Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid

Immunocapture-based fluorometric assay for the measurement of insulin-degrading enzyme activity in brain tissue homogenates

“…The fluorogenic peptide substrate used in this assay is normally cleaved by a number of related enzymes, such as NEP, ACE and ECE-1, which coexist in various tissue samples, thereby making the accurate measurement of individual enzymes problematic (Miners et al, 2008). We have demonstrated that immunocapture of IDE from brain tissue homogenates prior to the addition of the fluorogenic substrate allows accurate measurement of IDE activity in brain tissue samples.…”

“…We previously developed a fluorogenic immunocapture assay that specifically measures neprilysin (NEP) activity in brain tissue homogenates (Miners et al, 2008). The fluorogenic substrate used in the assay is cleaved by a number of enzymes.…”

Impact of Vitamin D on Amyloid Precursor Protein Processing and Amyloid-β Peptide Degradation in Alzheimer's Disease

“…NEP activity measurement was performed as published in Miners et al [19], with minor modifications, utilizing the antibody AF1182 (R&D Systems, Minneapolis, Minn., USA).…”

Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute?

“…Again, the assay conditions require inhibitors to prevent nonselective hydrolysis of the substrate, as well the addition of a Nep inhibitor (thiorphan, SCH39370) to demonstrate specificity. Miners et al (86) immunoprecipitated Nep before the addition of the fluorescent substrate, and this maneuver may enhance the specificity of the assay. This approach should be applicable to other peptidases provided the appropriate antibodies are available to pull down the enzyme and do not inhibit the active site.…”