A generic capture ELISA for recombinant proteins fused to glutathione S-transferase: validation for HPV serology.

An enzyme-linked immunosorbent assay (ELISA) system has been developed that uses glutathione crosslinked to casein as capture protein to bind recombinant protein antigens fused to N-terminal glutathione S-transferase (GST). The method allows simple and efficient immobilization and one-step purification of overexpressed recombinant antigens from crude lysates on ELISA plates coated with glutathione casein. Several antigens can be tested in parallel under the same conditions without the need to biochemically purify or renature the proteins. An additional undecapeptide epitope fused to the C-terminus of each antigen permits the detection and quantification of any full-length protein antigen bound to the ELISA plate with one single monoclonal antibody. The ELISA system was applied with four antigens to detect antibodies against E6 and E7 proteins of human papillomavirus types 16 and 18. Antibody reactivities of 164 sera from patients with cervical carcinoma and healthy individuals were in good agreement with those determined using a previously established capture ELISA with biochemically purified and renatured proteins as antigens although the GST capture ELISA was more sensitive with no loss of specificity. The GST capture ELISA could be adapted to provide standardized antibody assays for many protein antigens.

Antibodies against human papillomavirus type 16 and 18 E6 and E7 proteins in cervicovaginal washings and serum of patients with cervical neoplasia.

Serum antibodies against the E6 and E7 proteins of human papillomavirus (HPV) 16 and 18 are associated with cervical cancer. The aim of this study was to investigate the presence of local antibodies against HPV in cervicovaginal washings (CWs). In this study antibodies against the native HPV16 and HPV18 E6/E7 proteins were detectable in CWs (48%) and sera (29%) from patients with cervical cancer (n = 21) utilizing a sandwich protein enzyme-linked immunosorbent assay (ELISA). In paired CWs and sera from patients with cervical intraepithelial neoplasia (n = 38) and from healthy women (n = 22) no antibodies against these proteins were found. In 10 of 11 patients, the antibody response corresponded with the HPV type in the cervical smear and/or tumor tissue, which indicates the HPV type specificity of the assay. In 7 of 11 patients with antibody reactivity against HPV16 or HPV18 E6 and/or E7 proteins a higher level of antibody reactivity in the CWs than in the paired serum samples was found at similar inputs of total IgG. This suggests that the antibodies in the CWs against the investigated HPV proteins in these patients were locally produced.

Spontaneous regression of CIN and delayed-type hypersensitivity to HPV-16 oncoprotein E7.

We investigated delayed-type hypersensitivity to human papillomavirus (HPV) in women with cervical dysplasia or cancer. Women were challenged by skin tests with synthetic HPV-16 E7 oncoprotein peptides. 11 women were regressors (cleared disease without treatment) and 37 were progressors (required surgery). Antibodies to early antigens (markers for progression) were detectable in a higher proportion of cancer patients than all other patients, particularly progressors with cervical intraepithelial neoplasia (CIN). By contrast, cellular immunity to HPV-16 E7, measured by skin test, was significantly (p=0.0001) associated with clinical and cytological resolution of HPV-induced CIN, indicating that E7-specific T-helper cells have a role in control of HPV.

Absence of antibody against human papillomavirus type 16 E6 and E7 in patients with cervical cancer is independent of sequence variations.

It is not known whether the lack of antibody response against human papillomavirus (HPV) type 16 E6 and E7 among some cervical cancer patients is due to naturally existing sequence variations. In this study, naturally occurring HPV-16 E6 and E7 variants (including the prototype) were cloned, antigens were expressed by in vitro transcription and translation, and the humoral immune response of 34 HPV-16-positive cervical cancer patients was analyzed by radioimmunoprecipitation assay (RIPA). In addition, the RIPA results were compared with those of a sandwich-protein ELISA, to further substantiate antibody status. Sera lacking E6 reactivity by RIPA remained negative by protein ELISA. All E6 antigens (the prototype and the variants 350G¿L83V, 131G¿R10G/350G¿L83V, 335T¿H78Y/350G¿L83V, 345G¿Y81C/350G¿L83V, and African 2 ¿Af2) showed cross-reactivity by RIPA. The lack of HPV-16 E6 or E7 antibodies is independent of naturally occurring variants in cervical cancer patients. Thus, testing for HPV-16 E6 or E7 prototype antigens seems to be sufficient in serological assays.

Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in patients with head-and-neck squamous-cell carcinoma.

Human papillomaviruses (HPVs) have been recognized as an essential pathogenic factor in anogenital cancer. HPV DNA has also been found in a subgroup of head-and-neck squamous-cell carcinomas (HNSCCs), and a causative role of the virus in the development of these tumors has been suggested by the concomitant inactivation of the tumor-suppressor protein pRb. Using 4 second-generation ELISAs, we found antibodies against at least 1 of the oncoproteins E6 and E7 of the high-risk HPV types 16 and 18 in 11 of 92 sera (12%) taken from HNSCC patients at or near diagnosis, in 1 of 52 sera (2%) taken from HNSCC patients >6 months after diagnosis and in 10 of 288 sera (3. 5%) taken from sex- and age-matched healthy control individuals of the normal population. In 11 of the 12 seropositive HNSCC cases, antibodies were directed against HPV16 proteins. In patients, the HPV16 antibodies were mostly of high titer, and in 6 cases, antibodies against both HPV16 oncoproteins were present. Seven of the 8 HPV16 antibody-positive sera from the control group were of low titer, and none of the 10 antibody-positive sera reacted with both oncoproteins of the same HPV type. The HPV type of the antigens detected by the antibodies in HNSCC patients correlated well with that of the HPV DNA found in the tumor. Of 19 patients known to have HPV16 DNA-positive tumors, 7 (37%) also had HPV16 E6 and/or E7 antibodies. Our finding suggests that the antibodies were formed in an immune response against HPV E6 and E7 proteins expressed in the HNSCC and thus strongly supports the concept of a biologically active role of HPV in the development of a subgroup of HNSCC.

Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in cervical-carcinoma patients from Russia.

Certain human papillomaviruses (HPV), mainly types 16 and 18, have been widely recognized as an essential etiologic factor for the development of carcinoma of the uterine cervix. The early HPV proteins E6 and E7 are consistently expressed in the tumor cells, and cervical-carcinoma patients can develop antibodies against these oncoproteins. For cervical-carcinoma patients from Eastern Europe and Russia, detailed information on HPV DNA prevalence and HPV-specific immune responses is limited. The presence of HPV DNA in 128 Russian cervical-carcinoma tissues was determined: HPV16 DNA was found in 78% of the cases, HPV18 DNA in 14%, and no HPV-DNA in 10%. Using 4 recently developed sensitive and highly specific second-generation enzyme-linked immunosorbent assays, we also analyzed the prevalence of antibodies against HPV16 and -18 E6 and E7 proteins in sera from 95 cervical-carcinoma patients, from 61 female patients with non-HPV-associated tumors and from 83 female healthy controls. The strong association of E6 and/or E7 antibodies with cervical carcinoma was confirmed, with 36% seropositives in this group against only 2% in the control groups. The detected antibodies are highly HPV-type-specific since all 26 HPV16-E6- or -E7-antibody-positive patients had HPV16 DNA in their tumor and 6 out of the 8 HPV18-antibody-positive patients had HPV18 DNA. Antibody responses to HPV16 E6 and E7 appear to be dependent on clinical stage of the disease, with 21% seropositives found in FIGO stage I, 42% in stage II and 53% in stage III. Antibody response to HPV16 E6 is more frequent than to E7, especially in early stages.

Antibodies against early proteins of human papillomaviruses as diagnostic markers for invasive cervical cancer.

Cervical cancer is the most prevalent tumor in developing countries and the second most frequent cancer among females worldwide. Specific human papillomaviruses (HPVs) and, most notably, HPV types 16 and 18 are recognized as being causally associated with this malignancy. Antibodies against early HPV proteins E6 and E7 have been found more often in patients with tumors than in controls. Existing peptide enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-E6 and anti-E7 antibodies in human sera have low levels of sensitivity and specificity and thus are not suitable for use as diagnostic tools. Based on highly purified recombinant native proteins, we developed four sandwich ELISAs for the detection of antibodies against HPV type 16 and 18 E6 and E7 proteins. We demonstrate their sensitivities and high degrees of specificity for cervical cancer. Among a total of 501 serum specimens from unselected patients with invasive cervical cancer, 52.9% reacted positively in at least one of the four assays. In contrast, among 244 serum specimens from control subjects without cervical cancer, only 2 reactive serum specimens (0.8%) were found. For 19 of 19 antibody-positive patients, the HPV type indicated by seroreactivity was identical to the HPV DNA type found in the tumor, which also indicates a high degree of specificity for antibody detection with respect to HPV type. In a direct comparison of 72 serum specimens from patients with cervical cancer, 56% of the specimens reacted in at least one of the four protein ELISAs, whereas 40% reacted in at least one of seven peptide ELISAs covering the four antigens. These assays could be of value for the detection of invasive cervical cancer in settings in which cytology-based early tumor screening is not available, for the clinical management of patients diagnosed with cervical cancer, and for the immunological monitoring of E6 and E7 vaccination trials.

A general purification protocol for E7 proteins from "high- and low-risk" human papillomavirus types expressed in the yeast Schizosaccharomyces pombe.

A purification protocol was developed to obtain human papillomavirus (HPV) type 16 E7 protein expressed in the yeast Schizosaccharomyces pombe. Only three chromatographic steps were necessary to purify the unfused HPV 16 E7 protein to homogeneity (95-99%) as shown by silver staining after polyacrylamide gel electrophoresis. Approximately 0.8 mg of highly purified E7 was obtained from 5 x 10(10) yeast cells. The purified HPV 16 E7 phosphoprotein (Ser 31/32) was refolded and assayed for functionality. Binding to the proteins Rb1 and p107 in vitro and induction of DNA synthesis after microinjection into serum-deprived NIH 3T3 cells suggest that the E7 protein retains some of its biological activities. Most importantly, the purification strategy is also applicable for different HPV 16 E7 mutants and for E7 proteins from other HPV types such as HPV 18 and 11.

Application of a bovine cervical mucus penetration test.

One hundred seventy-eight ejaculates obtained from infertility patients were evaluated by routine semen analysis and by a bovine cervical mucus penetration test (BCMPT). A significant correlation (p less than 0.01) was observed between mucus penetration and both sperm count (r = 0.349) and sperm motility (r = 0.394). One hundred fifty-two of 178 patients (85%) had normal sperm counts (greater than 20 x 10(6)/ml). Of these patients, 68% had good (greater than 30 mm), 26% had questionable (21-30 mm), and 7% had abnormal (less than 20 mm) penetration values. One hundred sixty-one of 178 patients (90%) had normal sperm motilities (greater than 40%). Of these patients, 71% had good, 25% had questionable, and 4% had abnormal penetration values. Conversely, 46% and 18% of patients with abnormal sperm count and motility, respectively, had normal penetration values (greater than 30 mm). A significant relationship (p less than 0.05) was observed between the BCMPT and pregnancy problems not apparent by semen analysis data, and may prove to be a useful adjunct to the use of routine semen analysis in evaluating male fertility.