The high specificity of all four sandwich protein ELISAs for cervical cancer compared to those of the peptide ELISAs and ELISAs in which the protein is coupled directly to the plate is facilitated by the background control used to correct for reactions not specific for HPV proteins. The conditions in wells containing the E6-tag and E7-tag fusion proteins bound to the anti-tag antibody and in the control wells containing only the anti-tag antibody are very similar. For negative sera this results in the very low difference in absorption (net OD) observed for the group of control sera. During development of the assays we have seen that the high biochemical purities of the antigens and the anti-tag antibody and the capture principle both contribute significantly to the low level of reactivity of control sera. This allowed a stringent definition of low cutoff values even for sera of African origin, which are known to have high background reactivities in serological assays. Thus, even weakly reacting sera from patients with cervical cancer can be reliably distinguished from negative sera. Optimized renaturation protocols for the antigens were seen to drastically enhance the reactivities of positive sera. Thus, low cutoff values and a high proportion of antigen in the native conformation contribute to the high sensitivity.
However, despite this increased sensitivity, the four assays still detected disease in only 53% of the group of unselected patients with invasive cervical cancer. In Tanzania, HPV-16 DNA has been found in 38 to 45% of biopsy specimens from patients with cervical cancer, and HPV-18 DNA has been found in 25 to 32% of biopsy specimens from patients with cervical cancer (3
). In Mexico, 43% of tumors have been found to contain HPV-16 DNA (17
). It appears reasonable to assume that these type distributions are also valid for the large groups of patients with cervical cancer analyzed here. From the observed low rate of antibody cross-reactions between HPV-16 and HPV-18 early proteins and from the agreement of the HPV type specificity of serological and DNA data, we expect antibodies against E6 and E7 proteins from yet other HPV types to react only rarely in our assays. On the basis of these assumptions we estimate a serological HPV type-specific detection rate of 76 to 95% for HPV-16 DNA-positive patients with cervical cancer and 57 to 80% for HPV-18 DNA-positive Tanzanian patients with tumors.
The possibility that the sensitivities of our assays could be increased by technical improvements cannot be excluded. On the other hand, unreactive sera might reflect immunological nonreactivity to these HPV proteins, as has been discussed previously (24
). In view of the clear HPV type specificity of our assays, higher overall detection rates might also be obtained by the development of additional protein ELISAs for E6 and E7 proteins of the less prevalent carcinoma-associated HPV types such as HPV-45. Despite the sequence heterogeneity among the E6 and E7 proteins of different HPV types, the general purification and renaturation protocols developed for E6 and E7 should be applicable without major variation. We are analyzing whether equally sensitive multiplex assays that detect antibodies against E6 or E7 proteins from different HPV types simultaneously can be developed.
We started analyzing a small number of serum samples from patients with premalignant cervical lesions. The frequencies of HPV-16 E6- and/or HPV-16 E7-positive reactions were far lower with these sera than with the sera from patients with invasive cervical carcinoma, but some were clearly positive. Studies are in progress to determine the predictive value of E6- and E7-specific antibodies for the progression of such lesions. The high degree of association of the anti-E6 and anti-E7 antibodies with disease and the absence of these antibodies in the population without cancer strongly suggest that E6 and E7 are not expressed in sufficient amounts and/or are not at the appropriate site to be accessible to the immune system during primary and latent infections. By using the newly developed assays, the time of seroconversion during the development of cervical neoplasia can now be determined.
HPV-specific sandwich protein ELISAs with high degrees of sensitivity and specificity might be of value for the detection of invasive cervical cancer in developing countries where the logistics for systematic screening by cytology are difficult to establish. In addition, they might contribute to the management and follow-up of patients diagnosed with cervical cancer and also immunological monitoring of E6- and E7-specific vaccination trials.