Production and characterisation of monoclonal antibodies reactive with the E7 protein of human papillomavirus type 18 : including some applications for the characterisation of the human papillomavirus type 18 E7 protein expressed in cervical carcinoma cells and of recombinant human papillomavirus type 18 E7 protein expressed in insect cells : a report

Abstract

23 monoclonal antibodies were raised against a MS2-replicase/HPV 18 E7 fusion protein. 19 if the monoclonal antibodies reacted with two or more octapeptides derived from the amino acid sequence of the putative HPV 18 E7 protein. It was deduced that these antibodies react with 3 major linear epitopes in the N-terminal region of the protein. The results of competition immunoassays suggested that the four monoclonal antibodies which did not react with linear peptides also reacted with epitopes situated in the N-terminal region of the HPV 18 E7 protein.12 tested monoclonal antibodies immunoprecipitated a protein, whose molecular weight was consistent with the putative HPV 18 E7 protein, from HPV 18 containing but not HPV 16 containing cervical carcinoma cells. However, none of the monoclonal antibodies detected an HPV 18 specific protein by western blotting or indirect immunofluorescence and the data suggested that this was a result of inadequate sensitivity of these two detection methods.Two recombinant baculoviruses that encoded HPV 18 E7 protein were produced. 20 of the 23 monoclonal antibodies reacted with recombinant HPV 18 E7 protein expressed in insect cells by western blotting and indirect immunofluorescence. HPV 18 E7 protein was detected predominantly in the nucleus of recombinant baculovirus infected cells by indirect immunofluorescence.5 of 6 monoclonal antibodies immunoprecipitated a radiolabelled protein from extracts of HeLa cells that had been biosynthetically labelled with 32P, suggesting that at least some of the HPV 18 E7 protein in HeLa cells is phosphorylated. One of the antibodies, 15H4, did not precipitate 32P labelled HPV 18 E7 protein although it precipitated 35S labelled HPV 18 E7 protein suggesting that both phosphorylated and non-phosphorylated HPV 18 E7 protein occur in HeLa cells and that 15H4 reacts only with the non-phosphorylated form. Isoelectric focussing data using 15H4 and other monoclonal antibodies to detect HPV 18 E7 protein in extracts of insect cells that had been infected with an HPV 18 E7 recombinant baculovirus suggested that recombinant HPV 18 E7 protein is predominantly phosphorylated in insect cells.A sandwich ELISA for HPV 18 E7 protein was developed using a combination of several monoclonal antibodies and a rabbit antiserum raised against the MS2- replicase/HPV 18 E7 fusion protein. Using this assay, HPV 18 E7 protein was quantified in HPV 18 containing HeLa and C4-1 cells. The addition of hydrocortisone, but not progesterone, s-oestradiol or testosterone, resulted in a 50% increase in the concentration of HPV 18 E7 protein in HeLa and C4-1 cells. This effect of hydrocortisone was blocked by the glucocorticoid antagonist cortexolone.It was concluded that:- 1) HPV 18 E7 protein is antigenic; 2) antibodies to this protein can be used to detect and quantify HPV 18 E7 protein in HPV infected cells, and 3) monoclonal antibodies can be used to distinguish post-translationally modified (phosphorylated) from unmodified E7 protein.