J Vet Diagn Invest 4:447-449 (1992)
BRIEF COMMUNICATIONS Assessment of western immunoblotting for the confirmatory diagnosis of ovine scrapie and bovine spongiform encephalopathy (BSE) Jonathan B. Katz, Janice C. Pedersen, Allen L. Jenny, William D. Taylor Ovine scrapie and bovine spongiform encephalopathy (BSE) are members of a group of transmissible, genetically influenced, neurodegenerative diseases known variously as unconventional slow virus infections, prion diseases, or spon4-7,14 Laboratory diagnosis of these giform encephalopathies. human and animal diseases has, until recently, depended solely upon neurohistopathologic examination and labora2,4,9,14 The histopathologic approach tory rodent inoculation. is often inconclusive, particularly with autolyzed specimens, and the inoculation procedure may require many months before clinical and pathologic changes become manifest. Abnormal proteinase K (PK)-resistant forms of a highly conserved host encoded protein termed prion protein (PrP) have been found in the brains of humans and animals dying of 5,8,11,13 These proteins exhibit a spongiform encephalopathies. characteristic electrophoretic banding pattern in the range of 5,11,12 The presence of PK-resistant 15-34 kilodaltons (kD). RES PrP (PrP ) has been used as a biochemical marker for the 3,5,11-13 Our objective was to develop presence of these diseases. a diagnostic western immunoblot (WI) procedure to detect PrPRES and to compare WI with histopathology on known infected sheep and cattle brain specimens, negative control specimens from a closed flock, and specimens from sheep in which histopathologic findings were inconclusive. All sheep brains were first examined histopathologically. The following 4 findings were considered necessary for a positive diagnosis of scrapie: status spongiosis, neuronal vacuolation, neuronal necrosis, and astrocytosis.7,9 Two Britishorigin bovine brain stem specimens were derived from cattle that died of BSE in Great Britain. These had been histopathologically diagnosed as positive for BSE in that country (Dr. Mark Robinson, Agricultural Research Service, USDA, Pullman, WA, in cooperation with the Central Veterinary Laboratory, Weybridge, Surrey, Great Britain, personal communication). Ovine negative control brain stems were histopathologically negative and originated from lambs born into a closed flock with no clinical or epidemiological evidence of scrapie since its inception in 1974. Positive controls were derived from clinically and histopathologically positive animals in known scrapie-infected commercial flocks. Ovine and bovine specimens were randomly coded and tested by WI using a blind study design.
PrP and PrPRES were detergent extracted from 1.0-g specimens as previously described.5,8,11 Nearly all brains were tested in triplicate using separate brain stem, midbrain, and cerebellum samples. To adapt WI to a diagnostic laboratory environment, several technical modifications were made. Each tissue was homogenized in 20 ml of 25 mM TRISa buffer, pH 7.4, containing 10% (w/v) N-lauroyl sodium sarcosinate (sarcosyl) detergent.a The homogenate was centrifuged at 6 C for 30 minutes at 2,000 x g, and the resulting supernatant was ultracentrifuged at 6 C for 2 hours at 220,000 x g. All subsequent processing of the ultracentrifuge pellet was conducted in 1.7-ml microcentrifuge tubesb for reasons of speed and economy, and a microultracentrifugec was employed to reduce centrifugation times. The ultracentrifuge pellet was vortex disrupted and washed twice for 30 minutes each time with vigorous agitation at 37 C in a 25 mM TRIS buffer, pH 6.8, containing 10% (w/v) NaCl and 1% (w/v) sarcosyl. The residual particulates were collected after each wash by a 20minute 25,000 x g centrifugation. The washed pellet was then disrupted and treated with a high concentration of PK (75 µg/ml) d in wash buffer for 30 minutes at 37 C to eliminate all except PrP RES. Protease activity was terminated by adding 2.5 mM phenylmethylsulfonyl fluoride (PMSF).a The mixture was made to 80% (v/v) formic acid by adding 100% formic acid, incubated on ice for 2 hours, and dried under vacuum, leaving only a very small pellet to be dissolved and loaded onto the gel in sample loading buffer. 10 This procedure saved many hours by reducing washing and ultracentrifugation times and by exposing the washed material to a relatively high concentration of PK at 37 C for a short time. Formic acid treatment improved WI sensitivity about 4-fold as previously reported.5 Precast miniaturized 10-15% gradient polyacrylamide gelse were used for automated sodium dodecyl sulfate polyacrylTable 1. Comparison of histopathologic diagnoses with western immunoblots (WI) for the diagnosis of scrapie and bovine spangiform encephalopathy.
