Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.
Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and cerebrospinal fluid (CSF). The assay uses a neprilysin-specific antibody, previously used in a commercially available ELISA kit, to isolate and immobilise NEP from brain homogenates and CSF, prior to the addition of a fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)). This fluorogenic substrate is ordinarily cleaved by multiple enzymes. We have shown that without the immunocapture phase, even under reaction conditions reported to be specific for neprilysin - i.e. in the presence of thiorphan, at pH above 7 - the fluorogenic peptide substrate does not allow neprilysin activity in brain homogenates and CSF to be discriminated from that of other closely related enzymes. The specificity of the immunocapture enzyme activity assay was confirmed by >80% inhibition of substrate cleavage in brain homogenates and CSF in the presence of thiorphan. The assay allows high-throughput analysis and, critically, also ensures a high level of enzyme specificity even when assaying crude tissue homogenates or CSF.
Miners
James Scott
JS
Dementia Research Group, Institute of Clinical Neurosciences, Clinical Science at North Bristol, University of Bristol, Frenchay Hospital, Frenchay, Bristol BS16 1LE, UK. scott.miners@bristol.ac.uk
Verbeek
Marcel M
MM
Rikkert
Marcel Olde
MO
Kehoe
Patrick Gavin
PG
Love
Seth
S
D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D001923:Brain Chemistry; D002555:Cerebrospinal Fluid / Q000187:drug effects / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods; D006801:Humans; D015260:Neprilysin / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D015244:Thiorphan / Q000494:pharmacology; D013997:Time Factors
D005456:Fluorescent Dyes; D009841:Oligonucleotides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D015244:Thiorphan; D015260:Neprilysin