A direct radioimmunoassay for human epidermal growth factor receptor using ³²P-autophosphorylated receptor

A simple and reproducible radioimmunoassay of the epidermal growth factor (EGF) receptor which uses ³²P-labeled EGF receptor and anti-receptor monoclonal antibodies is reported. In vitro phosphorylation of A431 cell membranes with [γ-³²P]ATP in the presence of 20% dimethyl sulfoxide (which stimulates autophosphorylation of the EGF receptor) and 10 μm Na₃VO₄ (a potent inhibitor of phosphotyrosyl protein phosphatase) provides radiolabeled EGF receptor for radioimmunoassay without further purification. The most selective phosphorylation of the EGF receptor is achieved at ATP concentrations of 0.1 - 0.2 μm, which corresponds to the reported K_m value for the autophosphorylation reaction of the EGF receptor (W. Weber, P. J. Bertics, and G. N. Gill, 1984, J. Biol. Chem., 259, 14631-14939). The incorporation of ³²P into EGF receptors increases in proportion to the increase of ATP concentration up to 6 mol of labeled phosphate at 2.0 μm ATP. The label is entirely on tyrosine residues. The cell membranes can be stored at -70°C for 3 months without loss of immunoreactivity and autophosphorylating activity. Standard curves for the radioimmunoassay were constructed employing either A431 cell membranes or whole cell homogenates containing a known amount of EGF receptor. The assay can detect 7 × 10¹⁰ EGF receptor molecules or 20 ng of the receptor protein, and can quantitatively distinguish the difference in EGF receptor numbers between A431 cells and 29E2 and KB cells with 10-fold and 15-fold fewer receptors than A431 cells, respectively. 29E2 cells and KB cells express twofold more immunoreactive EGF receptors than EGF-binding sites. In contrast, A431 cells possess the same number of immunoreactive sites and receptor sites for EGF binding. To assess total EGF receptor expression, it is necessary to use a method which detects EGF receptors regardless their intrinsic kinase activity, or capacity to bind EGF. This radioimmunoassay detects immunoreactive receptor molecules, even those which do not bind EGF.