From the National Veterinary Services Laboratories, Science and Technology, Animal and Plant Health Inspection Service, US Department of Agriculture, Ames, IA 50010. Received for publication November 7, 1991. 447 Downloaded from vdi.sagepub.com at UCSF LIBRARY & CKM on March 13, 2015
448
Brief communications
Western immunoblots of proteinase K resistant PrP (PRPRES) from scrapie- and bovine spongiform encephalopathy (BSE)infected and uninfected sheep and cattle brains. Left to right: positive and negative scrapie controls, molecular weight (MW) markers, rabbit IgG control, 2 BSE-positive and 1 BSE-negative bovine specimen from Great Britain, and a strongly positive ovine specimen. Note the characteristic banding of PrPRES from both species. Rabbit origin anti-hamster PrP primary antibody was used at 1: 1,000 dilution. Figure 1.
amide gel electrophoresise and rapid semi-dry electroblotting of separated proteins onto 50-mm nitrocellulose squares.f,10 Immunodetection was performed using donated primary RES rabbit anti-hamster PrPRES or rabbit anti-synthetic PrP g,h,1,3,13 (1:1,000 dilutions of each) and a peptide antibodies commercial biotinylated anti-rabbit immunoglobulin G (IgG) antibody streptavidin-alkaline phosphatase conjugate system-d The anti-hamster PrPRES antibody had been prepared
using PrPRES extracted from brains of hamsters infected with the 263K strain of scrapie.13 The anti-synthetic PrPRES peptide antibody had been prepared using an immunogen made by coupling the peptide hapten to keyhole limpet hemocyanin (KLH) carrier protein. 1,3 The peptide corresponded to the 15 amino acids numbered 89-103 inclusive in the hamster PrPRES amino acid sequence.1,3 WI generated no false-positive diagnoses and 1 false-neg-
RES Figure 2. Western immunoblots of proteinase K resistant PrP (PrP ) from scrapie-suspect sheep brains. Left to right: positive and negative ovine controls, molecular weight markers, rabbit IgG control, PrP analyses of brain stem specimens from 4 sheep with inconclusive histopathologic findings (lanes l-4). Specimens in lanes 2-4 exhibit clearly detectable PrPRES. Rabbit origin anti-synthetic PrPRES peptide primary antibody was used at 1:1,000 dilution.
Downloaded from vdi.sagepub.com at UCSF LIBRARY & CKM on March 13, 2015
449
Brief communications Table 2. Western immunoblot (WI) results on histopathologitally inconclusive scrapie suspect sheep brains.
c. Model TL- 100 Ultracentrifuge, Beckman Instrument Corp., Palo Alto, CA. d. Proteinase K and Immunoselect Detection System, Life Technologies Corp., Gaithersburg, MD. e. Pharmacia LKB Biotechnology, Piscataway, NJ. f. Schleicher and Schuell, Keene, NH. g. Dr. Richard Rubenstein, Institute for Basic Research in Developmental Disabilities, New York State Department of Health, Staten Island, NY. h. Dr. Richard Race, Rocky Mountain Laboratories, National Institutes of Health, Department of Health and Human Services, Hamilton, MT. The use of a particular manufacturer’s product does not constitute an endorsement on behalf of the USDA.
References
ative diagnosis relative to histopathology (Table 1). WI results of brain stem, midbrain, and cerebellum samples were always consistent for any given animal, although positive brain stem and midbrain specimens often produced stronger signals than did the corresponding cerebellum. Both BSEpositive specimensh and a negative control were correctly identified by WI, even though these specimens were in a state of advanced autolysis when tested. The BSE PrPRES pattern appeared much like the ovine pattern (Fig. 1). The WI and histopathologic diagnoses of scrapie in sheep brains were in good agreement when at least 3 of the 4 pathologic criteria were observed (Table 2). As fewer criteria were met, that agreement weakened, but a number of histopathologically inconclusive specimens contained easily detectable levels of PrPRES (Fig. 2). WI analysis for PrPRES may help confirm a diagnosis of spongiform encephalopathy when histopathologic examination is inconclusive or unwarranted as a result of postmortem autolysis. Controlled animal inoculation studies will ultimately be helpful in resolving discrepant WI and histopathologic results and in determining the sensitivity limit of WI, which is particularly important for scrapie and BSE among the spongiform encephalopathies because the concentration of PrP and PrPRES is reportedly much lower in sheep and cattle than in the usual laboratory animal model species.4 Both methods together may provide greater diagnostic certitude than either method alone. WI may offer immunochemical verification or lack thereof to a diagnosis of BSE, should the presence of that disease be suspected in the United States.5,9
Sources and manufacturers a. Sigma Chemical Co., St. Louis, MO. b. PGC Scientifics, Gaithersburg, MD.
1. Barry R, Vincent M, Kent S, et al.: 1988, Characterization of prion proteins with monospecific antisera to synthetic peptides. J Immunol 140:1188-1193. 2. Carp RI, Callahan SM: 1991, Variation in the characteristics of 10 mouse-passaged scrapie lines derived from 5 scrapie-positive sheep. J Gen Virol 72:293-298. 3. Caughey B, Race R, Vogel M, et al.: 1988, In vitro expression in eukaryotic cells of a prion protein gene cloned from scrapie infected mouse brain. Proc Natl Acad Sci USA 85:4657-4661. 4. Eklund C, Kennedy R, Hadlow W: 1967, Pathogenesis of scrapie virus infection in the mouse. J Infect Dis 117: 15-22. 5. Farquhar CF, Somerville R, Ritchie LA: 1989, Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy. J Virol Methods 24:215-222. 6. Gajdusek D, Zigas V: 1957, Degenerative disease of the central nervous system in New Guinea: epidemic occurrence of “kuru” in the native population. N Engl J Med 257:974-978. 7. Hadlow W, Race R, Kennedy R, Eklund C: 1979, Natural infection of sheep with the scrapie virus. In: Slow transmissible diseases of the nervous system, ed. Pruisner S, Hadlow W, vol. 2, pp. 3-12. Academic Press, New York, NY. 8. Hilmert H, Diringer H: 1984, A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters. Biosci Rep 4:165-170. 9. Miller LD, Landgraf W, Taylor WD: 1985, Scrapie: procedures for laboratory diagnosis. Proc Annu Meet Am Assoc Vet Lab Diagn 28:417-420. 10. Prieur B, Russo-Marie F: 1988, An automated western blot analysis using the Phast system. Anal Biochem 172:338-343. 11. Serban D, Taraboulous A, DeArmond SJ, Pruisner SB: 1990, Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins. Neurology 40: 110-117. 12. Sklaviadis TK, Manuelidis L, Manuelidis EE: 1989, Physical properties of the Creutzfeldt-Jakob disease agent. J Virol 63: 1212-1222. 13. Rubenstein R, Kascsak R, Merz P, et al.: 1986, Detection of scrapie-associated fibril (SAF) proteins using an anti-SAF antibody in nonpurified tissue preparations. J Gen Virol 67:671681. 14. Wells GA, Scott AC, Johnson CT, et al.: 1987, A novel progressive spongiform encephalopathy in cattle. Vet Rec 121:419420.
