NR AKBJ

AU Robey,W.G.; Jackson,R.; Walters,R.L.; Brackett,J.M.; Harrington,C.A.; Killian,W.R.

TI Use of cerebrospinal fluid levels of 14-3-3 in predicting neurodegeneration in confirmed BSE symptomatic cattle

QU The Veterinary Record 1998 Jul 11; 143(2): 50-1

PT journal article

VT THE group of proteins named 14-3-3 have been reported to be cerebrospinal fluid (CSF) surrogate markers for Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE) in cattle (Hsich and others 1996, Lee and Harrington 1997, Zerr and others 1998). The elevation of this analyte in CSF in diseased animals could be, in principle, the basis of an antemortem diagnostic test for BSE. Currently the diagnosis of BSE is based on the postmortem detection of the PrPsc prion protein in neural tissue and a characteristic brain pathology (Oesch and others 1985, Wells and others 1987). This short communication presents the results of a study using a sensitive immunoblot assay, which show that the CSF 14-3-3 analyte is not a suitable marker to distinguish between normal cattle and cattle showing early symptoms of BSE.
CSF specimens were evaluated using a modification of a published immunoblot (Lee and Harrington 1997) and an ELISA specific for the 14-3-3 proteins. Both immunoassays utilised highly purified 14-3-3 (>95 per cent by SDS-PAGE) isolated from bovine brain (Boston and others 1982, Token and others 1990). The p30 band was the dominant forrn of 14-3-3 detected in this study and the antigen used to score the immunoblot results. The 14-3-3 antibodies used were either murine monoclonal antibodies (Setoguchi and others 1995) or polyclonal antipeptide antibodies. Both antibodies reacted comparably with 14-3-3. The Western-Light Plus (Tropix) immunoblot and chemiluminescent detection system was used as recommended by the manufacturer.
Samples of CSF were collected from cattle using three needle puncture methods. The first was from the lumbrosacral region on standing animals using minimal restraint. The CSF was drawn into a syringe using vacuum aspiration. The second method was from recumbent anaesthetised animals using a gravity drip procedure. The third method was aspiration of CSF from the cisterna magna of recumbent barbiturate euthanased animals. This method was used on all cattle showing clinical symptoms of BSE. Specimens were collected in polypropylene containers, chilled and frozen and stored at -70° as soon as possible.
The immunoassays used in this study were developed using CSF specimens obtained in the USA by the CSF aspiration and gravity drip collection methods. The aspiration method was developed as a field sample collection method to obtain 2 to 3 ml of specimen. The gravity drip method was used to obtain larger volume specimens. In the course of 14-3-3 analyte recovery studies using gravity drip specimens, the authors observed that the two methods of collection influenced the levels of 14-3-3 detected in the immunoblot as shown in Table 1. The distribution of values obtained for the aspirated CSF samples trend higher than those from the gravity collected samples showing the presence of an experimental procedure artefact. This variable was controlled in the 14-3-3 levels shown for 116 CSF samples aspirated from UK BSE-confirmed cattle relative to 144 CSF samples obtained from clinically normal cattle using the field aspiration method (Table 1). These results show that there was no statistical difference (Chi square analysis of categorical data) in the 14-3-3 levels in normal cattle and confirmed BSE cattle. A similar percentage of normal cattle had elevated levels of 14-3-3 as BSE-affected cattle. This equivalence was also observed in the optical density means and standard deviations of the ELISA values obtained from normal and confirmed BSE cattle (0.275 [0.356] v 0.417 [0.560]). The population differences between the 14-3-3 analyte levels in normal and BSE-affected cattle required to justify the use of this analyte as an antemortem screen for cattle entering the human food supply were not observed. The differences in these conclusions from those of other studies cited above could be explained by the small numbers of BSE and control animals examined in the previous studies, very early BSE disease status, undefined immunoblot sensitivity, sampling or storage artefacts, and level of cellular contamination. The first three reasons are, in the authors' opinion, the most likely explanations. In conclusion, the use of the 14-3-3 surrogate marker for the detection of BSE associated neurodegeneration in early symptomatic cattle is not justified.
