Proteinase K enhanced immunoreactivity of the prion protein-specific monoclonal antibody 2A11

Here, we report the development and further characterisation of a novel PrP-specific monoclonal antibody: 2A11. By Western blot analysis, 2A11 reacts with PrPC from a variety of species including cow, sheep, pig, hamster, rabbit, cat, dog, deer and mouse but fails to react with human, chicken and turtle PrP. Reactivity to PrPC in Western blot was found to be dependent on the redox state of the protein since binding of mAb 2A11 to its epitope was more effective in reducing conditions. 2A11 binding site was mapped within a region comprised by residues 171-179 (six octarepeats bovine PrP notation; 163-171 for the ovine PrP notation). Interestingly, in immunohistochemistry (IHC) analysis, immunoreactivity was greatly enhanced after proteinase K (PK) sample treatment, while little or no reaction was observed in non-PK-treated BSE samples and samples from healthy animals. Quantitative differences in reactivity to BSE prions after PK treatment were also observed, to a lesser extent, by Western blot analysis. Since definitive diagnosis of prion diseases rely on IHC assays of proteinase K-treated samples, the use of mAb 2A11 might contribute to reduce the occurrence of false positive detection due to incomplete proteinase K digestion.

[1]  S. Prusiner,et al.  Scrapie prions aggregate to form amyloid-like birefringent rods , 1983, Cell.

[2]  S. Prusiner,et al.  Experimental Scrapie in the Mouse: Electrophoretic and Sedimentation Properties of the Partially Purified Agent , 1980, Journal of neurochemistry.

[3]  K Wüthrich,et al.  NMR solution structure of the human prion protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[4]  K Wüthrich,et al.  NMR characterization of the full‐length recombinant murine prion protein, mPrP(23–231) , 1997, FEBS letters.

[5]  P E Wright,et al.  Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[6]  S. Prusiner,et al.  Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Tomoyuki N. Tanaka,et al.  Analyses of Gerstmann–Straussler syndrome with 102Leu219Lys using monoclonal antibodies that specifically detect human prion protein with 219Glu , 2000, Neuroscience Letters.

[8]  S. Prusiner,et al.  Rapid Acquisition of β-Sheet Structure in the Prion Protein Prior to Multimer Formation , 1998, Biological chemistry.

[9]  J. Collinge,et al.  Monoclonal antibodies inhibit prion replication and delay the development of prion disease , 2003, Nature.

[10]  M. Enari,et al.  Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Itohara,et al.  In Vivo Conversion of Cellular Prion Protein to Pathogenic Isoforms, as Monitored by Conformation-specific Antibodies* , 2001, The Journal of Biological Chemistry.

[12]  R. Carp,et al.  Immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen. , 1999, Journal of neurovirology.

[13]  Pauline M. Rudd,et al.  Antibodies inhibit prion propagation and clear cell cultures of prion infectivity , 2001, Nature.

[14]  R. Riek,et al.  Prion (PrPSc)-specific epitope defined by a monoclonal antibody , 1997, Nature.

[15]  C. Korth,et al.  Monoclonal antibodies specific for the native, disease-associated isoform of the prion protein. , 1999, Methods in enzymology.

[16]  M. Groschup,et al.  The use of monoclonal antibody epitopes for tagging PrP in conversion experiments. , 2000, Archives of virology. Supplementum.

[17]  K Wüthrich,et al.  Three-dimensional structures of prion proteins. , 2001, Advances in protein chemistry.

[18]  J. Liddell,et al.  A Practical Guide to Monoclonal Antibodies , 1991 .

[19]  K Wüthrich,et al.  NMR structure of the bovine prion protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Birkett,et al.  A monoclonal antibody that enables specific immunohistological detection of prion protein in bovine spongiform encephalopathy cases , 2001, Neuroscience Letters.

[21]  Witold K. Surewicz,et al.  Crystal structure of the human prion protein reveals a mechanism for oligomerization , 2002, Nature Structural Biology.

[22]  H. Wiśniewski,et al.  Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins , 1987, Journal of virology.

[23]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .

[24]  R. Riek,et al.  NMR structure of the mouse prion protein domain PrP(121–231) , 1996, Nature.

[25]  P. Lansbury,et al.  Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Miller,et al.  Preclinical Diagnosis of Scrapie by Immunohistochemistry of Third Eyelid Lymphoid Tissue , 2000, Journal of Clinical Microbiology.

[27]  S. Laboissiere,et al.  A prion protein epitope selective for the pathologically misfolded conformation , 2003, Nature Medicine.

[28]  Clemencia Pinilla,et al.  Mapping the Prion Protein Using Recombinant Antibodies , 1998, Journal of Virology.

[29]  M. Moser,et al.  Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE) , 1999, Acta Neuropathologica.

[30]  S. Prusiner,et al.  Molecular biology of prion diseases , 1991, Science.

[31]  W. Surewicz,et al.  Aggregation and fibrillization of the recombinant human prion protein huPrP90-231. , 2000, Biochemistry.

[32]  Tong Liu,et al.  Differential expression of cellular prion protein in mouse brain as detected with multiple anti-PrP monoclonal antibodies , 2001, Brain Research.

[33]  R. Somerville,et al.  Immunolocalization of the prion protein in scrapie affected rodent retinas , 1999, Neuroscience Letters.