Direct human papillomavirus E6 whole-cell enzyme-linked immunosorbent assay for objective measurement of E6 oncoproteins in cytology samples

Abstract

A novel, whole-cell enzyme-linked immunosorbent assay (ELISA) based on a non-type-specific anti-human papillomavirus (HPV) E6 antibody was tested on 182 residual cytological specimens. For samples with a designation of more severe than cervical intraepithelial neoplasia grade 3 (CIN3+), 83% tested positive for E6; in a subset with paired testing for E6 ELISA and HPV DNA, 72% tested E6 positive and 92% tested high-risk (HR)-HPV DNA positive (P = 0.2). Among the women with a less than CIN3 diagnosis, 31% and 47% tested positive for E6 and HR-HPV DNA, respectively (P = 0.0006).

Figures

Fig 1

Anti-E6 antibody binds specifically to HPV E6 protein. (A) ELISA results showing that anti-E6 antibody recognizes recombinant E6 proteins from HPV16 and -18 but not recombinant E7 proteins from HPV16 or -18, recombinant HPV16 L1 protein, or the 6×His tag. (B) Titration curves of recombinant HPV18 E6 protein detected by anti-E6 antibody in ELISA showing that the limits of detection range from 10 to 100 pg/ml. The data are presented as means and standard errors. (C) Immunoblot using cervical cancer cell line lysates. The top blot shows that anti-E6 antibody recognizes recombinant HPV18 E6 protein (right lane) and E6 protein from cell lysates from HeLa (HPV-positive) but not C33A (HPV-negative) cells. At the bottom is an immunoblot using anti-β-actin for the corresponding cell lysates. (D) Whole-cell ELISA using anti-E6 antibody detected E6 proteins from HeLa and SiHa cells expressing HPV18 and HPV16, respectively, with signal strengths dependent on the cell density. E6 protein was minimally detected in HPV-negative C33A cells.

Fig 2

Whole-cell ELISA using anti-E6 antibody. (A) Scatter dot plot of the individual absorbance signals of clinical samples in a whole-cell ELISA using anti-E6 antibody. E6 whole-cell ELISA was performed on cells from 182 cervical scrapes that were categorized into the following 4 groups: group 1, histology negative and HPV DNA negative (Neg/HPV−) (n = 62); group 2, histology negative and HPV DNA positive (Neg/HPV+) (n = 27); group 3, CIN1/2 (n = 51); and group 4, CIN3+ (n = 42). Lines show means and standard errors of the means (SEM). (B) Average absorbances obtained from the ELISA in panel A graphed as means and SEM. (C) Results from E6 whole-cell ELISA using 45 SurePath fresh samples obtained within 1 to 2 weeks of collection. The data are presented as percent positive rates for histological designations of CIN ≤ 1 (36 samples), CIN3 (6 samples), and cervical cancer (3 samples) and compared to the positive rates for HPV DNA.