Homogeneous insulin and C-Peptide sensors for rapid assessment of insulin and C-peptide secretion by the islets.
OBJECTIVE
Glucose-stimulated islet insulin or C-peptide secretion experiments are a fundamental tool for studying and assessing islet function. The goal of this work was to develop Ab-based fluorescent homogenous sensors that would allow rapid, inexpensive, near-instantaneous determinations of insulin and C-peptide levels in biological samples.


METHODS
Our approach was to use molecular pincer design (Heyduk et al., Anal Chem 2008;80:5152-5159) in which a pair of antibodies recognizing nonoverlapping epitopes of the target are modified with short fluorochrome-labeled complementary oligonucleotides and are used to generate a fluorescence energy transfer (FRET) signal in the presence of insulin or C-peptide.


RESULTS
The sensors were capable of detecting insulin and C-peptide with high specificity and with picomolar concentration detection limits in times as short as 20 min. Insulin and C-peptide levels determined with the FRET sensors showed outstanding correlation with determinations performed under the same conditions with enzyme-linked immunosorbent assay. Most importantly, the sensors were capable of rapid and accurate determinations of insulin and C-peptide secreted from human or rodent islets, verifying their applicability for rapid assessment of islet function.


CONCLUSIONS
The homogeneous, rapid, and uncomplicated nature of insulin and C-peptide FRET sensors allows rapid assessment of β-cell function and could enable point-of-care determinations of insulin and C-peptide.
Heyduk
Ewa
E
Edward A Doisy Department of Biochemistry and Molecular Biology, St Louis University Medical School, St Louis, Missouri, USA. heydukt@slu.edu
Moxley
Michael M
MM
Salvatori
Alison
A
Corbett
John A
JA
Heyduk
Tomasz
T
D000906:Antibodies; D001483:Base Sequence; D015374:Biosensing Techniques; D002096:C-Peptide / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D057230:Limit of Detection; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011384:Proinsulin / Q000032:analysis / Q000378:metabolism; D011863:Radioimmunoassay
D000906:Antibodies; D002096:C-Peptide; D007328:Insulin; D009838:Oligodeoxyribonucleotides; D011384:Proinsulin