Characterization of monoclonal antibodies specific to the activated ras p21 with aspartic acid at position 13.
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.
La Vecchio
J A
JA
E.I. Du Pont, Oncogene Research Group, Medical Products Department, North Billerica, Massachusetts 01862.
Hamer
P J
PJ
Ng
S C
SC
Trimpe
K L
KL
Carney
W P
WP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001224:Aspartic Acid; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009154:Mutation; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000276:immunology; D016283:Proto-Oncogene Proteins p21(ras)
D000911:Antibodies, Monoclonal; D011518:Proto-Oncogene Proteins; D001224:Aspartic Acid; D016283:Proto-Oncogene Proteins p21(ras)