Capillary electrophoresis-based immunoassay to determine insulin content and insulin secretion from single islets of Langerhans.
The development of a competitive immunoassay for insulin that is based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) is described. The assay uses fluorescein isothiocyanate-derivatized insulin (FITC-insulin) as a tracer and the Fab fragment of an anti-insulin monoclonal antibody as the immunoreagent. Reproducibility and reliability of the assay are improved when CZE capillaries are coated with a neutral hydrophilic polymer. The assay has an average relative standard deviation of 3.4% and a detection limit of 3 nM or about 6 fg injected. Using the assay, insulin content of single islets of Langerhans was determined to be 35 +/- 4 ng, which is in good agreement with literature values. The assay was also used to determine insulin secretion from single islets. During 5-min static incubations, 3 mM glucose elicited 0.8 +/- 0.2 ng of insulin release while 16 mM glucose elicited 3.2 +/- 0.5 ng of insulin release. These values are also in good agreement with previously reported results. These applications demonstrate the capability of rapidly determining a low level of protein in a complex biological microenvironment.
Schultz
N M
NM
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Huang
L
L
Kennedy
R T
RT
D000818:Animals; D004586:Electrophoresis / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D007118:Immunoassay; D007328:Insulin / Q000032:analysis* / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D007328:Insulin; D016650:Fluorescein-5-isothiocyanate; D005947:Glucose