Insulin binding to the analytical antibody sandwich pair OXI-005 and HUI-018: Epitope mapping and binding properties.

The insulin epitopes for two monoclonal antibodies (mAbs), OXI-005 and HUI-018, commonly used in combination for insulin concentration determination in sandwich assays, were determined using X-ray crystallography. The crystal structure of the HUI-018 Fab in complex with human insulin (HI) was determined and OXI-005 Fab crystal structures were determined in complex with HI and porcine insulin (PI) as well as on its own. The OXI-005 epitope comprises insulin residues 1,3,4,19-21 (A-chain) and 25-30 (B-chain) and for HUI-018 residues 7,8,10-14,17 (A-chain) and 5-7, 10, 14 (B-chain). The areas of insulin involved in interactions with the mAb are 20% (OXI-005) and 24% (HUI-018) of the total insulin surface. Based on the Fab complex crystal structures with the insulins a molecular model for simultaneous binding of the Fabs to PI was built and this model was validated by small angle X-ray scattering measurements for the ternary complex. The epitopes for the mAbs on insulin were found well separated from each other as expected from luminiscent oxygen channeling immunoassay results for different insulins (HI, PI, bovine insulin, DesB30 HI, insulin glargine, insulin lispro). The affinities of the OXI-005 and HUI-018 Fabs for HI, PI, and DesB30 HI were determined using surface plasmon resonance. The KD s were found to be in the range of 1-4 nM for the HUI-018 Fab, while more different for the OXI-005 Fab (50 nM for HI, 20 nM for PI and 400 nM for DesB30 HI) supporting the importance of residue B30 for binding to OXI-005.

Treatment with insulin analog X10 and IGF-1 increases growth of colon cancer allografts.

Obesity and type 2 diabetes are associated with an increased risk for development of certain forms of cancer, including colon cancer. The publication of highly controversial epidemiological studies in 2009 raised the possibility that use of the insulin analog glargine increases this risk further. However, it is not clear how mitogenic effects of insulin and insulin analogs measured in vitro correlate with tumor growth-promoting effects in vivo. The aim of this study was to examine possible growth-promoting effects of native human insulin, insulin X10 and IGF-1, which are considered positive controls in vitro, in a short-term animal model of an obesity- and diabetes-relevant cancer. We characterized insulin and IGF-1 receptor expression and the response to treatment with insulin, X10 and IGF-1 in the murine colon cancer cell line (MC38 cells) in vitro and in vivo. Furthermore, we examined pharmacokinetics and pharmacodynamics and monitored growth of MC38 cell allografts in mice with diet-induced obesity treated with human insulin, X10 and IGF-1. Treatment with X10 and IGF-1 significantly increased growth of MC38 cell allografts in mice with diet-induced obesity and we can therefore conclude that supra-pharmacological doses of the insulin analog X10, which is super-mitogenic in vitro and increased the incidence of mammary tumors in female rats in a 12-month toxicity study, also increase growth of tumor allografts in a short-term animal model.

Small-molecule agonists for the glucagon-like peptide 1 receptor.

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists.

A luminescent oxygen channeling immunoassay for the determination of insulin in human plasma.

The authors describe a homogeneous, sensitive, and rapid bead-based sandwich immunoassay with a broad analytical range for quantifying insulin in human plasma. The assay was performed as a 2-step reaction by incubating the sample with a mixture of biotinylated anti-insulin antibody and beads covalently coated with anti-insulin antibody for 1 h. This was followed by incubation with beads covalently coated with streptavidin for 30 min. After the incubation steps, light generated from a chemiluminescent reaction within the beads was quantitated. The assay was run in 384-well plates with a sample volume of 5 microL. The analytical range extended from 1 to 10,000 pM. Intra-assay precision (% coefficient of variation) ranged from 1.9% to 3.8% for various insulin concentrations. Interassay precision ranged from 4.6% to 7.3%. Assay detection limit was 0.3 pM. There was no interference from moderate hemolysis (with hemoglobin up to 375 mg/dL), bilirubin (up to at least 50 mg/dL), triglyceride (up to at least 1000 mg/dL), biotin (up to at least 7.7 ng/mL), or ascorbic acid (up to 100 mg/dL). However, gross hemolysis did affect the assay. Comparable results were obtained for plasma (ethylenediamine tetra-acetic acid, citrate, and heparin treated) and serum. The correlation with enzyme-linked immunosorbent assay (ELISA) was good (y = 1.25x + 1.19, R(2) = 0.98). This method is convenient and represents an alternative to ELISA.