Lijnen H R, Beelen V, Declerck P J, Collen D
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Thromb Haemost. 1993 Sep 1;70(3):491-4.
A bio-immunoassay (BIA) for the determination of staphylokinase (STA) activity in a plasma milieu has been developed. MA-7H11, a murine monoclonal antibody raised against STA, which has a high affinity for STA but does not interfere with the complex formation between plasmin(ogen) and STA or with plasminogen activation by STA, was coated on microtiter plates at a concentration of 4 micrograms/ml. STA-containing samples were incubated overnight at 4 degrees C and, after extensive washing, bound STA was quantitated by incubation with plasminogen (final concentration 0.5 microM) for 1 h at 37 degrees C, followed by determination of generated plasmin from the absorbance at 405 nm 10 min after addition of the chromogenic substrate S-2403 (final concentration 0.3 mM). Calibration curves constructed with natural (STAN) or recombinant (STAR) STA were linear between approximately 1 and 10 nM, with a lower detection limit of < or = 1 nM in buffer and in plasma of the human, baboon or hamster. Following bolus injection of STAR in hamsters, the disposition rate of STAR activity from plasma, determined with the BIA correlated very well (r = 0.98) with that of STAR-related antigen determined by ELISA, indicating that STAR is cleared in a functionally active form. The initial half-life was about 2 min, as determined with both methods. Following continuous intravenous infusion over 1 h in baboons, the plasma clearance of STAR activity, determined from the infusion rate and the steady-state plasma level of STAR activity, ranged between 45 and 62 ml/min for doses of STAR between 0.063 and 0.250 mg/kg.
已开发出一种用于测定血浆环境中葡萄球菌激酶（STA）活性的生物免疫测定法（BIA）。MA-7H11是一种针对STA产生的鼠单克隆抗体，它对STA具有高亲和力，但不干扰纤溶酶（原）与STA之间的复合物形成，也不干扰STA对纤溶酶原的激活，以4微克/毫升的浓度包被在微量滴定板上。含STA的样品在4℃下孵育过夜，经过大量洗涤后，通过与纤溶酶原（终浓度0.5微摩尔）在37℃下孵育1小时来定量结合的STA，然后在加入显色底物S-2403（终浓度0.3毫摩尔）10分钟后，从405纳米处的吸光度测定生成的纤溶酶。用天然（STAN）或重组（STAR）STA构建的校准曲线在大约1至10纳摩尔之间呈线性，在缓冲液以及人、狒狒或仓鼠的血浆中的检测下限为≤1纳摩尔。在仓鼠中静脉推注STAR后，用BIA测定的血浆中STAR活性的处置速率与通过ELISA测定的STAR相关抗原的处置速率相关性非常好（r = 0.98），表明STAR以功能活性形式被清除。两种方法测定的初始半衰期约为2分钟。在狒狒中持续静脉输注1小时后，根据输注速率和STAR活性的稳态血浆水平测定的STAR活性的血浆清除率，对于剂量在0.063至0.250毫克/千克之间的STAR，范围在45至62毫升/分钟之间。
