Immuno-histochemistry results with type-specific anti-E4 antibodies on cervical biopsies.

<p>HPV-16, HPV-18, and HPV-58 containing raft controls were positive with the appropriate anti-E4 antibodies in each experiment.</p><p>− = negative;</p><p>+ = positive.</p><p>WTS-PCR = whole tissue section PCR.</p><p>N/A = Not applicable (Tissues section not tested).</p>*<p>58 positive area is different to the area sampled by LCM (laser capture micro-dissection).</p>**<p>31 positive area lost from slide during immunostaining protocol.</p>***<p>differentiated layers lost from slide during immunostaining protocol.</p><p>() = weakly positive for this type.</p><p>CIN: cervical intraepithelial neoplasia.</p>£<p>All HPV types were detected by LCM-PCR as single type HPV infections in different CIN lesion areas.</p

Specificity of HPV type-specific antibodies against different HPV E1?E4 proteins by ELISA and Western blotting.

<p>Optical density measurements from ELISA on a panel of 10 recombinant maltose-binding E4 proteins (HPV-16, 18, 31, 33, 35, 39, 45, 52, 58, and 59) used to evaluate the specificity of on M16E4_35–42, R18E4_53–60 and R58E4_23–30 polyclonal antibodies (A) and MoAb16E4_35–42 monoclonal antibody (B). Cross-reactive TVG405 was used for comparison (C) and the relative abundance of the various MBP proteins is shown following staining with Coomassie blue (lower panel of C). Western blot results are shown as inserts under the corresponding graphs presenting the ELISA results.</p

Immunohistochemical staining of HPV E4 proteins in productive cervical lesions caused by different HPV types using MoAb16E4_35–42, R18E4_53–60, R58E4_23–30 or TVG405 antibodies.

<p>A) Scan of hematoxylin and eosin (H&E) stained biopsy 44 (genotype HPV-16, 31 by WTS-PCR) with areas of interest boxed in yellow. Detection of HPV-31 E4 in region of CIN1 (i) using TVG 405; MoAb16E4_35–42 antibody gave no staining on the same tissue section. HPV-16 E4 is detected using MoAb16E4_35–42 antibody in a region of CIN2 (ii) and confirmed using TVG405 on the same tissue section. B) Scan of H&E stained biopsy 62 (genotype HPV-58 by WTS-PCR and LCM-PCR) with area of interest boxed in yellow. Detection of HPV-58 E4 protein expression by R58E4_23–30 antibody in an HPV-58-infected region classified as CIN2. MoAb16E4_35–42 antibody gave no staining on the same tissue section indicating no cross-reactivity. C) Scan of H&E stained section biopsy 16 (genotype HPV-18 by WTS-PCR) with area of interest boxed in yellow. Detection of HPV-18 E4 protein expression using R18E4_53–60 antibody in an HPV-18-infected CIN1 lesion and confirmation by TVG405 staining regime 2 on the same tissue section. MoAb16E4_35–42 antibody gave no staining indicating no cross-reactivity. All sections were counterstained with 4′,6′-diamino-2-diamino-2-phenylindole (DAPI, blue).</p

Evaluation of antibody specificity using rafts containing HPV-16, 18 and 58.

<p>A) Raft sections containing HPV-16, -18 or -58 genomes were individually probed with MoAb16E4_35–42, R18E4_53–60 and R58E4_23–30 antibodies (red) and were counterstained with DAPI (blue). The different antibodies allowed type-specific detection of E4 and showed no cross-reactivity amongst the types tested. B) E4 protein expression was detected in HPV-16, -18 and -58 rafts after pre-treatment with solution D, pH 9.0 and autoclaved for 2 min, prior to incubation with MoAb16E4_35–42, R18E4_53–60 and R58E4_23–30 antibodies (red - upper panels). In the lower panels, sections were pre-treated in the same way prior to incubation with cross-reacting TVG405 or RE4 (green). All sections were counterstained with 4′,6′-diamino-2-diamino-2-phenylindole (DAPI, blue).</p

Evaluation of E4, MCM and L1 protein expression in HPV16, 18 and 58 rafts.

<p>(A) HPV-16 and 18 rafts were probed with cross-reactive TVG405 (green) and MCM (red) antibodies. The HPV-58 raft was stained with cross-reactive (RE4) rabbit sera (green) and MCM (red) antibody. The staining patterns are typical of those expected for high-risk HPV types. (B) Novel HPV-58 rafts were further probed with R58E4_23–30 (green) and HPV L1 (red) antibodies and compared with rafts containing HPV16 and 18 and stained with HPV L1 and MoAb16E4_35–42 and R18E4_53–60 respectively. The detection of L1 in a subset of the E4-positive cells was seen in each raft. All sections were counterstained with 4′,6′-diamino-2-diamino-2-phenylindole (DAPI, blue). The images were taken on a microscope using a 10x (A) or 40x (B) objective. The merged image (E4 green/MCM red) is shown on the right of the figure. L1 was detected in the superficial and mid-spinous cell layersp.</p