Pedersen A N, Høyer-Hansen G, Brünner N, Clark G M, Larsen B, Poulsen H S, Danø K, Stephens R W
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.
J Immunol Methods. 1997 Apr 11;203(1):55-65. doi: 10.1016/s0022-1759(97)00008-2.
ELISAs for urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have shown that tumor levels of these molecules are prognostic parameters in breast cancer as well as other types of cancer. These ELISAs measure the total amount of the given component, including preforms, active, inactive, and complex-bound forms. However, the amount of the active forms of a component may more closely reflect the ongoing level of proteolytic activity and thereby be particularly related to prognosis. Since the inactive complex between uPA and PAI-1 can only be formed the active forms of the individual components, we have developed a sensitive and specific uPA:PAI-1 complex ELISA consisting of a sandwich format with two monoclonal antibodies (MAbs) against PAI-1 as capture antibodies and three biotinylated MAbs against uPA as detector antibodies. The data were collected as kinetic measurements of bound alkaline phosphatase activity. A standard of uPA:PAI-1 complex could be specifically measured in the assay with a detection limit of 8 pg/ml and a linear relationship between signal and complex concentration up to 4 ng/ml. Neither free uPA nor free PAI-1 were detected by this assay and the addition to the internal standard of free PAI-1 in amounts up to 20 ng/ml or uPA did not reduce the detection of complex by the assay. This ELISA was applied to extracts from 20 individual breast cancers. Each tumor was extracted in two different buffers and the median concentration of uPA:PAI-1 complex in the optimal extraction buffer was 0.8 ng/mg protein, range 0.4-2.8 ng/mg protein. Extraction of the tumor tissue at a low pH prevented de novo formation of complex from free uPA and PAI-1 in the tissue without destabilizing preformed uPA:PAI-1 complex. During incubation of the assay plate at neutral pH further uPA:PAI-1 complex formation from free components in the extracts was blocked by p-nitrophenyl guanidinobenzoate (NPGB). Thus, the present assay selectively quantifies preformed complex in tumor extracts and will enable us, for the first time, to evaluate the potential prognostic value of the uPA:PAI-1 complex in cancer.
针对尿激酶型纤溶酶原激活物（uPA）和1型纤溶酶原激活物抑制剂（PAI-1）的酶联免疫吸附测定（ELISA）表明，这些分子在肿瘤中的水平是乳腺癌以及其他类型癌症的预后参数。这些ELISA检测给定成分的总量，包括前体、活性、非活性和复合物结合形式。然而，一种成分的活性形式的量可能更能准确反映蛋白水解活性的持续水平，因此与预后尤其相关。由于uPA和PAI-1之间的非活性复合物只能由各个成分的活性形式形成，我们开发了一种灵敏且特异的uPA:PAI-1复合物ELISA，它采用夹心形式，以两种抗PAI-1的单克隆抗体（MAb）作为捕获抗体，三种生物素化的抗uPA的MAb作为检测抗体。数据以结合碱性磷酸酶活性的动力学测量值形式收集。在该检测中可以特异性测量uPA:PAI-1复合物标准品，检测限为8 pg/ml，信号与复合物浓度在高达4 ng/ml范围内呈线性关系。该检测未检测到游离的uPA和游离的PAI-1，向内部标准中添加高达20 ng/ml的游离PAI-1或uPA不会降低检测对复合物的检测能力。这种ELISA应用于20个个体乳腺癌的提取物。每个肿瘤在两种不同缓冲液中进行提取，在最佳提取缓冲液中uPA:PAI-1复合物的中位浓度为0.8 ng/mg蛋白质，范围为0.4 - 2.8 ng/mg蛋白质。在低pH值下提取肿瘤组织可防止组织中游离的uPA和PAI-1重新形成复合物，同时不会破坏预先形成的uPA:PAI-1复合物。在中性pH值下孵育检测板期间，提取物中游离成分进一步形成uPA:PAI-1复合物被对硝基苯基胍苯甲酸酯（NPGB）阻断。因此，本检测可选择性地定量肿瘤提取物中预先形成的复合物，并将首次使我们能够评估uPA:PAI-1复合物在癌症中的潜在预后价值。
