Yousef George M, Polymeris Mary-Ellen, Grass Linda, Soosaipillai Antoninus, Chan Pak-Cheung, Scorilas Andreas, Borgoño Carla, Harbeck Nadia, Schmalfeldt Barbara, Dorn Julia, Schmitt Manfred, Diamandis Eleftherios P
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada.
Cancer Res. 2003 Jul 15;63(14):3958-65.
The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein (hK) gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5). KLK5 was shown to be differentially expressed at the mRNA level in breast and ovarian cancer. Until now, detection of hK5 protein in either biological fluids or tissues has not been described due to lack of suitable reagents and methods. The aim of this study was to develop immunological reagents and a sensitive and specific fluorometric immunoassay (ELISA) for hK5, to examine the presence of hK5 in human tissues and biological fluids, and to study the possible clinical utility of hK5 as a biomarker for endocrine-related malignancies. Recombinant hK5 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK5 antibodies. A sandwich-type microplate immunoassay (ELISA) was developed using these antibodies, coupled with a time-resolved fluorometric detection technique. The ELISA assay was then used to measure hK5 in various biological fluids, tissue extracts, and serum samples from normal individuals and patients with various malignancies. The hK5 ELISA immunoassay has a lower detection limit of 0.1 micro g/liter, is specific for hK5, and has no cross-reactivity with other homologous kallikreins. The dynamic range is 0.1-25 micro g/liter, and within-run and between-run coefficients of variation within this range are <10%. hK5 is found in many tissues, with the highest expression levels seen in the skin, breast, salivary gland, and esophagus. hK5 is present at relatively high levels in milk of lactating women. Whereas the levels of hK5 are almost undetectable in serum of normal individuals (male and female) and patients with diverse malignancies, higher concentrations were found in a proportion of patients with ovarian (69%) and breast (49%) cancer. High levels were also detected in ascites fluid from metastatic ovarian cancer patients and in ovarian cancer tissue extracts. In conclusion, we report development of the first immunofluorometric assay for hK5 and describe the distribution of hK5 in biological fluids and tissue extracts. Our preliminary data indicate that hK5 is a potential biomarker in patients with ovarian and breast cancer.
激肽释放酶家族是一组15个丝氨酸蛋白酶基因，聚集在19号染色体的13.4区带上。人激肽释放酶（hK）基因5（KLK5）是该家族的一员，编码一种分泌型丝氨酸蛋白酶（hK5）。研究表明，KLK5在乳腺癌和卵巢癌中的mRNA水平存在差异表达。到目前为止，由于缺乏合适的试剂和方法，尚未见在生物体液或组织中检测hK5蛋白的报道。本研究的目的是开发针对hK5的免疫试剂和灵敏特异的荧光免疫分析方法（ELISA），检测人组织和生物体液中hK5的存在，并研究hK5作为内分泌相关恶性肿瘤生物标志物的潜在临床应用价值。利用毕赤酵母表达系统生产并纯化重组hK5蛋白。该蛋白用作免疫原，制备小鼠和兔抗hK5多克隆抗体。使用这些抗体结合时间分辨荧光检测技术，开发了一种夹心型微孔板免疫分析方法（ELISA）。然后用该ELISA方法检测正常个体和各种恶性肿瘤患者的多种生物体液、组织提取物和血清样本中的hK5。hK5 ELISA免疫分析方法的检测下限为0.1μg/升，对hK5具有特异性，与其他同源激肽释放酶无交叉反应。动态范围为0.1 - 25μg/升，在此范围内批内和批间变异系数<10%。hK5在许多组织中都有发现，在皮肤、乳腺、唾液腺和食管中表达水平最高。哺乳期妇女乳汁中hK5含量相对较高。而在正常个体（男性和女性）以及各种恶性肿瘤患者的血清中几乎检测不到hK5水平，在一部分卵巢癌（69%）和乳腺癌（49%）患者中发现了较高浓度的hK5。在转移性卵巢癌患者的腹水中以及卵巢癌组织提取物中也检测到了高水平的hK5。总之，我们报道了首个针对hK5的免疫荧光分析方法的开发，并描述了hK5在生物体液和组织提取物中的分布情况。我们的初步数据表明，hK5是卵巢癌和乳腺癌患者的潜在生物标志物。
