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Novel bioassays for biomarkers of inflammation: serine proteases
Author(s) -
Korzus Gabriela A.,
Saedi Mo
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1121.14
Subject(s) - proteases , granzyme , cathepsin g , granzyme a , biochemistry , enzyme , serine , serine protease , granzyme b , pmsf , biology , elastase , chemistry , protease , microbiology and biotechnology , in vitro , cytotoxic t cell , perforin
Proteases, the largest and most diverse family of enzymes, are protein‐degrading enzymes involved in vital processes. Proteases from same class have similar substrate preferences, thus present a challenge for their functional analysis in complex biological samples. This is particularly true for serine proteases involved in inflammation, namely human neutrophil elastase (HNE), protease 3 (PR3), cathepsin G (CG), and cytotoxic T lymphocyte‐granule‐associated granzymes. The recent observations indicate that these enzymes function as specific regulators of the immune response, therefore, a need exists for functional assays to specifically measure their activity. We have developed such bioassays using a novel method whereby an antibody is immobilized on a solid surface used to capture the enzyme and assay its activity with fluorogenic substrates. We have used this assay format to develop assays for HNE, PR3 and granzyme B. For granzyme B assay we included an intermediate substrate, a modified pro‐urokinase (pro‐UK GrB ) containing a GrB recognition sequence upstream of active UK. All the assays are standardized with native enzymes. Typical detection range of enzymes varies from 3–50 pg/ml to 1–100 ng/ml. The three assays developed are versatile and can be used to detect enzyme activity in a variety of biological samples, including cell lysates, tissue extracts, body fluids, and for screening enzyme inhibitors.