Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor

Abstract

Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function.

Mutational analysis of the N-linked glycosylation sites of the human insulin receptor

“…Cell-surface expression was monitored by FACS analysis with mAb 83.7 [39]. All except one of the stable cell lines showed increases in fluorescence of more than 18-fold that of the parental CHO-K1 cell line ( Figure 2) and comparable with that of the CHO.T cell line (Table 1), which produces more than 250 000 insulin receptors per cell [34].…”

“…Cell lines expressing a high level of receptor were selected by FACS and were cloned by limiting dilution. The ELISA employed anti-(insulin receptor) monoclonal antibody (mAb) 83.7 as the capture antibody and biotin-labelled mAb 83.14 for detection [39]. In FACS analysis, cells were treated with mAb 83.7 followed by FITC-labelled antimouse secondary antibody from sheep.…”

Memory block: A consequence of conflict resolution

“…The details of the injection volume and injection procedure were described previously (Murakami et al, 2013). This antibody blocks the binding between insulin and insulin receptors (Soos et al, 1986;Taylor et al, 1987). The control experiments were performed by injection of IgG (4.4 µg ml −1 as its final concentration in Lymnaea saline; whole molecule, Jackson ImmonoResearch Laboratories, West Grove, PA, USA; Murakami et al, 2013).…”

The insulin receptor tyrosyl kinase phosphorylates holomeric forms of the guanine nucleotide regulatory proteins Gi and Go

“…The insulin receptor kinase was purified to apparent homogeneity using a 'single-step' immunoadsorption technique employing a monoclonal antibody raised against a pure human insulin receptor preparation. This has been described in detail elsewhere [22,23]. Purification of Gt and G, to apparent homogeneity was done as described in some detail by one of us [lo].…”