Kishi Tadaaki, Grass Linda, Soosaipillai Antoninus, Shimizu-Okabe Chigusa, Diamandis Eleftherios P
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada.
Clin Chem. 2003 Jan;49(1):87-96. doi: 10.1373/49.1.87.
The serine protease human kallikrein 8 (hK8; neuropsin), a new member of the human kallikrein family, was predicted to be secreted; thus, it is expected to be present in biological fluids. The aim of this study was to develop a sensitive and specific immunoassay for hK8 (hK8-ELISA) and establish the distribution of hK8 in tissue extracts and biological fluids.
Recombinant hK8 was produced in a baculovirus expression system and purified with a three-step chromatographic procedure. Purified hK8 was injected into mice and rabbits for antibody generation. A highly specific and sensitive sandwich-type immunoassay (ELISA) was developed using the rabbit and mouse antisera to hK8. The hK8-ELISA was then used to study the distribution of hK8 in various biological fluids and tissue extracts.
The dynamic range of the hK8-ELISA was 0.2 (detection limit) to 20 micro g/L, and imprecision (CV) was <10% within this range. This hK8-ELISA was specific for hK8 and had no detectable cross-reactivity with other members of the human kallikrein family. With this assay, hK8 was detected in tissue extracts of esophagus (highest concentrations), skin, testis, tonsil, kidney, breast, and salivary gland and in the biological fluids breast milk (highest concentrations), amniotic fluid, seminal plasma, and serum. Furthermore, in some cancer cell lines, the concentration of hK8 was regulated by steroid hormones.
We report for the first time production of recombinant hK8 protein, generation of antibodies, and development of a highly sensitive and specific immunoassay for quantification of hK8 in tissue extracts and biological fluids. This assay can be used to explore the potential of hK8 as a marker of cancer or other conditions.
丝氨酸蛋白酶人组织激肽释放酶8（hK8；神经蛋白酶）是人类组织激肽释放酶家族的新成员，预计可分泌；因此，预计它会存在于生物体液中。本研究的目的是开发一种针对hK8的灵敏且特异的免疫测定法（hK8 - ELISA），并确定hK8在组织提取物和生物体液中的分布。
重组hK8在杆状病毒表达系统中产生，并通过三步色谱法进行纯化。将纯化的hK8注射到小鼠和兔子体内以产生抗体。使用兔和小鼠抗hK8血清开发了一种高度特异且灵敏的夹心型免疫测定法（ELISA）。然后使用hK8 - ELISA研究hK8在各种生物体液和组织提取物中的分布。
hK8 - ELISA的动态范围为0.2（检测限）至20μg/L，在此范围内不精密度（CV）<10%。该hK8 - ELISA对hK8具有特异性，与人类组织激肽释放酶家族的其他成员无明显交叉反应。通过该测定法，在食管（浓度最高）、皮肤、睾丸、扁桃体、肾脏、乳腺和唾液腺的组织提取物以及生物体液母乳（浓度最高）、羊水、精浆和血清中检测到了hK8。此外，在一些癌细胞系中，hK8的浓度受类固醇激素调节。
我们首次报道了重组hK8蛋白的产生、抗体的生成以及开发了一种用于定量组织提取物和生物体液中hK8的高度灵敏且特异的免疫测定法。该测定法可用于探索hK8作为癌症或其他病症标志物的潜力。
