Studies on the establishment of a method to detect HPV 6b E7-specific antibodies of serum and cervical secretion from patients with condyloma acuminatum and its epidemiological significance

Abstract

To establish a method for detection of the human papi11omavirus (HPV) 6b E7-specific antibodies in serum and cervical secretion from patients with condyloma acuminatum (CA). A full-length HPV 6b E7 gene was amplified by PCR from the CA tissue to construct the recombinant plasmid pET32a(+)/HPV 6b E7. The expression of prokaryotic protein was analyzed by SDS-PAGE and Western blot, then purified with Ni-NTA Agarose affinity column and used as an diagnostic antigen for establishing indirect ELISA method, to detect specific serum IgG and specific cervical secretion sIgA from 56 CA patients, 81 healthy control. Sera from 43 cervical cancer was served as control. HPV 6b DNA from 56 CA patients was identified by PCR. Data showed that the nucleotide homology of cloned sequence was 99.5%, compared to the standard sequences of HPV 6b E7 gene (GenBank accession number: NC001355). A high level expression of E7 fusion protein was obtained in prokaryotic expression system (40 µg/ml). Based on HPV 6b E7 fusion protein being used as coating antigen, results from ELISA showed that the absorbance rates (A) of serum IgG from CA, cervix cancer and healthy control groups were 1.82 ± 0.48, 1.36 ± 0.39 and 1.39 ± 0.27, respectively. The level of IgG antibody in the serum of CA group was significantly higher than that in cervix cancer group and healthy control (P < 0.05). The A values of cervical secretion sIgA in CA and healthy control groups were 0.63 ± 0.26 and 0.53 ± 0.06, respectively, while the level of sIgA antibody in the cervical secretion of CA group was also significantly higher than that in healthy controls (P < 0.05). The positive rate of HPV 6b E7 DNA in CA tissue was 78.6% (44/56) by PCR method. When compared the results measured by PCR, the HPV 6b E7-specific IgG and sIgA antibodies by ELISA used to detect the patients infected with HPV 6b infection, showed that the sensitivity rates were 68.2% (30/44) and 54.6% (24/44) respectively, and the specificity were all 100% (12/12). Based on the serum and cervical secretion specific HPV 6b E7 antibodies from patients with CA to diagnose HPV 6b infection, results showed medium sensitivity and high specificity, and could further be used to investigate the epidemiological characteristics of HPV 6b infection.