Downloaded from vdi.sagepub.com at UCSF LIBRARY & CKM on March 13, 2015
BRIEF COMMUNICATIONS Assessment of western immunoblotting for the confirmatory diagnosis of ovine scrapie and bovine spongiform encephalopathy (BSE) Jonathan B. Katz, Janice C. Pedersen, Allen L. Jenny, William D. Taylor Ovine scrapie and bovine spongiform encephalopathy (BSE) are members of a group of transmissible, genetically influenced, neurodegenerative diseases known variously as unconventional slow virus infections, prion diseases, or spon4-7,14 Laboratory diagnosis of these giform encephalopathies. human and animal diseases has, until recently, depended solely upon neurohistopathologic examination and labora2,4,9,14 The histopathologic approach tory rodent inoculation. is often inconclusive, particularly with autolyzed specimens, and the inoculation procedure may require many months before clinical and pathologic changes become manifest. Abnormal proteinase K (PK)-resistant forms of a highly conserved host encoded protein termed prion protein (PrP) have been found in the brains of humans and animals dying of 5,8,11,13 These proteins exhibit a spongiform encephalopathies. characteristic electrophoretic banding pattern in the range of 5,11,12 The presence of PK-resistant 15-34 kilodaltons (kD). RES PrP (PrP ) has been used as a biochemical marker for the 3,5,11-13 Our objective was to develop presence of these diseases. a diagnostic western immunoblot (WI) procedure to detect PrPRES and to compare WI with histopathology on known infected sheep and cattle brain specimens, negative control specimens from a closed flock, and specimens from sheep in which histopathologic findings were inconclusive. All sheep brains were first examined histopathologically. The following 4 findings were considered necessary for a positive diagnosis of scrapie: status spongiosis, neuronal vacuolation, neuronal necrosis, and astrocytosis.7,9 Two Britishorigin bovine brain stem specimens were derived from cattle that died of BSE in Great Britain. These had been histopathologically diagnosed as positive for BSE in that country (Dr. Mark Robinson, Agricultural Research Service, USDA, Pullman, WA, in cooperation with the Central Veterinary Laboratory, Weybridge, Surrey, Great Britain, personal communication). Ovine negative control brain stems were histopathologically negative and originated from lambs born into a closed flock with no clinical or epidemiological evidence of scrapie since its inception in 1974. Positive controls were derived from clinically and histopathologically positive animals in known scrapie-infected commercial flocks. Ovine and bovine specimens were randomly coded and tested by WI using a blind study design.
PrP and PrPRES were detergent extracted from 1.0-g specimens as previously described.5,8,11 Nearly all brains were tested in triplicate using separate brain stem, midbrain, and cerebellum samples. To adapt WI to a diagnostic laboratory environment, several technical modifications were made. Each tissue was homogenized in 20 ml of 25 mM TRISa buffer, pH 7.4, containing 10% (w/v) N-lauroyl sodium sarcosinate (sarcosyl) detergent.a The homogenate was centrifuged at 6 C for 30 minutes at 2,000 x g, and the resulting supernatant was ultracentrifuged at 6 C for 2 hours at 220,000 x g. All subsequent processing of the ultracentrifuge pellet was conducted in 1.7-ml microcentrifuge tubesb for reasons of speed and economy, and a microultracentrifugec was employed to reduce centrifugation times. The ultracentrifuge pellet was vortex disrupted and washed twice for 30 minutes each time with vigorous agitation at 37 C in a 25 mM TRIS buffer, pH 6.8, containing 10% (w/v) NaCl and 1% (w/v) sarcosyl. The residual particulates were collected after each wash by a 20minute 25,000 x g centrifugation. The washed pellet was then disrupted and treated with a high concentration of PK (75 µg/ml) d in wash buffer for 30 minutes at 37 C to eliminate all except PrP RES. Protease activity was terminated by adding 2.5 mM phenylmethylsulfonyl fluoride (PMSF).a The mixture was made to 80% (v/v) formic acid by adding 100% formic acid, incubated on ice for 2 hours, and dried under vacuum, leaving only a very small pellet to be dissolved and loaded onto the gel in sample loading buffer. 10 This procedure saved many hours by reducing washing and ultracentrifugation times and by exposing the washed material to a relatively high concentration of PK at 37 C for a short time. Formic acid treatment improved WI sensitivity about 4-fold as previously reported.5 Precast miniaturized 10-15% gradient polyacrylamide gelse were used for automated sodium dodecyl sulfate polyacrylTable 1. Comparison of histopathologic diagnoses with western immunoblots (WI) for the diagnosis of scrapie and bovine spangiform encephalopathy.
