Novel epitopes identified by anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions

Abstract

Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform (PrP(Sc)) of a normal cellular protein (PrP(C)). Shared sequence identity of PrP(Sc) with PrP(C) has limited the detection sensitivity of immunochemical assays, as antibodies specific for the disease-causing PrP(Sc) isoform have not been developed. Here we report the generation of three new monoclonal antibodies (MAbs) to PrP, which were isolated following immunization of Prnp(0/0) Balb/cJ mice with highly purified PrP(Sc) isolated from brain lipid rafts. Epitope mapping using synthetic PrP peptides revealed that the three MAbs bind different epitopes of PrP. The DRM1-31 MAb has a conformational epitope at the proposed binding site for the putative prion conversion co-factor "protein X." The DRM1-60 MAb binds a single linear epitope localized to the β2-α2 loop region of PrP, whereas DRM2-118 binds an epitope that includes sequences within the octarepeat region and near the site of N-terminal truncation of PrP(Sc) by proteinase K. Our novel anti-PrP MAbs with defined PrP epitopes may be useful in deciphering the conformational conversion of PrP(C) into PrP(Sc).

FIG. 1.

Direct-binding ELISA results from the initial screening of a cell-fusion experiment using splenocytes of PrP^Sc-immunized mice. A microtiter plate was coated with purified PrP^Sc-DRM following PK digestion. Since the majority of wells of a screening plate are not expected to contain hybridomas secreting specific antibody, the plate background was calculated as the average (dashed line) relative luminescence units (RLU), 0.15×10⁶ RLU for the plate shown.

FIG. 2.

ELISA binding of five MAbs—(A) DRM1-15, (B) DRM1-26, (C) DRM1-31, (D) DRM1-60, and (E) DRM2-118—to titrated recombinant PrP harboring different species sequences: recSHaPrP(90–231), open red circle; recMoPrP(89–230), open blue diamond; recOvPrP(23–231), open pink triangle; and recHuPrP(90–231), open green square. Data points and error bars represent mean and standard error of the mean (SEM) from three independent experiments. Concentration of MAb at 1 μg/mL.

FIG. 3.

Western blot detection of PrP^C in 40 μg of normal brain homogenates with the five MAbs. Homogenates were prepared from uninfected whole Syrian hamster brain (SHa); whole mouse brain (Mo); sheep cortex (Ov); horse cortex (Eq); cow cortex (Bo); pig cortex (Po); whole human brain (Hu); and white-tailed deer obex (Ce). For each MAb, 2 μg/mL was used. Molecular masses of protein standards are shown in kilodaltons (kDa).

FIG. 4.

Amino acid sequences for the variable regions of DRM1–15, DRM1-26, DRM1-31, DRM1-60, and DRM2-118 kappa light and heavy chains. The deduced amino acids, from the first (FWR-1) to the end of the fourth framework region (FWR-4) are shown. The three complementarity determining regions (CDRs) are shaded, J-region is shown in bold, amino acids coded for by the primers are underlined, and framework regions (FWR-1, FWR-2, FWR-3, and FWR-4) are indicated.

FIG. 5.

Epitope mapping of MAbs DRM1-31 (A), DRM1-60 (B), and DRM2-118 (C) using overlapping BoPrP peptides in direct-binding ELISAs. Peptides spanned 12 amino acids, with 7-residue overlaps, corresponding to BoPrP(25–241). Dashed line represents 2× background activity. Bar represents mean±SEM from two independent experiments.

FIG. 6.

Detection of PrP^C and PrP^Sc in DRMs by MAb DRM1-31 (A), DRM1-60 (B), and DRM2-118 (C) by Western blots. DRM samples were left undigested (–) or digested with PK (+). For each lane, 1 μg/mL of each MAb was used to detect 8 μg of brain DRM protein. Molecular masses of protein standards are shown in kilodaltons (kDa).

FIG. 7.

Effect of disulfide bond disruption in PrP on MAb binding. Scrapie-infected hamster brain homogenates were treated with (+) and without PK (-) in the presence (+) or absence (-) of 10% 2-mercaptoethanol (2-ME) to reduce the single disulfide bond present in PrP. Brain homogenates were loaded at 10 μg/lane and DRM MAb used at 1 μg/mL. Molecular masses of protein standards are shown in kilodaltons (kDa).

FIG. 8.

Location of DRM MAb binding mapped onto space-filled models of PrP^C. The amino acids that contribute to the epitope of DRM1-31 from each of the three peptides clusters (^Bo170NQVYYRPVDQYS, red; ^Bo180NFVHDC, green; and ^Bo225EQMCITQY, blue) are shown in the left panel. Amino acids ^Bo170NQVYYRPVDQYS (red) that contribute to DRM1-60 MAb binding are depicted in the center panel. Amino acids GWGQ (purple) that contribute to DRM2-118 binding are shown in the right panel. Space-filled models used for DRM1-31 and DRM1-60 from Protein Database (PDB) file 1DX0 and for DRM2-118 from PDB file 2KUN.