Comparative immunohistochemical reactivity of monoclonal and polyclonal antibodies to H-ras p21 in normal and neoplastic tissues of rodents and humans

• Published in: Oncogene Vol. 4; no. 2; p. 203
• Main Authors: Ward, J M, Perantoni, A O, Santos, E
• Format: Journal Article
• Language: English
• Published: England 01-02-1989
• Subjects: Animals; Antibodies, Monoclonal; Cell Line, Transformed; Harvey murine sarcoma virus; Humans; Immunoblotting; Immunohistochemistry; Mice; Neoplasms - analysis; Neoplasms, Experimental - analysis; Proto-Oncogene Proteins - analysis; Proto-Oncogene Proteins - immunology; Proto-Oncogene Proteins p21(ras); Rats; Sarcoma, Experimental - analysis
• ISSN: 0950-9232

Five mouse monoclonal antibodies (mES 2, 4, 8, 13 and 20) produced against bacterially expressed BALB ras p21 and five other monoclonal or polyclonal antibodies (Cetus rabbit or mouse pan p21, RAP-5, Triton Ha-ras, Y13-259) to H-ras p21 were used for comparative immunohistochemical detection of H-ras p21 by the avidin biotin peroxidase-complex technique in selected normal fixed tissues and tumors from rats, mice, and humans. Nine of these antibodies were immunoreactive with cell membranes and cytoplasm of harvey sarcoma virus-induced sarcoma cells, but usually only with Bouin's fixed tumors. A few normal tissues were immunoreactive with some of the antibodies except for many immunoreactive tissues found with mES 13. Although mES 13 stained sarcoma cells on the cell membrane, a prominent granular staining, which appeared to be mitochondrial or lysosomal, was seen in many normal and neoplastic rodent tissues and in normal human colon, colon polyps, and carcinomas. Interpretation of positive immunoreactivity with any antibody was sometimes difficult due to nonspecific (background) staining. The cellular pattern of specific reactivity (membrane or granular) and inhibition of immunoreactivity by absorption of the antisera with H-ras p21 was therefore important. Western blots with BALB transforming (Lys 12) p21 expressed in E. coli revealed reactivity of all antibodies except for RAP-5. In addition, immunoblots of solubilized proteins from the EJ cell line with RAP-5 indicated reactivity of this monoclonal antibody with proteins of several different molecular weights. RAP-5 also never specifically immunoreacted with cell membranes of normal or malignant cells including EJ cells. Interpretation of these findings in comparison with those in published reports of H-ras p21 localization in fixed tissue sections is discussed, including the importance of fixative, specific antibody and controls.