Kapprell Hans-Peter, Maurer Andreas, Kramer Florian, Heinrich Boris, Buenning Carsten, Narvaez Alfredo, Kalbacher Hubert, Flad Thomas
Abbott GmbH & Co.KG, Wiesbaden-Delkenheim, Germany.
Assay Drug Dev Technol. 2011 Oct;9(5):549-53. doi: 10.1089/adt.2010.0360. Epub 2011 Jun 15.
Protease impurities in raw materials used in enzyme immunoassays can impair assay performance. This risk may be greatly decreased if incoming protein-based raw materials are controlled for protease impurities or if protease inhibitors are used in the assay formulations. As many different proteases might occur in protein raw materials, it is desirable to have a general test for protease contamination. With the help of a fluorescence resonance energy transfer peptide library containing about 2.5 million peptides, we have succeeded in establishing such a system, with sensitivity in the nanogram range for known proteases. Protease contamination was found to differ between different raw materials and was correlated with assay performance. Protease activity in contaminated raw materials could be suppressed to various degrees with different chemical inhibitors or by thermal treatment. This technology is suited for the control of incoming protein-based raw materials used for enzyme immunoassays, as well as for the optimization of the use of protein inhibitors or thermal treatment of protein-based raw materials for the inactivation of proteases.
酶免疫测定中使用的原材料中的蛋白酶杂质可能会损害测定性能。如果对 incoming 的基于蛋白质的原材料进行蛋白酶杂质控制，或者在测定配方中使用蛋白酶抑制剂，这种风险可能会大大降低。由于蛋白质原材料中可能会出现许多不同的蛋白酶，因此需要进行蛋白酶污染的通用测试。借助一个包含约 250 万个肽的荧光共振能量转移肽库，我们成功建立了这样一个系统，对已知蛋白酶的灵敏度在纳克范围内。发现不同原材料之间的蛋白酶污染情况不同，并且与测定性能相关。受污染原材料中的蛋白酶活性可以通过不同的化学抑制剂或热处理在不同程度上得到抑制。这项技术适用于对用于酶免疫测定的 incoming 基于蛋白质的原材料进行控制，以及用于优化蛋白质抑制剂的使用或对基于蛋白质的原材料进行热处理以灭活蛋白酶。
