TSE Diagnostics: Recent Advances in Immunoassaying Prions

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of rare fatal neurodegenerative diseases, affecting humans and animals. They are believed to be the consequence of the conversion of the cellular prion protein to its aggregation-prone, β-sheet-rich isoform, named prion. Definite diagnosis of TSEs is determined post mortem. For this purpose, immunoassays for analyzing brain tissue have been developed. However, the ultimate goal of TSE diagnostics is an ante mortem test, which would be sensitive enough to detect prions in body fluids, that is, in blood, cerebrospinal fluid, or urine. Such a test would be of paramount importance also for screening of asymptomatic carriers of the disease with the aim of increasing food, drugs, and blood-derived products safety. In the present paper, we have reviewed recent advances in the development of immunoassays for the detection of prions

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent

Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

BACKGROUND: An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrP(Sc)) from different species (human, bovine, deer and mouse). METHODOLOGY/PRINCIPAL FINDINGS: Degradation of the PrP(Sc) isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrP(Sc) were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrP(Sc), the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl(2) were 105 °C ± 0.5 °C and pH 6.5 ± 0.2, respectively. CONCLUSIONS/SIGNIFICANCE: Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrP(Sc)

Anti-idiotypic antibodies

In certain cases, anti-idiotypic antibodies that recognize an antigen-combining site of an antibody can mimic the structure and/or function of certain nominal antigens. This feature makes them particularly useful if conventional experimental approaches fail to fulfil expectations, especially when the molecule of interest is infectious, toxic or difficult to isolate and purify. We suggest the application of an anti-idiotype concept to the field of prion biology, with the aim of evoking a humoral immune response against the pathological isoform of the prion protein (PrPsc). Different ways to induce anti-idiotypic responses were studied in mice and chickens using various forms of V5B2, a PrPsc-specific monoclonal antibody we have described previously. The preparation of anti-idiotypic monoclonal antibodies was achieved with well-defined strategies of immunization, selection and subsequent characterization. Our results demonstrate that it is possible to induce a strong anti-idiotypic immune response against the V5B2 monoclonal antibody in both xenogeneic and syngeneic experimental systems. From the competition seen between polyclonal and monoclonal anti-iditypic antibodies and the original immunogen, the PI peptide, and even more importantly, the ultimate target antigen, PrPsc, we conclude that selected antibodoes bind to the antigen-combining site of hte V5B2 monoclonal antibody and might even resemble the PrPSc-specific epitope. The involvement of both antigen-combining sites in the interaction between V5B2 and the most promising monoclonal anti-idiotypic antibody was further supported by molecular docking. The results of the present study not only provide an example of the successful production of Ab2 monoclonal antibodies based on a well planned strategy for selection, but should also provide a new experimental approach that is applicable to the field of prion diseases

SDS-PAGE analyses of mouse (moScFv) and humanized (huScFv) scFv V5B2.

<p>Cell lysates were analyzed before (−) and after (+) IPTG induction, along with purified scFvs (e). Molecular masses of protein standards in kDa are indicated on the left. Similar electrophoretic pattern was obtained with all humanized scFv variants.</p

Immunohistochemistry of the PrP^Sc deposits in the cerebellum of a sCJD patient (upper three figures).

<p>Immunolabeling was performed with whole mAb V5B2, murine scFv (moScFv) and humanized scFv (huScFv) of V5B2. The arrow marks PrP^Sc plaques, while the triangle marks the diffuse, synaptic PrP^Sc deposition. On the lower three figures immunolabeling was performed on the cerebellum of the CJD negative patient.</p

Western blotting of the recombinant human PrP 23–226 (PrP226*) and of the recombinant human PrP23–231 (HuPrP).

<p>Reaction with the whole mAb V5B2 is compared to reactions with murine scFv (moScFv) and humanized scFv (huScFv) of V5B2. mAb 6H4 was used as a control antibody. Approximate molecular weights are in kilodaltons.</p

Identity and similarity scores of the V5B2 humanized variable domain frameworks with HhumκI and HumIII human antibody frameworks calculated by ClustalW.

<p><i>X</i> is the number of identical framework residues; <i>Y</i> is the total number of framework residues.</p

Amino acid sequence alignments.

<p>Alignment of light chain (Vl) and heavy chain (Vh) variable regions of the murine V5B2 scFv (moScFv), two humanized (LLHmut1, LLHmut2) and final humanized version (huScFv) of mAb V5B2 with human consensus sequences of light chain κ subgroup I (HhumκI) and heavy chain subgroup III (HumIII). The dashes represent unchanged amino acids. CDRs are underlined. Amino acids are numbered according to Kabat <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015783#pone.0015783-Kabat1" target="_blank">[36]</a>.</p