On-line competitive immunoassay based on capillary electrophoresis applied to monitoring insulin secretion from single islets of Langerhans.
An automated on-line competitive immunoassay based on capillary electrophoresis (CE) was utilized to monitor secretion of insulin from single islets of Langerhans stimulated by glucose and tolbutamide. In the instrument, fluorescein isothiocyanate-labeled insulin (FITC-insulin), monoclonal anti-insulin and perifusate of single islets were mixed on-line while islets were exposed to different levels of glucose and tolbutamide. Insulin released from single islets competed with FITC-insulin for antibody binding sites. Therefore, the amounts of bound and free FITC-insulin were modulated by insulin released from islets. The bound and the free FITC-insulin were separated by CE every 3 s and the bound over free ratio (B/F) was measured. Insulin levels were obtained by comparing B/F with calibration curves obtained under the same conditions except that the islet perfusate was replaced with various concentrations of insulin. Patterns of insulin secretion stimulated by glucose and tolbutamide observed were comparable to what has been seen previously using radioimmunoassay or enzyme-linked immunoassay. This on-line competitive immunoassay system provided a fast and direct way to measure insulin release from single islets. The effects of temperature on antibody-antigen reaction rate and binding equilibrium were also studied.
Tao
L
L
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Aspinwall
C A
CA
Kennedy
R T
RT
D000818:Animals; D001667:Binding, Competitive; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005947:Glucose / Q000494:pharmacology; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D066298:In Vitro Techniques; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D013696:Temperature; D013997:Time Factors; D014044:Tolbutamide / Q000494:pharmacology
D007328:Insulin; C031573:insulin, fluorescein-isothiocyanated-; D014044:Tolbutamide; D016650:Fluorescein-5-isothiocyanate; D005947:Glucose