Generation of antibodies against bovine recombinant prion protein in various strains of mice

Abstract

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, belong to a group of neurodegenerative disorders affecting humans and animals. To date, definite diagnosis of prion disease can only be made by analysis of tissue samples for the presence of protease-resistant misfolded prion protein (PrP(Sc)). Monoclonal antibodies (MAbs) to the prion protein provide valuable tools for TSE diagnosis, as well as for basic research on these diseases. In this communication, the development of antibodies against recombinant bovine prion protein (brecPrP) in four strains of mice (BALB/c, ND4, SJL, and NZB/NZW F(1)) is described. Immunization of autoimmunity-prone NZB/NZW F(1) and SJL mice with brecPrP was applied to overcome self-tolerance against the prion protein. ND4 and SJL mice did not develop an immune response to brecPrP. BALB/c mice produced antibody titers of 1:1,000 to 1:1,500 in an enzyme-linked immunosorbent assay (ELISA), while NZB/NZW F(1) mice responded with titers of 1:7,000 to 1:11,000. A panel of 71 anti-brecPrP MAbs recognizing continuous and discontinuous epitopes was established from BALB/c and NZB/NZW F(1) mice. Seven anti-brecPrP MAbs reacted with both the cellular form of PrP and protease K-resistant PrP(Sc) from sheep brain in Western blot assays. The epitope specificity of these MAbs was determined, and applicability to immunohistochemical detection of prions was studied. The MAbs generated will be useful tools in the development of TSE immunochemical diagnosis and for research. This is the first report of the development of anti-PrP MAbs by use of autoimmune NZB/NZW F(1) mice as an alternative approach for the generation of PrP-specific MAbs.

FIG. 1.

Recombinant bovine prion protein purification and verification. (A) Profile of purification of His-tagged brecPrP by Ni²⁺ affinity chromatography. OD 280, optical density at 280 nm; Im, imidazole. (B) SDS-PAGE of purified bovine recPrP under nonreducing conditions. The single band indicates that the purified protein does not contain intermolecular disulfide bonds. MWM, molecular weight markers. (C) Verification of the sequence of purified brecPrP by Western blotting with MAbs VMRD F89/160.1.5 and VMRD F99/97.6.1. The values between panels B and C are molecular masses.

FIG. 2.

ELISA evaluation of mouse sera after immunization with brecPrP. (A to D) Reactivity of anti-brecPrP sera from different strains of mice against brecPrP. Note the pronounced immune responses of BALB/c and NZB/NZW F₁ mice. (E and F) Reactivity of anti-brecPrP sera from NZB/NZW F₁ and BALB/c mice against moPrP. Note the lack of cross-reaction of BALB/c anti-brecPrP sera with moPrP and the presence of anti-moPrP reactivity in anti-brecPrP NZB/NZW F₁ sera. Antibody titers were evaluated 1 week after the last immunization with brecPrP conjugated with KLH. Control sera were obtained 7 days before the first immunization and did not contain the anti-brecPrP and anti-moPrP antibodies, as determined by ELISA (data are not presented). OD₄₁₄, optical density at 414 nm.

FIG. 3.

Comparison of reactivities of NZB/NZW F₁ and BALB/c mouse anti-brecPrP sera with proteinase K-resistant PrP^Sc by Western blot assay. PrP^Sc was extracted from scrapie-affected sheep brain tissue homogenate as described in Materials and Methods. Serial dilutions of proteinase K-treated sheep brain PrP^Sc were loaded onto a 15% polyacrylamide gel starting with the equivalent of 25 μg of brain tissue and analyzed by Western blot assay. Anti-brecPrP sera from NZB/NZW F₁ and BALB/c mice were used at a 1/2,000 dilution. The source of the anti-brecPrP serum used in the Western blot analysis is indicated on the right. The values on the left are molecular mass markers.

FIG. 4.

Epitope mapping of polyclonal anti-brecPrP antibodies after development of a humoral immune response in BALB/c and NZB/NZW F₁ mice. Aligned amino acid sequences of bovine, sheep, and mouse PrPs are presented. Amino acid positions are numbered according to bovine PrP. Bars above the sequence indicate the locations of antigenic epitopes in brecPrP. Data were combined from experiments with three sera from each group of animals.

FIG. 5.

Western blot reactivity of anti-brecPrP MAbs with ovine PrP. (A) Examples of immunoblot analysis of normal and scrapie-affected sheep brain tissues, with and without proteinase K treatment, with MAbs M2188 and M2281. Hybridoma tissue culture fluids were used at a 1:1 dilution. (B) Immunoblot detection of proteinase K-resistant scrapie-affected sheep PrP^Sc with MAbs M2182 to M2283 (used at 2 μg/ml). Anti-PrP MAb F89/160.1.5 (VMRD) was used as the positive control at a 1/300 dilution. Lane MWM contained molecular weight markers. The values on the left are molecular masses in kilodaltons.

FIG. 6.

Immunohistochemistry of PrP^Sc in formalin-fixed tissues of sheep with scrapie. MAb M2197 selectively labels pathological PrP^Sc aggregates. (A, B, and C) Sections of the obex of the medulla oblongata. Note the abundant bright red chromogen accumulation in the dorsal motor nuclei of the vagus nerve (A, bar = 400 μm) and the higher magnification of scrapie-specific perineuronal and neuropil staining (B, bar = 40 μm). No PrP^Sc-associated chromogen accumulated within anatomically matched scrapie-free sheep tissue (C, bar = 40 μm). (D, E, and F) Sections of a retropharyngeal lymph node. Note the bright red chromogen deposits in the follicle germinal center of scrapie-affected sheep (D, bar = 200 μm; E, bar = 80 μm). No staining occurred in a retropharyngeal lymph node from a scrapie-free sheep (F, bar = 200 μm).