Acknowledgements. - The authors thank Dr Danny Matthews and the Central Veterinary Laboratory of the Ministry of Agriculture, Fisheries and Food, UK and Dr Margaret Good and the Department of Agriculture, Food and Forestry, Republic of Ireland, for providing CSF from confirmed BSE cattle. The Morinaga Institute of Biological Science, Yokohama, Japan, provided the murine monoclonal antibodies; the polyclonal antipeptide antibodies were provided by Santa Cruz Biotechnology, Santa Cruz, California. The authors would also like to thank Dr Sarah Moyle and Dr Shiva Khalafpour and the Guildhay Technical Support Team and Dr Tracey Colpitts and the Abbott Venture Support Team for their technical assistance. Dr M. Kato and Dr S. Hashizume are thanked for helpful discussions.
References
BOSTON, P. F.. JACKSON, P., KYNOCH, P. A. M. & THOMPSON, R. J. (1982) Journal of Neurochemistry 38, 1474
HSICH, G., KENNEY, K., GIBBS, C. J., LEE, K. H. & HARRINGTON, M. G. (1996) New England Journal of Medicine 335, 924
LIEE, K. H. & HARRINGTON, M. G. (1997) Veterinary Record 140,206 OESCH, B., WESTAWAY, D., WALSCHLI, M., McKINLEY, M. P., KENT, S. B., AEBERSOLD, R., BARRY, R. A., ThMPST, P., TEPLOW, D. B.. HOOD, L. E., PRUSINIER, S. B. & WEISSMANN, C. (1985) Cell 40, 735
SETOGUCHI, Y., KATO, M., SHOJI, M., HONJOH, T., KAMEI, M., SUGITANI, M, SATO, S., HASHIZUME, S., HANAGIRI, T., YOSItltMATSU, T., NAKANISHI, K.. YASUMOTO, K.. NAGASHIMA, A., NAKAHASHI, H., SUZUKI, T., IAIAI, T., SHIRAHATA, S. & NOMOTO, K. (1995) Human Antibodies and Hybridomas 6, 137
TOKEN. A., ELLIS, C., SELLERS, L & AITKEN, A. (1990) European Journal of Biochemistry 191, 421
WELLS, G. A. H., SCOTT. A. C., JOHNSON. C. T., GUNNING, R. F., HANCOCK, R. D., JEFFREY, M., DAwSON, M. & BRADLEY, R. (1987) Veterinary Record 121, 419
ZERR, I., BODEMER, M., GEFELLER, 0., OTTO. M., POSER, S., WILTFANG, J., WINIOL, 0., KRETZSCHMAR, H. k & WEBER, T. (1998) Annals of Neurology 43, 32
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TABLE 1: Levels of 14-3-3 in bovine CSF determined by semi-quantitative immunoblot
Estimated 14-3-3 concentration (ng/ml) and pefoentages of cattle per category*
sam- identity Collection method Total number 0-10 10-30 30-50 >50
USA normal Gravity drip 75 67 29 3 1
USA normal Vacuum aspiration 37 16 32 14 38
UK/Ireland normal+ Vacuum aspiration 144 16 35 23 26
UK/Ireland BSE# Vacuum aspiration 116 20 47 18 15
* Estimates were based on p30 band intensity from 10-fold CSF oonco'ltrates relative to intensities of purified bovine 14-3-3 standards (0.10,30 and 50 ng/ml)
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present on esen PVDF immunoblot nnsmbnsne Detection sensitivity by angan dilution was 0-2 ng/ml The >50 ng/ml p30 bands were et-onally intense.
Category assignments were made independently by treee experienced laboratory pensonnel similarly to Zerr and otners (1998)
+ Normal catie CSF aimoimes were obtained ftom UK/Irish fanna tisat are historically free of BSE. lmmunonlstooi'e'nstry and histopathology were not pe,fomad
# Symptomology and histopathology OOnlim,ed by fleid veterinarians and scientists of MAFF and DAFF

ZR 8

MH Animal; Biological Markers/cerebrospinal fluid; Cattle; Cattle Diseases/*diagnosis; Cerebrospinal Fluid Proteins/*analysis; Encephalopathy, Bovine Spongiform/*diagnosis; Immunoblotting

AD W.G. Robey, R.L. Walters, J.M. Brackett, C.A. Harrington (harrica.add@notes.abbott.com), W. R. Killian, Abbott Diagnostics Division, Department 9RB, Building AP2O, Abbott Laboratories, North Chicago. IL 60064. USA, R. Jackson, Guildhay Limited, Guildford, Surrey GU I 4UG, Correspondence to Dr Killian

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