NR AOXU
AU Peoc'h,K.; Frobert,Y.; Volland,H.; Boquet,D.; Sato,T.A.; Clement,G.; Grassi,J.; Laplanche,J.L.; Creminon,C.
TI Production of monoclonal antibodies against 14-3-3 protein and their use for CJD diagnosis
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-40
PT Konferenz-Poster
AB
The detection of 14-3-3 protein in the CSF is a WHO criterion of probable sporadic CJD. Regarding different studies (1,2), the performances of detection appear quite variable. The improvement of the sensitivity is of main interest for the ante-mortem diagnosis of Creuztfeldt-Jakob disease. We describe here new anti-14-3-3 monoclonal antibodies, which proved to be suitable for detecting the 14-3-3 protein in human CSF using western-blot or a newly developed enzyme immunoassay.
Monoclonal antibodies (mAbs) were raised by immunising Biozzi mice with murine recombinant 14-3-3 protein. Immunological response was monitored in mouse serum using conventional enzyme immunoassay by checking the capacity of polyclonal antibodies to bind biotin-labelled recombinant protein (3).The mouse presenting the highest titer was selected for producing monoclonal antibodies. After fusion of B lymphocytes with NS1 myeloma cells, 70 hybridoma cells were stabilized and 20 of them were selected to be expanded as ascitic fluids in nude mice.
mAbs were systematically tested for their capacity to detect human CSF 14-3-3 in western-blot experiments. Until now, three of them were proven to be suitable for this application.In addition, in view to establish two-site immunometric assays (sandwich immunoassay) allowing measurement of the 14-3-3 protein in human samples, we have tested all possible combinations between solid-phase immobilized and biotin-labelled antibodies. This leads to the identification of mAbs allowing specific detection of recombinant 14-3-3. The detection limit of these assays is below 100 pg/ml as estimated with standard curves established with the recombinant protein.
In conclusion, these new methods represent promising tools to improve the ante-mortem diagnosis of human prion diseases.
References.
1) Geschwind MD et al, Arch Neurol. 2003 ;60(6):813-6. 2) Beaudry P et al, Dement Geriatr Cogn Disord. 1999 ;10(1):40-6. 3) Grassi J et al, J.Immunol.Methods 1989 ;123:193-210.
AD K. Peoc'h, J.-L. Laplanche, Service de Biochimie et Biologie Moléculaire, Hôpital Lariboisière, Paris, France; Y. Frobert, H. Volland, D. Boquet, J. Grassi, C. Créminon, CEA, Service de Pharmacologie et d'Immunologie, CEA/Saclay, Gif sur Yvette , France; T.-A. Sato, Molecular Oncology, dpt of Otolaryngo/Pathology, Columbia university, USA; G. Clément, Laboratoire d'ImmunoAllergie Alimentaire, INRA-CEA, SPI, Gif sur Yvette, France
SP englisch
PO Deutschland