Prion protein-specific antibodies that detect multiple TSE agents with high sensitivity

Abstract

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94-233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays.

Figure 1. Four distinct binding regions are identified using Pepscan analysis.

Antibodies were tested using solid-phase, PrP-based peptide Pepscan analysis. Y-axis represents absorbance; x-axis corresponds to the peptide set. Each peptide set is composed of 15 amino acids, whereby set number 1 starts with the residue 1 in PrP; peptide set 2 starts at residue 2 in PrP and so on. Positive reactions between antibodies and surface-bound peptides resulted in an increase in absorbance (405 nm) which could be discriminated from background/‘noise’ readouts. Here we show increased absorbance indicative of the core binding regions from individual representatives of the four distinct antibody groups identified.

Figure 2. Partial alignment of PrP sequences studied in this work.

Reference sequences were obtained from UniProt and aligned using ClustalX using the sheep sequence as the reference. Segments of the sequence corresponding to the core epitopes for the 4 groups of antibodies are shown in bold with additional flanking sequence for reference. Where amino acids are the same as sheep this is indicated with a dash; where amino acids differ these are explicitly stated, or a gap is left where no amino acids align.

Figure 3. Binding of ROS-FH6 (compared to ROS-DE3) to mammalian PrP is affected by the sequence.

Immunoblots of recOvPrP residues 25–233 ovine sequence (lane 1), recOvPrP residues 94–233 ovine sequence (lane 2), murine (Mur) PrP residues 23–230 murine sequence (lane 3), hamster (Ham) residues 23–231 hamster sequence (lane 4), bovine (Bov) PrP residues 25–241 bovine sequence (lane 5) and human (Hum) PrP residues 23–230 human sequence(lane 6) when probed with ROS-DE3 and ROS-FH6. ROS-DE3 cross reacts with all species of mammalian PrP tested, whereas ROS-FH6 failed to bind to murine and hamster PrP. Molecular weight markers are shown in kDa. Exposure time is shown in minutes (m).

Figure 4. ROS-BC6 requires arginine (R) at codon 154 for binding to C1 PrP^C.

Representative image of mature-length (FL) and C1 PrP^C (C1, deglycosylated forms) extracted from brain of seven sheep of either ARQ/AHQ (lane 1), ARQ/ARQ (lane 2) or AHQ/AHQ (lanes 3–7) PRNP genotypes. Membrane was probed with ROS-BC6 (0.3 µg/ml) simultaneously with anti-tubulin (T, 0.01 µg/ml). ROS-BC6 binds specifically to mature-length and C1 PrP in brain from ARQ/AHQ and ARQ/ARQ sheep but not from AHQ/AHQ sheep. Molecular weight markers are shown in kDa, exposure time was 10 minutes.

Figure 5. Testing the mAb specificity for different TSE agents.

PrP^Sc was extracted from the brain of a range of host animals infected with different TSE agents, treated with PK and detected using SDS-PAGE and immunoblotting with the new mAbs and selected commercially available antibodies. Natural sheep (Shp) scrapie (lane 1), goat scrapie (lane 2), CH1641 scrapie (lane 3), sheep (Shp) BSE (lane 4), goat BSE (lane 5), cow (Bov) BSE (lane 6), deer BSE (lane 7) and atypical scrapie - frontal cortex (FC), cerebellum (C) and medulla (M) (lanes 8, 9 and 10 respectively). Molecular weight masses are shown in kDa. Each immunoblot shows antibody concentration (µg/ml) and exposure time to film (minutes, m).

Figure 6. Different profiles of PrP^d staining are evident when brain sections from TSE-infected sheep, goat, cow and deer were stained with ROS-IH9.

Labelling for PrP^d in the brain (indicated by brown staining) from several ruminant species infected with different TSE agents using the antibody ROS-IH9 (0.0625 µg/ml). Experimental classical scrapie in sheep specifically the dorsal motor nucleus of the vagus nerve (DMNV, panel A); CH1641 scrapie in sheep (cerebellum, panel B); cattle BSE in sheep (DMNV, panel C); atypical scrapie in sheep (cerebellum, panel D); goat BSE in goat (DMNV, panel E); cattle BSE in red deer (cerebellum, panel F); classical scrapie in goat (hypothalamus, panel G); natural BSE in cattle (spinal tract of the trigeminal nerve, panel H). Scale bar = 200 µm.

Figure 7. ROS-BH1 stains PrP^d deposited after 87V infection in mice in a more sensitive manner compared to 6H4.

Staining of PrP^Sc during 87V (scrapie) infection in the cortical hippocampus, the cortex and the CA2 region of the hippocampus in mice using ROS-BH1 (0.2 µg/ml, panels A, D and G), 6H4 (3.0 µg/ml, panels B, E and F) and the IgG1 isotype control antibody, TNP (0.2 µg/ml, panels C, F and I). Panels, G H and I are higher magnification images of the CA2 region. Neuroanatomical landmarks are identified as follows: Fi – fimbria; Th - thalamus; DG - dentate gyrus; CA2 - CA2 region of the hippocampus; CA1 - CA1 region of the hippocampus; Cx - cortex; V - ventricle and S - septum. Scale bars are indicated in µm. ROS-BH1 gave more sensitive detection of PrP^d in this assay compared to 6H4 whilst retaining an identical specificity and profile of staining to that of 6H4.