Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment

HTRF measurement of dopamine and bromocriptine effects on insulin secretion in cells and islets.

<p><b>(A)</b> Increasing concentrations of dopamine (DA) caused dose-dependent inhibition of GSIS in INS-1E cells, which was best fit to a sigmoidal curve (IC₅₀ = 1.28 ± 0.06 μM, R² = 0.93). <b>(B)</b> Similarly, treatment of wildtype mouse islets with 10 μM DA significantly and comparably inhibited GSIS (p<0.001) by 70.7 ± 6.8%. Consistent with a role for dopaminergic signaling as a negative mediator of GSIS, treatment of islets with10 μM bromocriptine inhibited GSIS by 67.4 ± 8.1%. For INS-1E cell-based and mouse islet experiments (<b>Panels A and B</b>, respectively), data are represented as % maximal insulin secretion based on mean HTRF values ± SEM from n≥3 independent experiments. For all panels, HTRF measurements were performed in 96-well plates with INS-1E cell secretion experiments performed in triplicate and mouse islet experiments performed in hextuplicate.</p

Advantages of the HTRF-based insulin assay compared to ELISA and RIA approaches.

<p>Here we show the main advantages of an HTRF insulin assay over comparable RIA and ELISA-based methods. Though all three methods are similarly sensitive and specific for insulin detection, the homogenous nature of the HTRF assay eliminates numerous reagents and mixing, washing and blocking steps, making the assay shorter, less expensive and more amenable to medium and high-throughput screens.</p

Validation of HTRF insulin assay conditions.

<p><b>(A)</b> pH significantly affected the ratiometric HTRF signal across a range of human insulin concentrations [F(2, 40) = 21.35, p<0.001]. Antibody incubation at pH 7 yielded the greatest HTRF signal compared to other pHs (p<0.001). <b>(B)</b> 2 h antibody incubation was sufficient to produce a robust HTRF signal, with longer antibody incubation times (12 and 48 h) further increasing HTRF signal. <b>(C)</b> There was a temperature-dependent difference in HTRF values when antibodies were incubated for 2 h at room temperature (RT, 25°C) versus 37°C [F(1,29) = 16.57, p<0.001] with higher signal observed at RT. <b>(D)</b> There was no significant difference in HTRF signal between human, rodent, porcine and bovine insulin across a range of concentrations (0.01–10 nM; p>0.05). <b>Panels A-C:</b> Data are represented as the mean emitted HTRF ratio ± SEM. <b>Panel D:</b> Data are represented as %ΔF of the HTRF signal for the respective species. For all panels, the data are from experiments performed in triplicate in 384-well plates.</p

Comparison between HTRF and ELISA insulin detection assays.

<p>Supernatants collected from glucose-stimulated INS-1E cells (20 mM glucose, 90 min, 37°C) were measured concurrently with HTRF or ELISA insulin assays. The respective HTRF and ELISA assay-derived insulin concentration values were plotted. A linear regression curve of the data showed close correlation of the insulin values from the two methods (slope = 1.15 ± 0.16, R² = 0.84). Results are represented as mean insulin concentrations ± SEM performed in triplicate in 3 independent experiments.</p

Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) to Analyze the Disruption of EGFR/HER2 Dimers: A NEW METHOD TO EVALUATE THE EFFICIENCY OF TARGETED THERAPY USING MONOCLONAL ANTIBODIES*

In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation