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Background: Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also involved in the development of other tumours (skin, head and neck). Serological survey of the HPV infections is important to better elucidate their natural history and to disclose antigen determinants useful for vaccine development. At present, the analysis of the HPV-specific antibodies has not diagnostic value for the viral infections, and new approaches are needed to correlate the antibody response to the disease outcome. The aim of this study is to develop a novel ELISA, based on five denatured recombinant HPV16 proteins, to be used for detection HPV-specific antibodies. Methods: The HPV16 L1, L2, E4, E6 and E7 genes were cloned in a prokaryotic expression vector and expressed as histidine- tagged proteins. These proteins, in a denatured form, were used in ELISA as coating antigens. Human sera were collected from women with abnormal PAP smear enrolled during an ongoing multicenter HPV-PathogenISS study in Italy, assessing the HPV- related pathogenetic mechanisms of progression of cervical cancer precursor lesions. Negative human sera were collected from patients affected by other infectious agents. All the HPV-positive sera were also subjected to an avidity test to assess the binding strength in the antigen-antibody complexes. Results: Most of the sera showed a positive reactivity to the denatured HPV16 proteins: 82% of the sera from HPV16 infected women and 89% of the sera from women infected by other HPV genotypes recognised at least one of the HPV16 proteins. The percentages of samples showing reactivity to L1, L2 and E7 were similar, but only a few serum samples reacted to E6 and E4. Most sera bound the antigens with medium and high avidity index, suggesting specific antigen-antibody reactions. Conclusion: This novel ELISA, based on multiple denatured HPV16 antigens, is able to detect antibodies in women infected by HPV16 and it is not genotype-specific, as it detects antibodies also in women infected by other genital HPVs. The assay is easy to perform and has low cost, making it suitable for monitoring the natural history of HPV infections as well as for detecting pre-existing HPV antibodies in women who receive VLP-based HPV vaccination. Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:6 http://www.infectagentscancer.com/content/1/1/6 Escherichia coli (E. coli). The assay uses the recombinant Background Human Papillomaviruses (HPVs) infect cutaneous, geni- viral L1 and L2 capsid proteins, E6 and E7 oncoproteins, tal, and respiratory epithelia in tissue-specific manner. and non-structural E4 protein, all in denatured form. The Papillomaviruses are non-enveloped viruses with a dou- assay has been tested to monitor the antibody response in ble-strand DNA genome of 8 Kb. The genome is encapsi- women enrolled in the HPV-PathogenISS cohort to study dated in an icosahedral structure of 55 nm-diameter, the HPV-related pathogenetic mechanisms of CIN pro- composed of the L1 and L2 proteins, which are the major gression in HIV-negative and HIV-positive women . and minor capsid protein, respectively. The HPV16 genome codes for several non-structural proteins: E1, E2, Results and discussion E4, E5, E6 and E7. E1 and E2 are essential in viral tran- Analysis of the HPV genotypes scription and replication, E4 binds cytokeratins and is Before testing the 99 sera for the presence of HPV antibod- involved in modification of the cytoskeleton network; E5 ies, DNA samples from cervical smears of the same affects cellular receptors of growth factors, whereas E6 and patients were analysed for the presence of HPV DNA, fol- E7 are the major transforming proteins . lowed by virus genotyping. HPV sequences were found in all the samples, but the identification of a specific HPV HPV infections are widespread in the general population, genotype was possible only in 66 samples. In the present and viral infection is closely associated to both benign study these 66 cases were considered (Table 2). Among and malignant lesions [2,3]. Studies performed by several the 18 genotypes detected, 12 (66.7%) were "high risk" groups have recently established that only few of the over HPV (HR-HPV), 1 (genotype 90) was of "indeterminate 30 genital HPV genotypes described, are important risk risk" and 5 were "low risk" HPV (LR-HPV) according to factors for developing high-grade of cervical intraepithe- the epidemiological classification of Munoz and co-work- lial neoplasia (CIN3) and cervical cancer (CC), with a ers . The most common HPV genotypes in descending high prevalence of genotype 16 [4,5]. Serological studies order of frequency were: HPV16 (33.3%), HPV35 (9.1%), are important to understand the natural history of HPV HPV31 (7.5%), HPV6-18-52-66 (6%). HPV16 was the infections, and during the past 15 years, efforts have been most prevalent genotype; half of the infections were asso- made to develop reliable genotype-specific serological ciated to the HR 16, 35, 31 genotypes, whereas HPV18 assays. Most of the sero-epidemiological studies have was found in only 6% of the women. The prevalence of focused on confirming the relationship between the pres- HPV16 is similar to that previously reported in Italy [13- ence of HPV antibodies and the detection of anogenital 17] whereas that of the other genotypes is very different, cancers or their precursors. The majority of these studies probably due to the small number of samples analysed. have used either virus like particles (VLPs) or E6/E7 onco- protein-based serological assays, while other HPV pro- Analysis of specific antibodies in HPV patients teins have been used as antigens less frequently [2,6,7]. To study the HPV-specific antibody response, several ELI- SAs were set up in parallel using the recombinant HPV16 The main conclusion from these sero-epidemiological L1, L2, E4, E6 and E7 proteins as coating antigens. Figure studies is that the antibody response to HPV16 proteins 1 shows the SDS-PAGE analysis of the proteins expressed does not invariably occur during a natural HPV infection. in E. coli and purified. The protein pattern shows bands of For example a humoral immune response to VLPs is expected molecular mass; the level of protein purity is induced in about half of the women with normal cytology over 95%. The proteins were inoculated into mice to gen- and HPV DNA presence in their cervical epithelium. The erate specific hyper-immune sera. All the viral proteins VLP-specific antibodies are neutralizing and genotype- showed high immunogenicity, inducing specific antibod- specific and have been crucial for the development of pre- ies reacting with the corresponding antigen in ELISA, ventive HPV vaccines [8,9]. An anti-HPV16 E6/E7 Immunoprecipitation and in Western blotting experi- response has been predominantly found in patients with ments (data not shown). advanced CC, but it has also been found in control patients, making doubtful the use of serology in cancer In the attempt to identify HPV specific and pan-reactive prediction . However prospective cohort studies using antibodies, we set up an ELISA using the HPV16 recom- a widespread panel of viral antigens are urgently needed binant proteins in denatured form, with carefully deter- to improve our understanding on HPV seroconversion in mined cut-off values (see below). Among the 66 sera patients with and without cervical lesions, as well as to examined, 22 derived from women infected by HPV16, study the dynamics of antigen-specific HPV antibodies in while 44 cases from women infected by other HPV geno- relation to the clinical outcome of the viral infection. types. The results of a representative multiple HPV-ELISA are shown in Figure 2. The results of seroreactivity to the This paper reports the development of an in-house ELISA single HPV16 proteins, related to the HPV genotype data, system, based on five HPV16 proteins expressed in are listed in Table 2. The highest number of positive reac- Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:6 http://www.infectagentscancer.com/content/1/1/6 Table 1: HPV primers used in PCR amplification of the HPV 16 genes. Primer DNA sequence Gene target Nucleotide positions in NCBI no NC_001526 L1_Sfor 5-GCCGTCGACATGTCTCTTTGGCTGCCTAGTAGGGCCA-3' Late protein L1 5637–5663 L1_Srev 5'- GCCGTCGACGATTTGTAGTAAAAATTTGCGTCC-3' Late protein L1 7027–7050 L2_Bfor 5'-GCGCGGATCCATGcgacacaaacgttctgcaa -3' Late protein L2 4235–4256 L2_Hrev 5'-GCGCAAGCTTCTAGGCAGCCAAAGAGACATC -3' Late protein L2 5636–5656 E6_Bfor 5'-GCGCGGATCCATGCACCAAAAGAGAACTGC3' Early protein E6 83–102 E6_Hrev 5'-GCGCAAGCTTTTACAGCTGGGTTTCTCTACGTG -3' Early protein E6 537–559 E4_Bfor 5'-GCGCGGATCCATGTATTATGTCCTACATCTGTGTTT -3' Early protein E4 3332–3357 E4_Hrev 5'-GCGCAAGCTTCTATGGGTGTAGTGTTACTATTA-3' Early protein E4 3597–3619 The start and stop codons are bolded while the restriction sites (Sal I, Bam HI, and Hind III) are underlined tivity was observed against L1, L2 and E7, while only a few account for the cross-reactivity among the sera of women sera had antibodies to E6 and E4. Only few sera were able infected by different HPV genotypes. to react with all the HPV16 antigens and were collected from women infected by HPV16, 31, 35, 6, 6b, 11 and 90 The percentage of negative sera, shown in Figure 3, is 18% genotypes. These genotypes are correlated to some extent and 11% respectively in the HPV16 and in the other HPVs ; HPV31 and 35 belong to the same viral species as group. This finding could suggest that HPV16 is less HPV16; HPV6, 6b and 11 belong to species related to immunogenic than the other HPV genotypes, at least as HPV16, whereas HPV90 belongs to a species only dis- far as it concerns the humoral response against linear tantly related to HPV16. The reactivity to E6 was always epitopes revealed by our immunoassay. A different immu- associated with reactivity to other viral antigens as well. nogenicity of HPV genotypes could account for the mark- Some sera reacted only to one antigen, which was L1, L2, edly different prevalence of HPV genotypes in different E4 or E7. Among the 22 sera from HPV16-positive geographic regions . Taken together, these results sug- women, 18 (82%) were positive at least to 1 HPV16 pro- gest that our multiple ELISA system, based on denatured tein, while the rest (18%) were negative to all proteins. HPV16 proteins, could be used for detection of HPV-spe- The negative result indicates a complete lack of antibody cific antibodies in women infected by different viral gen- response in the patient, although we cannot exclude that otypes, although a larger number of sera needs to be the presence of mutations in the genome of infecting virus analysed for its standardization. could determine the lack of antibody reactivity. Avidity of the HPV antibodies Among the 44 sera of women infected by other HPV gen- The Avidity Index (AI) of specific immunoglobulins is a otypes, 39 (89%) reacted at least to one of the five HPV16 useful parameter for diagnosis of primary or past infection proteins, and only 5 (11%) did not react at all. Of all the in a large number of viral diseases . It is well known 66 sera analysed, 57 (86%) were reactive at least to one that after an infection or an immunization, the titer of HPV16 protein, and 9 sera (14%) were entirely negative. serum specific immunoglobulin as well as the antibody The reproducibility of the assay was high as indicated by affinity to the antigen, increase . Several studies have the inter-assay Coefficient of Variation (CV) of about 5%. shown that early after an infection serum antibodies have The response of the different sera to the same antigen is an AI lower than 40%; during the infection the AI values variable and the differences might reflect the different progressively increase with time after infection, achieving stage of viral infections. an AI higher than 60% [21-24]. This parameter is meas- ured by assaying the resistance of antigen-antibodies com- Figure 3 shows the percentage of sera positive to each pro- plexes to denaturing substances . To evaluate the tein in the two groups of sera: 1) HPV16 and 2) other specificity of each antigen-antibody complex in this HPV- HPVs. In the first group, the percentage of sera reactive to ELISA, we determined the AI in 32 sera positive to L1, 36 L2 (64%) is higher than that reactive to L1 (50%) or E7 to L2, 19 to E4, 22 to E6 and 41 to E7. The HPV-specific (50%). In the second group the percentage of sera reacting mouse hyper-immune sera and the commercial mono- to E7 (68%) is higher than that reactive to L2 (52%) or clonal antibodies described in Methods were used as a L1(45%). Of note the percentage of sera reacting to E7 is control of the assay. The AI values of the hyper-immune higher in the group infected by other HPVs rather than in sera were: 44% (anti-L1), 50% (anti-L2), 78% (anti-E4), the HPV16 group. These data suggest that linear and 26% (anti-E6) 46% (anti-E7). The anti-L1 and anti-E7 immunogenic epitopes are present in these proteins; these mAb AIs were 3% and 5% respectively; the anti-His mAb linear epitopes are shared by several genotypes, and AI was 20%. The AI results of the human sera are summa- Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:6 http://www.infectagentscancer.com/content/1/1/6 Table 2: HPV Genotypes and Serostatus. rised in Table 3. Most samples showed an AI higher than Serum n° HPV Sero-reactivity to HPV16 Proteins 40% against L1, L2, E7 and E4, but not E6. In the HPV16 group, 40 samples showed an AI higher than 40%, and L1 L2 E4 E6 E7 the AI was below 40% in only 7 samples. In the second group (other HPVs), 85 samples showed an AI higher 1 16 P PP PP than 40%, and 18 samples had AI lower than 40%. Most 216 P P 3 16 P PP PP serum samples positive to E6 showed a low AI. Summariz- 416 P ing, four out of five HPV16 proteins tested as antigens seem to be able to bind antibodies with high avidity (AI 616 P P 7 16 P PP PP >40%). These results suggest that L1, L2, E4 and E7 in 816 P P P denatured form could be considered as valid antigens to 916 P P P be used in immunoassays. This is not the case of the 10 16 P 11 16 P HPV16 E6 protein, the linear epitopes of which are recog- 12 16 P P P nised with weak avidity by the antibodies. The presence of 13 16 P P immunoglobulins with high avidity in the HPV sera 14 16 15 16 P might suggest that most of the women with abnormal 16 16 P PAP test enrolled in this cohort have been infected for a 17 16 P prolonged time. This issue merits further investigation, 18 16 P because comprehensive data on the appearance of HPV- 19 16 P P P 20 16 specific antibodies to each viral protein and on the varia- 21 16 P P P P P tion of their avidity have not been reported before. 22 16 23 35 P 24 35 P P P P P Conclusion 25 35 In this study, an in-house ELISA based on the recom- 26 35 P binant HPV16 L1, L2, E4, E6 and E7 proteins in denatured 27 35 P 28 35 P P P form has been developed and used for the parallel detec- 29 31 tion of specific antibodies in sera collected from women 30 31 P with abnormal PAP smears. The results show that this 31 31 P P P assay has an increased sensitivity compared to the tests 32 31 P P P P P 33 31 P based only on one viral protein, resulting in detection of 34 6 P a higher number of positive sera. The assay is not geno- 35 6 P PP PP type-specific but it can be used to detect antibodies raised 36 6 P P 37 6 P P against genital HPVs other than type 16. This suggests that 38 52 P P P P the 5 proteins belonging to different HPV genotypes share 39 52 P linear epitopes, as could be deduced from the high level 40 52 P 41 52 P P of amino acid identity of these proteins. By comparing the 42 18 P amino acid sequences of the HPV16 L1, L2, E4, E6 and E7 43 18 P P P to those of the other genotypes found in our study, the 44 18 45 18 P highest identity (>65%) is found among the L1 proteins. 46 66 P P The homology among the E7 proteins is over 50%, similar 47 66 P P to that of the E6 proteins, in contrast to L2, where homol- 48 66 P P P ogy is less than 50%. The sequence identity among the E4 49 66 50 53 P P P P proteins of related genotypes is less than 40%, and even 51 53 P less among distantly related genotypes (data not shown). 52 53 P P 53 51 P P P 54 51 P The specificity of the antigen-antibody interactions in the 55 56 ELISA was indirectly measured by the avidity test, where 56 56 P immunoglobulins non-specifically bound to denatured 57 81 P P P P 58 81 P P P P proteins were removed by the urea treatment. As dis- 59 6b P P P P P cussed above, most of the HPV-specific antibodies bound 60 11 P P P P P to the 5 antigens had AI higher than 40%, implicating spe- 61 39 P P 62 42 P P cific antigen-antibody reactions, even in the sera of 63 54 P P women infected with HPV other than type 16. These 64 58 P P P P results suggest that the ELISA combined with the Avidity 65 73 P P P P Index might improve the diagnostic value of serology sur- 66 90 P P P P P vey in HPV infections. P, positive test; empty cells represent negative tests. Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:6 http://www.infectagentscancer.com/content/1/1/6 M E4 E6 E7 L1 L2 A Figure 1 nalysis of purified proteins by SDS-PAGE Analysis of purified proteins by SDS-PAGE. The puri- fied HPV16 proteins E4, E6, E7, L1 and L2 were run on a polyacrilamide gel electrophoresis and stained by Coomassie blue. Each protein is indicated on the top of the correspond- ing lane. The weight of the molecular mass markers (lane M) is indicated on the left of the figure. Due to the limited number of sera tested in the present study, it is evident that this ELISA system needs validation in large-scale population studies, especially for the detec- tion of HPV pan-reactive antibodies in people infected by ELISA Figure 2 based on HPV16 L1, L2, E4, E6 and E7 coating antigens genotypes different from HPV16. Nevertheless, in view of ELISA based on HPV16 L1, L2, E4, E6 and E7 coating the known cross-reactivity between the most common antigens. Detection of specific immunoglobulins to the HPV genotypes circulating in Europe [5,25], this ELISA HPV16 proteins in sera of women infected by HPV 16 should be useful in monitoring the pre-existing serostatus (empty dots) or by other HPVs (black dots). The protein of women who receive HPV vaccination with the recently used as coating antigen is indicated on each panel. In abscissa is reported the number of each serum as listed in Tab.2; in introduced prophylactic VLP-based vaccines. ordinate is reported the optical density of each reaction. In conclusion, the results of the ELISA introduced in this study are encouraging even though further investigations are needed to evaluate its usefulness in population screen- form technologies, which are useful when high numbers ing for HPV infections. Moreover the assay is easy to set up of samples need to be processed [32,33]. since the recombinant proteins expressed and purified from E. coli are cost-effective and easy-to-standardize sero- Methods logical reagents [27-30]. The Histidine tail of these pro- DNA constructs teins allows a one-step purification procedure  and The HPV16 genes were generated by PCR on pMHPV16d the purified proteins are readily applicable to novel plat- plasmid containing the complete virus genome (Acces- Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:6 http://www.infectagentscancer.com/content/1/1/6 Table 3: Avidity Index in the two serum groups. HPV16 HPV16 Group: L1 L2 E4 E6 E7 Tot Other HPVs High AI > 40 10 14 6 10 40 Low AI < 40 1 5 1 7 Other HPVs Group: High AI > 40 21 22 11 3 28 85 Low AI < 40 2142 18 50% 64% 18% 23% 50% 18% HPV16 45% 52% 27% 32% 68% 11% Other HPVs sion number NCBI NC_001526) , kindly provided by A. Venuti, using specific primers carrying restriction sites useful for cloning in pQE-30 expression vector (QIA- Co Figure 3 mparison of the percentages of sero-reactivity Comparison of the percentages of sero-reactivity. GEN), and were expressed as MRGS (H) tag proteins The percentages of sera reacting to the HPV16 L1, L2, E4, E6 (Table 1). Cloning and expression of E7 (297 nt) were and E7 proteins are shown; the percentages of the HPV16 previously described by Accardi et al. 2005. A truncated serum group are in white bars and those the other HPVs form of L1 gene (1416 nt), was cloned into the restriction serum group are in black bars. The percentages of negative site SalI, the expressed protein starts at the second methio- sera in the two groups (Neg) are also given. nine of the L1 coding region and stops before the nuclear localization signal. The L2 (1422 nt), E6 (477 nt) and E4 (288 nt) genes were cloned between the restriction sites BamHI-HindIII of the vector. The plasmids were main- tained in E. coli JM109 strain. The authenticity of the con- structs was confirmed by DNA sequencing. The deduced amino acid sequences of the inserts were compared to used as secondary antibody. The immune-complexes were those of the HPV16 (NC_001526), showing 100% iden- revealed by chemiluminescence (Amersham Bioscience). tity. HPV genotype identification Protein expression, purification, SDS-PAGE, and Western- HPV genotyping was performed by sequence analysis. blot analysis DNA was extracted from the cells of cervical smears by The recombinant proteins were purified using a denatur- QIAamp DNA mini kit (QIAGEN). To assess DNA integ- ing protocol: bacterial cells transformed by L1, L2, E4, and rity, the beta-globin gene was