Mechanisms of heat-induced antigen retrieval: Analyses in vitro employing SDS-PAGE and immunohistochemistry
Mechanisms of heat-induced antigen retrieval: Analyses in vitro employing SDS-PAGE and immunohistochemistry
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DOI：
10.1369/jhc.4c6323.2005
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发表时间：
2005-01-01
影响因子：
3.2
通讯作者：
Okada, Y
中科院分区：
文献类型：
--
作者：
Yamashita, S;Okada, Y
在这项研究中，我们研究了热诱导抗原修复的机制，使用分析程序，包括SDS-PAGE，Western印迹和免疫组织化学。在存在或不存在25 mM CaCl 2的情况下，用4%甲醛处理五种蛋白质，然后在各种条件下加热，除去甲醛后，在SDS-PAGE上分析。甲醛在蛋白质中产生分子间和分子内交联。在高温下加热裂解这些交联在所有pH范围内检查（pH 3.0，6.0，7.5，9.0），并产生几乎相同的电泳作为天然蛋白质。用含有CaCl 2的甲醛处理的蛋白质显示出相似的电泳模式，在没有加热或在pH 6.0和pH 9.0在存在或不存在10 mM EDTA下加热后观察到。蛋白质印迹分析表明，可溶性形式的β-肌动蛋白（单体和低聚物）和纤连蛋白存在于脱蜡小鼠子宫切片高压灭菌15分钟的提取物中，但不存在于未加热标本的提取物中。10种抗原中的9种，与它们的等电点无关，在pH 9.0下加热的切片中表现出比在pH 6.0下加热的切片中强得多的免疫反应。在pH 6.0下的第二次加热显著降低了在pH 9.0下煮沸的抗原的免疫染色，但在pH 9.0下的第三次加热后恢复了免疫染色。这些结果表明，热诱导的抗原修复的主要机制是破坏的交联和pH值是一个必要的因素，适当的重折叠的表位。
In this study, we examined the mechanism of heat-induced antigen retrieval using analytical procedures involving SDS-PAGE, Western blotting, and immunohistochemistry. Five proteins were treated with 4% formaldehyde in the presence or absence of 25 mM CaCl2, then heated under various conditions after removal of formaldehyde and analyzed on SDS-PAGE. Formaldehyde produced inter- and intramolecular cross-links in the proteins. Heating at high temperatures cleaved these cross-links at all pH ranges examined (pH 3.0, 6.0, 7.5, 9.0) and produced almost the same electrophoregrams as the native proteins. Proteins treated with formaldehyde containing CaCl2 showed similar electrophoretic patterns, observed without heating or after heating at pH 6.0 and pH 9.0 in the presence or absence of 10 mM EDTA. Western blot analyses demonstrated that the soluble forms of beta-actin (monomer and oligomers) and fibronectin were present in extracts from deparaffinized mouse uterine sections autoclaved for 15 min but not in extracts from unheated specimens. Nine of ten antigens, independent of their isoelectric points, exhibited much stronger immunoreaction in the sections heated at pH 9.0 than in those heated at pH 6.0. The second heating at pH 6.0 significantly decreased the immunostaining of the antigens that had been boiled at pH 9.0, but the immunostaining was recovered after a third heating at pH 9.0. These results suggest that the main mechanism of heat-induced antigen retrieval is disruption of the cross-links and that pH is an essential factor for a proper refolding of epitopes.