Development of a fluorescence resonance energy transfer peptide library technology for detection of protease contaminants in protein-based raw materials used in diagnostic assays

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.

MeSH terms

  • Animals
  • Drug Contamination*
  • Drug Discovery
  • Fluorescence Resonance Energy Transfer / methods*
  • Humans
  • Peptide Hydrolases / analysis
  • Peptide Hydrolases / metabolism*
  • Peptide Library*
  • Peptides / metabolism
  • Protease Inhibitors / metabolism*
  • Proteins / analysis
  • Proteins / metabolism
  • Quality Control
  • Sensitivity and Specificity
  • Sulfones / metabolism
  • Trypsin / analysis
  • Trypsin / metabolism*
  • Trypsin Inhibitors / metabolism


  • Peptide Library
  • Peptides
  • Protease Inhibitors
  • Proteins
  • Sulfones
  • Trypsin Inhibitors
  • 4-(2-aminoethyl)benzenesulfonylfluoride
  • Peptide Hydrolases
  • Trypsin