Gentry L E, Lawton A
Virology. 1986 Jul 30;152(2):421-31. doi: 10.1016/0042-6822(86)90144-3.
Site-specific antibodies were generated against the erbB protein and epidermal growth factor (EGF) receptor by immunizing rabbits with a synthetic peptide corresponding to amino acid residues 285-296 of the predicted AEV-H erbB protein sequence. This peptide region lies within the tyrosine kinase domain of erbB and EGF receptor. Antibodies directed against this region readily identified native and denatured forms of the erbB gene product and EGF receptor as demonstrated by immuneprecipitation and immunoblot analysis. The anti-peptide antibody immuneprecipitated a functional EGF binding receptor molecule. Scatchard analysis demonstrated a KD for 125I-labeled EGF binding of 40 nM, a value consistent with that of detergent solubilized EGF receptor. Immuneprecipitates, though able to bind EGF, were unable to transfer phosphate from gamma-labeled ATP in a standard phosphorylation reaction. In detergent solubilized extracts of crude A431 microsomes, the anti-peptide antibody inhibited in a dose dependent manner the autophosphorylation of EGF receptor as well as receptor mediated phosphorylation of exogenously added substrates. In addition, this anti-peptide antibody reduced the overall level of tyrosine kinase activity present in microsomes prepared from AEV-transformed erythroblasts. This site-specific antisera should be useful for understanding the role of EGF receptor and erbB tyrosine kinase activity and their link with cell proliferation.
通过用对应于预测的AEV-H erbB蛋白序列氨基酸残基285-296的合成肽免疫兔子，产生了针对erbB蛋白和表皮生长因子（EGF）受体的位点特异性抗体。该肽区域位于erbB和EGF受体的酪氨酸激酶结构域内。如免疫沉淀和免疫印迹分析所示，针对该区域的抗体能够轻易识别erbB基因产物和EGF受体的天然和变性形式。抗肽抗体免疫沉淀出一种功能性EGF结合受体分子。Scatchard分析表明，125I标记的EGF结合的KD为40 nM，该值与去污剂溶解的EGF受体的值一致。免疫沉淀物虽然能够结合EGF，但在标准磷酸化反应中无法从γ标记的ATP转移磷酸。在粗制A431微粒体的去污剂溶解提取物中，抗肽抗体以剂量依赖性方式抑制EGF受体的自磷酸化以及受体介导的外源添加底物的磷酸化。此外，这种抗肽抗体降低了从AEV转化的成红细胞制备的微粒体中存在的酪氨酸激酶活性的总体水平。这种位点特异性抗血清对于理解EGF受体和erbB酪氨酸激酶活性的作用及其与细胞增殖的联系应该是有用的。
