A sensitive proximity ligation assay for active PSA
A sensitive proximity ligation assay for active PSA
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DOI：
10.1515/bc.2006.096
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发表时间：
2006-06
期刊：
影响因子：
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通讯作者：
Lei Zhu;H. Koistinen;Ping Wu;A. Närvänen;Edith Schallmeiner;S. Fredriksson;U. Landegren;U. Stenman
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文献类型：
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作者：
Lei Zhu;H. Koistinen;Ping Wu;A. Närvänen;Edith Schallmeiner;S. Fredriksson;U. Landegren;U. Stenman
摘要前列腺特异性抗原（PSA）是一种广泛应用的前列腺癌标志物。PSA测试的效用是有限的，因为它们无法区分前列腺癌与良性前列腺增生和前列腺炎等非恶性疾病。在循环中，PSA以各种复合和游离形式存在，其中一些的特异性测定可用于提高PSA测试的诊断准确性。我们先前已经鉴定了与酶活性PSA特异性结合的肽，并且使用这样的肽，我们已经开发了用于这种形式的PSA的免疫肽测定法。然而，该检测的灵敏度太低，无法测量临床重要水平的活性PSA。最近建立了一种新的用于蛋白质分析的灵敏免疫测定法，称为邻位连接测定法。在这里，我们描述了一个敏感的实施邻位连接测定，它利用PSA结合肽和抗体作为探针来检测活性PSA。该检测方法的灵敏度为0.07 μg/l，比我们以前的检测方法低约10倍。它不与无活性的proPSA或高度相似的激肽释放酶hK 2交叉反应。我们的研究结果表明，一个高度敏感的免疫肽测定法可以开发使用邻位连接。这一原则应有助于建立其他蛋白酶活性形式的特异性测定。
Abstract Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 μg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.