Immunocapture-based fluorometric assay for the measurement of insulin-degrading enzyme activity in brain tissue homogenates.
Internally quenched fluorogenic substrates are commonly used for measuring enzyme activity in biological samples and allow high sensitivity and continuous real-time measurement that is well suited for high throughput analysis. We describe the development and optimisation of an immunocapture-based assay that uses the fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)) and allows the specific measurement of insulin-degrading enzyme (IDE) activity in brain tissue homogenates. This fluorogenic substrate can be cleaved by a number of enzymes including neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1) and angiotensin-converting enzyme (ACE), as well as IDE, and we have previously shown that discrimination between these individual enzymes is not readily achieved in tissue homogenates, even in the presence of selective inhibitors and pH conditions. We tested a panel of IDE antibodies to isolate and capture IDE from brain tissue homogenates and found that immunocapture with antibody to the inactive domain of IDE prior to the addition of fluorogenic substrate allows sensitive (linear at 156-2500ng/ml) and specific measurement of IDE activity and negligible cross-reactivity with NEP, ACE or ECE-1. This assay should allow the measurement of IDE enzyme levels in a variety of biological tissues and may be useful in study of diseases such as Alzheimer's disease and insulin-dependent diabetes.
Miners
James Scott
JS
Dementia Research Group, Institute of Clinical Neurosciences, Clinical Science at North Bristol, University of Bristol, Bristol BS16 1LE, UK. scott.miners@bristol.ac.uk
Kehoe
Patrick Gavin
PG
Love
Seth
S
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001665:Binding Sites / Q000276:immunology; D001921:Brain / Q000201:enzymology*; D001923:Brain Chemistry / Q000502:physiology*; D003429:Cross Reactions / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology / Q000503:physiopathology; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay / Q000379:methods; D006801:Humans; D007339:Insulysin / Q000032:analysis* / Q000378:metabolism; D009448:Neurochemistry / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011237:Predictive Value of Tests; D051381:Rats; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D000906:Antibodies; D005456:Fluorescent Dyes; D010455:Peptides; D007339:Insulysin