Isolation of human‐p53‐specific monoclonal antibodies and their use in the studies of human p53 expression

Abstract

The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-1 cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with j-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognised by the antibodies. At least two previously unrecognised epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-I) cells cause expression of human p53 recognised by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.

Oncogenic human papillomavirus E6 proteins target the discs large tumour suppressor for proteasome-mediated degradation

“…Equal amounts of proteins were separated by SDS ± PAGE and transferred to nitrocellulose. The levels of remaining HAtagged DLG or p53 were determined by immunoblot analysis using either the anti-HA monoclonal antibody (BoehringerMannheim) or a pool of anti p53 monoclonal antibodies, pAb1801, 1802 and 1803 (Banks et al, 1986). Blots were developed using the Amersham ECL technique according to the manufacturer's instructions.…”

Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth

“…p53-null (10)1 mouse cells and p53-null Saos-2 human cells were transfected with plasmids expressing p53, HPV-18 E6 and HPV-18 E6*. After 24 h cell extracts were made and p53 protein levels were ascertained by Western blot analysis using a pool of the anti-p53 antibodies pAb1801, 1802 and 1803 (Banks et al, 1986). The results obtained are shown in Figure 6.…”

“…Proteins were separated by PAGE and then transferred to nitrocellulose. The blot was probed with a cocktail of the mouse anti-p53 monoclonal antibodies, pAb1801, 1802 and 1803, (Banks et al, 1986) followed by anti-mouse biotin conjugate (Dako), and avidin-peroxidase (Dako), before developing with the Amersham ECL detection system according to the manufacturer's instructions.…”

Tumour associated mutants of p53 can inhibit transcriptional activity of p53 without heterooligomerization

“…Empty pCG does not contain any insert ± the poly (A) signal follows directly the CMV promoter. p53 was detected with monoclonal antibodies pAb421 (Harlow et al, 1981, epitope between amino acids 372 ± 381), pAb1801 (Banks et al, 1986, epitope between amino acids 46 ± 55), pAb240 (Gannon et al, 1990, epitope between amino acids 210 ± 214) and B17 (Legros et al, 1994, epitope between amino acids 16 ± 25). The last was generous gift from Dr Thierry Soussi.…”