From the National Veterinary Services Laboratories, Science and Technology, Animal and Plant Health Inspection Service, US Department of Agriculture, Ames, IA 50010. Received for publication November 7, 1991. 447 Downloaded from vdi.sagepub.com at UCSF LIBRARY & CKM on March 13, 2015
448
Brief communications
Western immunoblots of proteinase K resistant PrP (PRPRES) from scrapie- and bovine spongiform encephalopathy (BSE)infected and uninfected sheep and cattle brains. Left to right: positive and negative scrapie controls, molecular weight (MW) markers, rabbit IgG control, 2 BSE-positive and 1 BSE-negative bovine specimen from Great Britain, and a strongly positive ovine specimen. Note the characteristic banding of PrPRES from both species. Rabbit origin anti-hamster PrP primary antibody was used at 1: 1,000 dilution. Figure 1.
amide gel electrophoresise and rapid semi-dry electroblotting of separated proteins onto 50-mm nitrocellulose squares.f,10 Immunodetection was performed using donated primary RES rabbit anti-hamster PrPRES or rabbit anti-synthetic PrP g,h,1,3,13 (1:1,000 dilutions of each) and a peptide antibodies commercial biotinylated anti-rabbit immunoglobulin G (IgG) antibody streptavidin-alkaline phosphatase conjugate system-d The anti-hamster PrPRES antibody had been prepared
using PrPRES extracted from brains of hamsters infected with the 263K strain of scrapie.13 The anti-synthetic PrPRES peptide antibody had been prepared using an immunogen made by coupling the peptide hapten to keyhole limpet hemocyanin (KLH) carrier protein. 1,3 The peptide corresponded to the 15 amino acids numbered 89-103 inclusive in the hamster PrPRES amino acid sequence.1,3 WI generated no false-positive diagnoses and 1 false-neg-
RES Figure 2. Western immunoblots of proteinase K resistant PrP (PrP ) from scrapie-suspect sheep brains. Left to right: positive and negative ovine controls, molecular weight markers, rabbit IgG control, PrP analyses of brain stem specimens from 4 sheep with inconclusive histopathologic findings (lanes l-4). Specimens in lanes 2-4 exhibit clearly detectable PrPRES. Rabbit origin anti-synthetic PrPRES peptide primary antibody was used at 1:1,000 dilution.
Downloaded from vdi.sagepub.com at UCSF LIBRARY & CKM on March 13, 2015
449
Brief communications Table 2. Western immunoblot (WI) results on histopathologitally inconclusive scrapie suspect sheep brains.
c. Model TL- 100 Ultracentrifuge, Beckman Instrument Corp., Palo Alto, CA. d. Proteinase K and Immunoselect Detection System, Life Technologies Corp., Gaithersburg, MD. e. Pharmacia LKB Biotechnology, Piscataway, NJ. f. Schleicher and Schuell, Keene, NH. g. Dr. Richard Rubenstein, Institute for Basic Research in Developmental Disabilities, New York State Department of Health, Staten Island, NY. h. Dr. Richard Race, Rocky Mountain Laboratories, National Institutes of Health, Department of Health and Human Services, Hamilton, MT. The use of a particular manufacturer’s product does not constitute an endorsement on behalf of the USDA.