PCR amplified in each sam- E7 plasmids were lysed in a Phosphate buffer (100 mM ples by the primers PCO3 and PCO4 . To determine Na HPO 300 mM NaCl, 10 mM Tris, 1% Triton X-100, the presence of HPV DNA, each DNA sample was sub- 2 4 pH 8), containing 8 M urea, while bacterial cells trans- jected to PCR amplification by the consensus primers formed by E6 plasmids were lysed in Phosphate buffer, GP5+/GP6+, which amplify a L1 region . When the containing 6 M Guanidine-HCl. Proteins were purified by PCR result was negative and the serological test was posi- affinity chromatography on Ni-NTA resin and eluted by a tive, the DNA samples were further subjected to a semi- pH gradient. The fractions containing the proteins were nested PCR using the MY11-GP6+ primers in the first pooled, adjusted to a neutral pH by adding Tris pH 8.8 round [37,38], and the GP5+/GP6+ couple of primers in and stored in urea buffer at -30°C, until use. Protein con- the second round. Amplicons were detected by agarose gel centration was determined by standard method (BC pro- electrophoresis, eluted by GFX PCR-DNA gel-band purifi- tein assay, BIORAD). To evaluate the purity, each protein cation kit (Amersham Bioscience) and sequenced using samples of L1, L2, E4, E6 and E7 were denatured in SDS- the consensus primers GP5+/GP6+. The nucleotide loading buffer (25 mM Tris-HCl pH 6.8, 5 % β-Mercapto- sequences were aligned to the Gene-Bank Database ethanol, 2% SDS, 50% glycerol), separated in pre-cast gel sequences, using the BLAST program of the National Nu-PAGE MES-SDS (INVITROGEN), stained by Gel Code Center for Biotechnology Information (NCBI) server . Blue Stain Reagent (PIERCE). The proteins were identified A sequence identity of 98% was considered significant for by Western blot using the monoclonal anti poly-Histidine the identification of the HPV genotype. The genotypes antibody Clone HIS1 (Sigma-Aldrich). A peroxidase-con- identified in the samples were HPV 6, 11, 16, 18, 31, 35, jugate goat anti-mouse IgG (H+L) (SBA-INC USA) was 39, 42, 51, 52, 53, 54, 56, 58, 66, 73, 81 and 90. Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:6 http://www.infectagentscancer.com/content/1/1/6 Human sera and control sera as the arithmetic mean of the absorbance values of the The sera analysed in this study were collected from negative sera, plus two standard deviation (SD). After the women enrolled in the HPV-PathogenISS study. The first cut-off calculation, the values of outlier serum sam- women had an abnormal PAP smear as requested for the ples were excluded, and the cut-off was re-calculated study design . Only 99 women, of the 244 enrolled, [43,44]. To control the reproducibility of the test, ten rep- gave the informed consent for serum taking. Serum sam- resentative control human sera, the anti-poly-Histidine ples were kept at -30°C, until used for analyses. Negative mAB and the hyperimmune serum specific to each HPV sera were randomly chosen among sera of individuals protein were always enclosed in each ELISA run. Each infected by other pathogens. As positive controls specific serum was assayed in duplicate and the mean of the hyperimmune sera were used, obtained either in mice or absorbance value was taken as the final readout; the ELISA in rabbits by injection of the recombinant purified HPV16 for each HPV protein was repeated three times. To evalu- L1, L2, E4, E6 and E7 proteins. The use of hyperimmune ate the human serum variability, Coefficient of Variation anti-E7 polyclonal antibody has been reported before [40- (CV = SD/Mean × 100) was calculated . All statistical 42]. The immunisation protocol was as previously tests were performed by Microsoft Office Excel software. described in Di Bonito et al. . Competing interests ELISA and avidity test The author(s) declare that they have no competing inter- The denatured HPV16 recombinant proteins (0.25 μg/ ests. well) were adsorbed in carbonate buffer (pH 9.4) into Polysorp microtiter plates (NUNC) at 4° O/N. After a Authors' contributions blocking step of 2 h at 37°C in phosphate buffer saline PDB conceived the study, participated in acquisition, (PBS) containing 3% Non Fat Dry Milk (NFDM), the analysis and interpretation of data and in drafting the plates were incubated with 100 μl of human sera diluted manuscript. FG, SM, MGD