免疫印迹分析诊断抗层粘连蛋白 γ-1 型天疱疮。
Diagnosis of anti-laminin γ-1 pemphigoid by immunoblot analysis.
机构信息
Department of Dermatology and Allergology, Philipps-University, Marburg, Germany.
Kurume University Institute of Cutaneous Cell Biology, Kurume, Fukuoka, Japan.
出版信息
J Eur Acad Dermatol Venereol. 2019 Apr;33(4):735-741. doi: 10.1111/jdv.15170. Epub 2018 Jul 30.
BACKGROUND
Anti-laminin-γ1 (lam-γ1) pemphigoid, a recently described immunobullous disorder sharing immune serological features of bullous pemphigoid and epidermolysis bullosa acquisita (EBA), is characterized by the detection of serum IgG autoantibodies against the lam-γ1 chain, a 200 kDa heterotrimeric component of the dermal-epidermal junction (DEJ).
OBJECTIVE
The aim of the study was to develop an easy-to-perform and reliable assay for the serological detection of anti-lam-γ1 IgG autoantibodies. The clinical appearance alone is not sufficient to establish diagnosis of anti-lam-γ1 pemphigoid and rather requires immune serological evidence of (i) IgG reactivity against the dermal portion of salt-split human skin; (ii) exclusion of IgG against other components of the DEJ; and (iii) IgG reactivity with a 200 kDa protein of dermal extracts by immunoblot analysis (IB).
METHODS
The sera of 55 patients with anti-lam-γ1 pemphigoid were tested by IB with two recombinant heterotrimers, laminin 111 (lam-111) and laminin 421 (lam-421), as well as with a recombinant lam-γ1 chain monomer. Additionally, a total of 41 control sera from patients with EBA (n = 15), psoriasis vulgaris (PV; n = 14), and healthy controls (HC; n = 12) were tested.
RESULTS
Immunoblot analysis revealed a positive reactivity with lam-111 and/or lam-421 in 46/55 (84%) of anti-lam-γ1 pemphigoid sera. Moreover, 8/9 of the initially non-reactive sera were positive with the lam-γ1 monomer, leading to an overall sensitivity of 98.2%. Analyses of 41 control sera with the three lam-γ1 recombinants led to a specificity of 88%. Specifically, 3/15 EBA sera, 1/14 PV serum and 1/12 HC serum reacted with the lam-γ1 monomer while only the 3 EBA sera reacted with lam-421.
CONCLUSIONS
Here we show a novel two-step IB assay using the two recombinant laminin trimers and lam-γ1 chain monomer for the detection of anti-lam-γ1 serum IgG with high sensitivity and specificity. This assay will facilitate the diagnosis and further characterization of this disease.
背景
抗层粘连蛋白-γ1（lam-γ1）天疱疮是一种最近描述的免疫性大疱性疾病，具有大疱性类天疱疮和获得性大疱性表皮松解症（EBA）的免疫血清学特征，其特征是检测到针对 lam-γ1 链的血清 IgG 自身抗体，lam-γ1 链是真皮表皮连接（DEJ）的 200 kDa 异三聚体成分。
目的
本研究旨在开发一种简单易行、可靠的血清学检测抗 lam-γ1 IgG 自身抗体的方法。仅凭临床表现不足以确定抗 lam-γ1 天疱疮的诊断，而需要免疫血清学证据证明：（i）对盐裂人皮肤真皮部分的 IgG 反应性；（ii）排除对 DEJ 其他成分的 IgG 反应性；和（iii）免疫印迹分析（IB）中与真皮提取物 200 kDa 蛋白的 IgG 反应性。
方法
用两种重组异三聚体，层粘连蛋白 111（lam-111）和层粘连蛋白 421（lam-421）以及重组 lam-γ1 链单体对 55 例抗 lam-γ1 天疱疮患者的血清进行 IB 检测。此外，还检测了 41 例 EBA 患者（n=15）、寻常型银屑病（PV；n=14）和健康对照（HC；n=12）的对照血清。
结果
免疫印迹分析显示，46/55（84%）例抗 lam-γ1 天疱疮血清与 lam-111 和/或 lam-421 呈阳性反应。此外，9/9 例最初非反应性血清与 lam-γ1 单体呈阳性反应，总体敏感性为 98.2%。用三种 lam-γ1 重组体分析 41 例对照血清，特异性为 88%。具体而言，15 例 EBA 血清中有 3 例、14 例 PV 血清中有 1 例和 12 例 HC 血清中有 1 例与 lam-γ1 单体反应，而只有 3 例 EBA 血清与 lam-421 反应。
结论
本文展示了一种使用两种重组层粘连蛋白三聚体和 lam-γ1 链单体的新型两步免疫印迹分析检测抗 lam-γ1 血清 IgG 的方法，具有高灵敏度和特异性。该检测方法将有助于该病的诊断和进一步表征。
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