Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies.
Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies.
复制标题
钙增强免疫荧光对天疱疮抗体的敏感性。
DOI：
10.1111/1523-1747.ep12471587
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Berger,RS
中科院分区：
文献类型：
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作者：
Matis,WL;Anhalt,GJ;Diaz,LA;Rivitti,EA;Martins,CR;Berger,RS
检测天疱疮和类天疱疮自身抗体的间接免疫荧光（IF）通常使用猴食管（ME）作为底物，磷酸盐缓冲盐水（PBS）作为稀释剂进行。本研究的目的是使用人皮肤（HS）作为底物，在所用缓冲液的变化下评价比较IF滴度。在与天疱疮或类天疱疮血清孵育以进行间接IF之前，将底物（ME或HS）在PBS、三乙酸盐缓冲盐水（TAS）、含5 mM CaCl+2的TAS（TAS-Ca+2）和含1 mM EDTA的PBS或TAS中孵育。我们检测了11例寻常型天疱疮（PV）、10例巴西落叶型天疱疮（BPF）和4例大疱性类天疱疮患者的血清。在20/21份天疱疮血清中，终点间接IF滴度在正常皮肤上以TAS-Ca+2最高。6份血清（2份PV和4份BPF）的终点比ME和PBS获得的终点高5倍稀释度。6份血清（3份PV和3份BPF）高出4倍稀释度，7份血清（3份PV和4份BPF）高出2-3倍稀释度，2份PV血清与两种底物/缓冲液相当。在间接IF之前用EDTA预孵育任一组织完全消除PV和BPF抗体结合。组织与PV或BPF血清孵育后暴露于EDTA不影响间接IF滴度。在Ca+2的存在下，抗原对浓度为0.001%的胰蛋白酶具有抗性;然而，在不存在添加的Ca+22的情况下，抗原被0.0001%的胰蛋白酶破坏。在大疱性类天疱疮血清中未观察到这些差异;所有4种血清的终点间接IF滴度相似。这项研究表明，天疱疮自身抗体的间接IF检测的灵敏度显着增加，通过使用Ca+2补充缓冲液对人皮肤。这一发现也可能对设计用于纯化和/或检测天疱疮抗原的程序有影响。
Indirect immunofluorescence (IF) to detect pemphigus and pemphigoid autoantibodies is commonly performed with monkey esophagus (ME) as substrate and phosphate-buffered saline (PBS) as a diluent. The purpose of this study was to evaluate comparative IF titers using human skin (HS) as substrate with variations in the buffers employed. Substrates (ME or HS) were incubated in PBS, Tris- acetate-buffered saline (TAS), TAS with 5 mM CaCl+2(TAS-Ca+2), and PBS or TAS with 1 mM EDTA, prior to incubation with pemphigus or pemphigoid sera for indirect IF. We examined sera from 11 patients with pemphigus vulgaris (PV), 10 patients with Brazilian pemphigus foliaceus (BPF), and 4 patients with bullous pemphigoid. In 20 of 21 pemphigus sera, endpoint indirect IF titers were highest on normal skin with TAS-Ca+2. Six sera (2 PV and 4 BPF) had endpoints that were 5 double dilutions higher than the endpoints obtained with ME and PBS. Six sera (3 PV and 3 BPF) were 4 double dilutions higher, 7 sera (3 PV and 4 BPF) were 2–3 double dilutions higher, and 2 PV sera were equivalent with both substrate/buffers. Preincubation of either tissue with EDTA prior to indirect IF abolished PV and BPF antibody binding completely. Exposure to EDTA after the tissue was incubated with PV or BPF sera did not affect indirect IF titers. In the presence of Ca+2, the antigen was resistant to trypsin in concentrations of 0.001%; however, iii the absence of added Ca+22 it was destroyed by 0.0001% trypsin. These differences were not observed with bullous pemphigoid sera; all 4 sera had similar endpoint indirect IF titers. This study shows a significant increase in the sensitivity of indirect IF assays for pemphigus autoantibodies by the use of Ca+2-supplemented buffers on human skin. This finding may also have implications for procedures designed to purify and/or detect pemphigus antigens.