性，尽管先前的研究表明大多数血清与0亚基反应。然而，在使用a3亚基G结构域重组蛋白的研究中，没有一种CP血清，包括纯化层粘连蛋白5中与a3亚基反应的血清，与任何重组蛋白发生反应。这种与重组蛋白的负反应性的原因尚不清楚。结论：纯化的层粘连蛋白5免疫印迹法虽然灵敏度低于常规免疫沉淀法，但仍可作为诊断抗层粘连蛋白5 CP的有效方法。(C) 2003年日本皮肤病研究学会。爱思唯尔爱尔兰有限公司出版。版权所有。
Background: Anti-laminin 5 cicatricial pemphigoid (CP) is a mucosal-dominant subepithetial blistering disease characterized by IgG anti-basement membrane zone autoantibodies, that bind to dermal side of 1 M NaCl. split skin and immunoprecipitate laminin 5. Laminin 5 is an epidermis-specific extracellutar matrix consisting of alpha3, beta3 and gamma2 subunits. Recent studies have suggested that autoantibodies of anti-laminin 5 CP recognize the G domains of alpha3 subunit. Objective: We examined the reactivity of anti-laminin 5 CP by immunoblotting using purified laminin 5 and recombinant proteins of alpha3 subunit. Method: We first examined the reactivity of anti-laminin 5 CP by immunoblotting using purified laminin 5. To further investigate the epitopes in the G domains of alpha3 subunit, we produced recombinant proteins of G1-2, G1-3, G2-3, G3-5 domains, that covered entire G domain, and examined the reactivity of anti-laminin 5 CP sera with these recombinant proteins by immunoblotting. Results: By immunoblotting using purified laminin 5, 7 of 21 anti-laminin 5 CP sera reacted with alpha3 subunit, while 8 sera reacted with 3 subunit and one serum reacted with gamma2 subunit. Two sera reacted with both alpha3 and beta3 subunits, while seven sera did not show positive reactivity. This result indicates that the reactivity of anti-laminin 5 CP sera is much more heterogeneous, although the previous studies suggested that most sera reacted with 0 subunit. However, in the studies using recombinant proteins of G domains of a3 subunit, none of the CP sera, including the sera reactive with a3 subunit in purified laminin 5, reacted with any recombinant proteins. The reason for this negative reactivity with the recombinant proteins is not clear. Conclusion: The immunoblotting using purified laminin 5 should be useful technique for the diagnosis of anti-laminin 5 CP, although the sensitivity was less than conventional immunoprecipitation analysis. (C) 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Iretand Ltd. All rights reserved.