Detection of human papillomavirus oncoprotein E7 in liquid-based cytology.

The selection and characterization of a set of mouse mAbs against high-risk human papillomavirus (HPV) E7 oncoprotein and the development of protocols for immunocytochemistry (ICC) are described here. A large number of antibodies raised towards HPV16 and 18 E7 were tested for high-risk specificity by ELISA using a panel of HPV E7 proteins. Antibodies detecting low-risk E7 were discarded, resulting in 38 high-risk HPV E7-specific antibodies. The corresponding epitopes were mapped using overlapping HPV E7 fragments displayed on phage particles. Functionality in ICC against formalin-fixed cervical cancer cell lines was demonstrated for ten mAbs; their high-risk specificity was confirmed by Western blot analysis and ICC on transiently transformed cells expressing high- or low-risk HPV E7. These mAbs were specific for one or several of the high-risk strains HPV16, 18, 31, 35 and 45. Specific E7 staining of liquid-based cytology (LBC) samples was demonstrated for seven mAbs and optimized protocols were established. The E716-41 and E718-79 mAbs demonstrated particularly strong and specific staining of cells stored in LBC fluid for at least 6 months. It is proposed that the high-risk HPV E7 staining protocols established in this study may have the potential to be included in a complementary test for the detection and identification of malignantly transformed cells, in for example atypical squamous cells of undetermined significance samples.

TD-12 workshop report: characterization of monoclonal antibodies to neuron-specific enolase.

Twelve antibodies to neuron-specific enolase (NSE) have been evaluated by four working groups. Human brain γγ-enolase, neuroblastoma-derived αγ-enolase, and recombinant γγ-enolase were used to determine antibody specificity and binding kinetics. All antibodies were found to be specific for the γ-isoform. It was possible to assign 11 of the antibodies to at least five epitope groups based on cross-inhibition experiments, QCM and SPR technology, and immunoassay combinations. Antibodies 9601 and 9602 showed the highest affinity for both native and recombinant γγ-enolase. Immunometric assays for both γγ- and αγ-enolase could be made by pairing 9601 with most of the other ISOBM antibodies. Antibodies differed in their ability to recognize native αγ-enolase, native γγ-enolase, and recombinant γγ-enolase. Some immunometric assay combinations appear to favor the detection of heterodimeric αγ-enolase over the homodimeric γγ-enolase. Although the majority of the antibodies failed to detect human NSE or recombinant NSE in Western blots, mAb 9601 recognized both, while E17 and 18E5 were specific for human NSE.

Phage display for site-specific immunization and characterization of high-risk human papillomavirus specific E7 monoclonal antibodies.

The paper describes a method to use filamentous phage to display specific regions of proteins for immunization in order to direct the immune response towards a pre-defined region of the protein. The method called site-specific immunization (SSI) was evaluated using the E7 protein of oncogenic (high-risk) human papillomavirus (HPV) type 16 as a model system. This protein consists of sequence blocks also present in other viral and cellular proteins and in the corresponding protein of low-risk HPVs. A fragment of the HPV16 E7 oncoprotein specific for a group of high-risk viruses was identified by sequence comparison and displayed on filamentous phages in fusion with the major phage coat protein VIII. The recombinant phages triggered an immune response in mice against the full-length HPV16 E7 protein. Fusion of B-lymphocytes from the immunized animals with myeloma cells resulted in three hybridomas producing monoclonal antibodies (MAbs) with reactivity against the endogenous E7 protein. The specificity of the MAbs for the HPV16 E7 protein in cancer cell lines was confirmed by Western blot analyses and immunocytochemistry. The epitope of each MAb was roughly mapped by determining the reactivity against overlapping E7 fragments displayed on phage particles. The mimotopes of the MAbs were further determined by biopanning against a randomized peptide library displayed on phage and found to be unique for a sub-set of high-risk HPV E7 proteins. The combination of different phage display techniques for immunization and epitope mapping was efficient for generation and characterization of highly specific MAbs.