References
ative diagnosis relative to histopathology (Table 1). WI results of brain stem, midbrain, and cerebellum samples were always consistent for any given animal, although positive brain stem and midbrain specimens often produced stronger signals than did the corresponding cerebellum. Both BSEpositive specimensh and a negative control were correctly identified by WI, even though these specimens were in a state of advanced autolysis when tested. The BSE PrPRES pattern appeared much like the ovine pattern (Fig. 1). The WI and histopathologic diagnoses of scrapie in sheep brains were in good agreement when at least 3 of the 4 pathologic criteria were observed (Table 2). As fewer criteria were met, that agreement weakened, but a number of histopathologically inconclusive specimens contained easily detectable levels of PrPRES (Fig. 2). WI analysis for PrPRES may help confirm a diagnosis of spongiform encephalopathy when histopathologic examination is inconclusive or unwarranted as a result of postmortem autolysis. Controlled animal inoculation studies will ultimately be helpful in resolving discrepant WI and histopathologic results and in determining the sensitivity limit of WI, which is particularly important for scrapie and BSE among the spongiform encephalopathies because the concentration of PrP and PrPRES is reportedly much lower in sheep and cattle than in the usual laboratory animal model species.4 Both methods together may provide greater diagnostic certitude than either method alone. WI may offer immunochemical verification or lack thereof to a diagnosis of BSE, should the presence of that disease be suspected in the United States.5,9
Sources and manufacturers a. Sigma Chemical Co., St. Louis, MO. b. PGC Scientifics, Gaithersburg, MD.
1. Barry R, Vincent M, Kent S, et al.: 1988, Characterization of prion proteins with monospecific antisera to synthetic peptides. J Immunol 140:1188-1193. 2. Carp RI, Callahan SM: 1991, Variation in the characteristics of 10 mouse-passaged scrapie lines derived from 5 scrapie-positive sheep. J Gen Virol 72:293-298. 3. Caughey B, Race R, Vogel M, et al.: 1988, In vitro expression in eukaryotic cells of a prion protein gene cloned from scrapie infected mouse brain. Proc Natl Acad Sci USA 85:4657-4661. 4. Eklund C, Kennedy R, Hadlow W: 1967, Pathogenesis of scrapie virus infection in the mouse. J Infect Dis 117: 15-22. 5. Farquhar CF, Somerville R, Ritchie LA: 1989, Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy. J Virol Methods 24:215-222. 6. Gajdusek D, Zigas V: 1957, Degenerative disease of the central nervous system in New Guinea: epidemic occurrence of “kuru” in the native population. N Engl J Med 257:974-978. 7. Hadlow W, Race R, Kennedy R, Eklund C: 1979, Natural infection of sheep with the scrapie virus. In: Slow transmissible diseases of the nervous system, ed. Pruisner S, Hadlow W, vol. 2, pp. 3-12. Academic Press, New York, NY. 8. Hilmert H, Diringer H: 1984, A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters. Biosci Rep 4:165-170. 9. Miller LD, Landgraf W, Taylor WD: 1985, Scrapie: procedures for laboratory diagnosis. Proc Annu Meet Am Assoc Vet Lab Diagn 28:417-420. 10. Prieur B, Russo-Marie F: 1988, An automated western blot analysis using the Phast system. Anal Biochem 172:338-343. 11. Serban D, Taraboulous A, DeArmond SJ, Pruisner SB: 1990, Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins. Neurology 40: 110-117. 12. Sklaviadis TK, Manuelidis L, Manuelidis EE: 1989, Physical properties of the Creutzfeldt-Jakob disease agent. J Virol 63: 1212-1222. 13. Rubenstein R, Kascsak R, Merz P, et al.: 1986, Detection of scrapie-associated fibril (SAF) proteins using an anti-SAF antibody in nonpurified tissue preparations. J Gen Virol 67:671681. 14. Wells GA, Scott AC, Johnson CT, et al.: 1987, A novel progressive spongiform encephalopathy in cattle. Vet Rec 121:419420.
Downloaded from vdi.sagepub.com at UCSF LIBRARY & CKM on March 13, 2015