participated in acquisition, 1:50 in 1% NFDM-PBS, at 37°C for 1 hr. The specific anti- analysis and interpretation of data, LA participated in gen-antibody complexes were detected by a peroxidase- acquisition, analysis and interpretation of data and criti- conjugated goat anti-human IgG (H+L) (SBA INC. USA), cally revised the manuscript, MB, SC; LM, AA, MC, partic- using the TMB (VECTOR) as a substrate. After 30 min, the ipated in acquisition of the data; KS participated in design enzymatic reaction was stopped by adding 50 μl of 1 M of the study and critically revised the manuscript. CG con- sulphuric acid/well. Optical density was read at 450 nm. ceived the study, participated in its design, coordination, Washing steps were done with 200 μl/well of PBS contain- in acquisition of funding and helped to draft of the man- ing 0.05 % Tween 20. The Monoclonal anti-poly-Histi- uscript. dine antibody was used as control of the coating step. All authors have read and approved the final version of The avidity test was performed as described by Roque- the manuscript. Afonso et al. . Serum samples were processed in par- allel by a traditional ELISA and by an ELISA enclosing a 6 Acknowledgements *HPV-PathogenISS Study Group: L Leoncini and M Alderisio, Unità M urea-wash step. After incubation of serum samples with Citoistopatologica, Centro Nazionale di Epidemiologia, Sorveglianza e Pro- the antigens, the wells were washed three times (5 min mozione della Salute, ISS, Roma, Italy; S Costa D Santini M De Nuzzo, each) with a PBS wash-buffer containing or not 6 M urea. Dipartimento di Ginecologia e Ostetricia, Azienda Ospedaliera S Orsola Each reaction was done in duplicate. A final fourth PBS- Malpighi, Bologna, Italy; L Di Bonito, D Bonifacio, F Zanconati, UCO Anato- wash was done in all wells. The Avidity Index (AI) was cal- mia Patologica, Istopatologia e Citodiagnostica, Ospedale Maggiore, Tri- culated as follows: AI = (absorbance reading with urea este, Italy; M Galati, Ginecologia e Ostetricia, IFO, Istituto Regina Elena, wash/absorbance reading without urea wash) × 100. The Rome, Italy; F Sesti, E Piccione, and A Criscuolo, Istituto di Ginecologia, HPV- specific hyperimmune sera to L1, L2, E6, E7 and E4, Università di Tor Vergata, Rome, Italy; A Benedetto, P Paba, C Favalli, Lab- oratory of Clinical Microbiology and Virology, University Hospital "Policlin- the commercial anti-Histidine mAb (clone HIS-1, Sigma- ico Tor Vergata", Rome, Italy; EA Casolati, and M Valieri, Clinica Ostetrica Aldrich), anti- HPV16 L1 mAb (clone CamVir-1, Cymbus e Ginecologica, Istituto Scienze Biomediche, Ospedale Luigi Sacco, Milano, Biotechnology) and the anti-HPV16E7 mAb (clone 8C9, Italy; Di Carlo, IFO, Istituto San Gallicano, Unità Operativa MST/HIV, Zymed Laboratories INC) were used as controls. Rome, Italy. Cut-off definition and statistical analysis We are grateful to Mr. Armando Cesolini for helping us in animal immuni- To determine the cut-off values for each antigen, 40 con- zation. We thank Dr. Aldo Venuti for kind providing the pMHPV16d plas- trol human sera were tested in ELISA against each of the mid. This work was supported by grants from the Italian Ministry of Health (Ministero della Salute): Ricerca Corrente, 2002; Fasc.OG/C; Ricerca Final- five HPV recombinant proteins. The assay included the izzata, Progetto strategico: "Strategie di immunoterapia contro genotipi di endpoint titre of the corresponding hyper-immune HPV HPV oncogeni e non oncogeni" 03/01/G/56, 2002–2004; AIDS National sera as a positive control. The cut-off value was calculated Page 7 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:6 http://www.infectagentscancer.com/content/1/1/6 Project: Co-infections, opportunistic Infections and AIDS-associated 19. Hedman K, Lappalainen M, Söderlund M, Hedman L: Avidity of IgG in serodiagnosis of infectious diseases. Rev Med Microbiol 1993, tumour, Rif. 50F/F, 2004. 4:123-129. 20. 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Grabowska K, Wang X, Jacobsson A, Dillner J: Evaluation of cost- precision rations of different strategies for ELISA measure- ment of serum antibody levels. J Immunol Methods 2002, 271:1-15. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes)
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