RAPID COMMUNICATION |
An Alternative Pretreatment Procedure in Animal Transmissible Spongiform Encephalopathies Diagnosis Using PrPsc Immunohistochemistry
Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Laboratoire d'Etudes et de Recherches en Pathologie Bovine et Hygiène des Viandes, Unité Agent Transmissible Non Conventionnel (ATNC), Lyon, France
Correspondence to: Dr. Anna Bencsik, AFSSA, Laboratoire d'Etudes et de Recherches en Pathologie Bovine et Hygiène des Viandes, Unité ATNC, 31 Avenue Tony Garnier, 69364 Lyon, Cedex 07-France. E-mail: a.bencsik{at}lyon.afssa.fr
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: bovine spongiform encephalopathy diagnosis antigen retrieval immunohistochemistry prion scrapie
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Remarkably, because antibodies recognizing only the pathological form (PrPsc) are not available, several pretreatments (a combination of chemical, heating, and enzymatic treatments) are essential for PrPsc IHC because it enhances antigen retrieval, while at the same time suppresses the recognition of normal cellular PrP (PrPc). Although very efficient, it may be interesting to use only chemical pretreatments. In particular, PrPsc IHC may not be only easier, less expensive, and quicker but also potentially as efficient as using the current pretreatment. For this reason, we compared the PrPsc detections obtained using classical and only chemical pretreatments on brain samples originating from BSE- or scrapie-affected animals as well as from unaffected animals. We showed that, in all tested species, PrPsc is detectable in both cases as well, without generating any unspecific background staining. Here we demonstrate the possibility to use this new pretreatment at least with the scrapie-associated fibrils (SAF)-84 antibody on brain samples for diagnostic purposes in animal TSEs with very good efficacy, despite evident increase of adhesion of brain slices.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PrP IHC Pretreatments
Brain slices were first dewaxed and rehydrated in graded alcohols. We used the combination of pretreatments already published (Debeer et al. 2001,2002
) that consisted of 98100% formic acid bath for 10 min at RT, followed by hydrated autoclaving for 20 min at 121C in water (Prestige Medical; Blackburn Lane, UK), and finally a digestion using proteinase K at 20 µg/ml (Roche Diagnostics; Meylan, France) for 15 min at 37C.
Additionally, we analyzed pretreatments performed at RT and based on a chemical sequence defined as follows: 10-min incubation in a solution of KMnO4 0.5% (in 0.1 M PBS, pH 7.0), three washes in distilled water, then 2-min incubation in a bath of Na2S2O5 1%, a wash in distilled water, and finally a 10-min incubation in a solution of sarkosyl (N-lauroylsarcosine) 0.1%, NaOH 75 mM, NaCl 2%. After a 5-min wash in tap water, the slides were ready for the PrP IHC procedure.
PrP Immunohistochemistry
PrPsc IHC was then performed as described previously, using SAF-84 monoclonal primary antibody (0.5 µg/ml in PBS 0.1 M, pH 7.4/0.1% Triton X-100) (Demart et al. 1999; Debeer et al. 2001
,2002
). Without any modification for the usual pretreatment, the chemical-only pretreatment required a little adjustment that was indeed interesting to note. Because KMnO4 reagent strongly inhibits endogenous peroxidase, there was no need for that separate inhibition step in the case of the chemical pretreatment. Final revelation was achieved using diaminobenzidine intensified with NiCl2 (Biosys GmbH; Karben, Germany). Finally, the sections were dehydrated, mounted using Eukitt (Microm; Franchville, France), and observed under a microscope (Olympus; Rungis, France) coupled to an image analysis workstation (Biocom; Les Ulis, France). The omission of the primary antibody that was substituted by control serum was used to check the nonspecific background staining in scrapie and BSE cases. Specificity of positive PrPsc immunolabeling was also assessed using the scrapie- and BSE-negative brains. These pretreatment procedures were repeated five times on these brain sections.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Interestingly, this new pretreatment compared with the usual pretreatment repeatedly gave a more intense and contrasted immunolabeling using SAF-84 monoclonal antibody in both species in each of the different brain areas analyzed. For two sheep, we repeatedly observed equal immunolabeling intensity with both pretreatments, without obvious intensification as seen in the five other cases.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To explore their possibilities we applied these pretreatments on brain slices prepared routinely for TSE diagnostic purpose. In the present analysis, we showed that PrP IHC analysis using only the chemical pretreatments retained all its abilities to satisfactorily detect PrPsc at cellular and histological levels (midbrain and brainstem regions) in both bovine and ovine species affected with TSEs. Furthermore, it regularly showed stronger staining in the bovine samples. In sheep staining it was at least of equal intensity, and an enhancement of PrP detection was obvious in five of seven cases. This may reflect the known variety of scrapie strains as well as the various PrP genotypes found in this species.
