Additional Text

Gene Name

INSR

Uniprot ID

P06213

Gene ID

3643

Purification

Purified

Antibody Clonality

Recombinant Monoclonal

Storage Note

Store at 4⁰C for up to 3 months. For longer storage, aliquot and store at -20⁰C.

Short Description

This chimeric rabbit antibody was made using the variable domain sequences of the original murine IgG2a format, for improved compatibility with existing reagents, assays and techniques.

Application Notes

The original format of this antibody (mouse IgG2a) was generated, and its binding to the HIR was characterized via RIA, Western blot, and immunoprecipitation assays. Its epitope was characterized through competition/inhibitory experiments (Soos et al., 1986; PMID: 2427071). This antibody was used to study the internalization of the HIR (Paccaud et al., 1992; PMID: 1618809), demonstrating that it facilitated constitutive internalization of the HIR via clathrin-coated pits, independent of receptor kinase activation or autophosphorylation. This process reflected the natural homeostasis of HIR on the cell surface and was mediated by the receptor's interaction with the clathrin machinery. The Fab format of the antibody was created and exhibited the same internalization rates as the full antibody, confirming that the process was not influenced by inter- or intramolecular cross-linking (Paccaud et al., 1992; PMID: 1618809). In an in vitro setting, a radiolabeled 125I version of this antibody strongly bound the Rhesus monkey brain capillary insulin receptor in an IHC assay. It demonstrated an ED50 of 0.09 µg/ml (0.60 nM) toward isolated human brain capillaries from autopsy, with a calculated KD of 0.45 +/- 0.10 nM. In vivo, the 125I-labeled version crossed the blood-brain barrier (BBB) rapidly via receptor-mediated transcytosis, with approximately 40% of its brain distribution volume attributed to postvascular compartments, as shown by capillary depletion methods. Pharmacokinetic analysis revealed an initial rapid clearance phase within 15 minutes of intravenous injection, followed by stable plasma concentrations with a mean residence time of 7-16 hours. This enabled delivery of 3-4% of the injected dose to the brain, with preferential uptake in gray matter (Pardridge et al., 1995; PMID: 7667183). This antibody was further studied in in vitro experiments with hCMEC/D3 cells, a human brain endothelial cell model. A fluorescently labeled version was used to confirm its functionality via flow cytometry, demonstrating successful binding to insulin receptor-expressing cells. To explore its potential for drug delivery, the antibody was conjugated to PDMS-b-PMOXA polymersomes, and fluorescence correlation spectroscopy (FCS) validated the conjugation efficiency. Cellular uptake and intracellular localization of the conjugated and free antibody were visualized through confocal laser scanning microscopy. Specificity for receptor-mediated uptake was confirmed in a competitive inhibition assay, where excess free mAb blocked the polymersome uptake (Dieu et al., 2014; PMID: 24929212). This antibody effectively bound multiple mutant human INSRs expressed on the cell surface of 3T3-L1 adipocytes (a mouse-derived cell line transfected with human mutant INSR constructs) in vitro. It induced receptor autophosphorylation, measured using an immunocapture assay with myc-tagged receptors, and selectively activated the Akt pathway, as shown by Western blotting for phospho-Akt.