La Vecchio J A, Hamer P J, Ng S C, Trimpe K L, Carney W P
E.I. Du Pont, Oncogene Research Group, Medical Products Department, North Billerica, Massachusetts 01862.
Oncogene. 1990 Aug;5(8):1173-8.
The ras proto-oncogenes encode membrane bound proteins (p21) which are structurally distinct from the proteins encoded by the activated transforming ras genes. These activated ras genes have been identified in various human tumors as well as their preneoplastic lesions such as colorectal tumors (20-40%), pancreatic carcinomas (95%), lung carcinomas (20-30%), myelodysplasia (40%) and acute myeloid leukemia (30%). The activation of ras p21 is due to amino acid substitutions at positions 12, 13 or 61 of the p21 protein. This report describes two monoclonal antibodies designated D129 and D146 raised against a synthetic peptide corresponding to amino acids 5-16 of ras p21 activated by the substitution of aspartic acid for glycine at position 13. D129 and D146 react specifically with the peptide with the aspartic acid substitution at position 13, but not with the peptide with valine at position 13 or the peptide containing the normal glycine at position 13. Western blot analysis demonstrates that D129 and D146 react specifically with p21 extracted from transformed NIH3T3 fibroblast lines containing aspartic acid at position 13. These studies also demonstrate that D146 is able to detect the activated p21 with aspartic acid at position 13 that is shed into the culture media. Studies demonstrate that MAb D146 specifically immunoprecipitates the cellular p21 with aspartic acid at position 13 from transformed NIH3T3 cells, whereas D129 cannot immunoprecipitate the activated p21. Using a sandwich ELISA format, D146 is able to detect the p21 with position 13 aspartic acid from cell extracts and culture fluids. The ability of D146 to function in the ELISA format raises the possibility that this assay maybe a quick and effective way of determining the presence of activated p21 with aspartic acid at position 13 in human fluids and tissues.
原癌基因ras编码膜结合蛋白（p21），其结构与活化的转化型ras基因所编码的蛋白不同。这些活化的ras基因已在各种人类肿瘤及其癌前病变中被鉴定出来，如结直肠癌（20%-40%）、胰腺癌（95%）、肺癌（20%-30%）、骨髓发育异常（40%）和急性髓系白血病（30%）。ras p21的活化是由于p21蛋白第12、13或61位氨基酸的替换。本报告描述了两种单克隆抗体，命名为D129和D146，它们是针对一种合成肽产生的，该合成肽对应于ras p21的第5-16位氨基酸，其中第13位的甘氨酸被天冬氨酸替换而活化。D129和D146与第13位有天冬氨酸替换的肽特异性反应，但不与第13位有缬氨酸的肽或第13位含有正常甘氨酸的肽反应。蛋白质印迹分析表明，D129和D146与从含有第13位天冬氨酸的转化NIH3T3成纤维细胞系中提取的p21特异性反应。这些研究还表明，D146能够检测到第13位有天冬氨酸且释放到培养基中的活化p21。研究表明，单克隆抗体D146能从转化的NIH3T3细胞中特异性免疫沉淀第13位有天冬氨酸的细胞p21，而D129不能免疫沉淀活化的p21。采用夹心酶联免疫吸附测定（ELISA）形式，D146能够从细胞提取物和培养液中检测到第13位有天冬氨酸的p21。D146在ELISA形式中发挥作用的能力增加了这样一种可能性，即该检测方法可能是一种快速有效的方法，用于确定人体液和组织中第13位有天冬氨酸的活化p21的存在。