Based on strong oxidation of the brain sections followed by a neutralization stage and the increase of the accessibility of PrP epitopes with a last detergent step, this pretreatment is cheaper and accessible for every routine histological laboratory. Because IHC labeling intensity appeared to be increased, it would also be possible to increase SAF-84 dilution, which represents an extra cost. However, there is a noticeable constraint found in the performance of this new pretreatment, which is a great incidence on the tissue slices adhesion. It is possible to play on some subtle changes in the composition of the chemical solutions (Onnasch et al. 2005) to avoid a too-strong tissue-loss effect, as well as using glass slides that allow a strong adhesion of the brain sample or, if necessary, 3-aminopropyltriethoxysilane-coated slides (Rentrop et al. 1986
). Actually, this chemical sequence is quite drastic and leads to a certain fragility of the sections that may represent an obstacle, but even if this method may not work with every PrP antibody, or in every species, in our study it worked as well in mice experimentally affected with TSEs (data not shown).
Thus, even if additional studies may be warranted to test it with other PrP antibodies, and even if it requires some technical cautions, we clearly showed the potentiality of this new method of PrPsc IHC, using SAF-84 antibody. This study opens new possibilities in the diagnosis of ruminant TSEs as an amplifying effect is also suggested.
![]() |
Acknowledgments |
---|
The authors would like to thank Dr. Eoin Monks, without whom this study would not have been possible. The chemical pretreatment was developed by E. Monks, A. Roche, and A. Church (unpublished data), Department of Agriculture, Food and Rural Development, Central Veterinary Research Laboratory, Abbotstown, Castleknock, Dublin, Ireland.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bolton DC, McKinley MP, Prusiner SB (1982) Identification of a protein that purifies with the scrapie prion. Science 218:13091311[Medline]
Caramelli M, Ru G, Casalone C, Bozzetta E, Acutis PL, Calella A, Forloni G (2001) Evidence for the transmission of scrapie to sheep and goats from a vaccine against Mycoplasma agalactiae. Vet Rec 148:531536
Casalone C, Zanusso G, Acutis PL, Ferrari S, Capucci L, Tagliavini F, Monaco S, et al. (2004) Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc Natl Acad Sci USA 101:30653070
Debeer S, Baron T, Bencsik A (2003) Neuropathological characterisation of French bovine spongiform encephalopathy cases. Histochem Cell Biol 120:513521[CrossRef][Medline]
Debeer S, Baron T, Bencsik A (2002) Transmissible spongiform encephalopathies diagnosis using PrPsc immunohistochemistry on fixed but previously frozen brain samples. J Histochem Cytochem 50:611616
Debeer SOS, Baron T, Bencsik AA (2001) Immunohistochemistry of PrPsc within bovine spongiform encephalopathy brain samples with graded autolysis. J Histochem Cytochem 49:15191524
Demart S, Fournier JG, Creminon C, Frobert Y, Lamoury F, Marce D, Lasmezas C, et al. (1999) New insight into abnormal prion protein using monoclonal antibodies. Biochem Biophys Res Commun 265:652657[CrossRef][Medline]
Foster JD, Wilson M, Hunter N (1996) Immunolocalisation of the prion protein (PrP) in the brains of sheep with scrapie. Vet Rec 139:512515
Miller JM, Jenny AL, Taylor WD, Race RE, Ernst DR, Katz JB, Rubenstein R (1994) Detection of prion protein in formalin-fixed brain by hydrated autoclaving immunohistochemistry for the diagnosis of scrapie in sheep. J Vet Diagn Invest 6:366368[Medline]
Onnasch H, Callanan JJ, Sammin DJ, McElroy MC, Basset HF (2005) A survey for TSE in meat and bone meal-fed Irish pigs. Vet Rec in press
Orge L, Simas JP, Fernandes AC (2000) Similarity of the lesion profile of BSE in Portuguese cattle to that described in British cattle. Vet Rec 147:486488
Rentrop M, Knapp B, Winter H, Schweizer J (1986) Aminoalkylsilane-treated glass slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions. Histochem J 18:271276[CrossRef][Medline]
Taylor DM, Brown JM, Fernie K, McConnell I (1997) The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue. Vet Microbiol 58:167174[CrossRef][Medline]
Van Everbroeck B, Pals P, Martin J-J, Cras P (1999) Antigen retrieval in prion protein immunohistochemistry. J Histochem Cytochem 47:14651470
Wood JLN, McGill IS, Done SH, Bradley R (1997) Neuropathology of scrapie: a study of the distribution patterns of brain lesions in 222 cases of natural scrapie in sheep, 19821991. Vet Rec 140:167174
|