Preparation, characterization and properties of monoclonal antibodies against intact H-ras p21 proteins.
Monoclonal antibodies against intact normal (Gly12) and transforming (Lys12) BALB p21 ras proteins have been prepared and characterized. Their reactivity against the purified antigen proteins as well as against cellular ras proteins present in cell lines transformed by various alleles of ras genes was determined. Although some of the antibodies recognized preferentially either the normal or the activated form of BALB p21, none of them recognized exclusively either one. Deletion fragments of Ha-ras proteins were used in ELISA to determine the region of the protein encompassing the epitope recognized by each antibody. Two regions (amino acid stretches 23-69 and 89-106) accounted for the epitopes of about 75% of the mAbs raised in this study, suggesting that they encompass the most exposed and immunogenic domains in native ras p21 proteins. The affinity of these antibodies for p21 was higher than that of other ras mAbs described previously and allowed for (i) more quantitative immunoprecipitation of ras proteins, (ii) the use of selected mAbs in immunoaffinity purification of ras proteins and (iii) improved immunohistochemical detection of p21 in fixed tissues (Ward et al., accompanying manuscript). Finally, one of the mAbs described here appeared to recognize specifically a related antigenic molecule besides ras p21.
Sorrentino
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V
Laboratory of Molecular Microbiology, NIAID, Bethesda, Maryland 20892.
Nebreda
A R
AR
Alonso
T
T
Santos
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D002846:Chromatography, Affinity; D000939:Epitopes / Q000032:analysis; D005260:Female; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000276:immunology; D016283:Proto-Oncogene Proteins p21(ras)
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011518:Proto-Oncogene Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)