NR ADLL
AU Diringer,H.; Rahn,H.C.; Bode,L.
TI Antibodies to protein of scrapie-associated fibrils
QU Lancet 1984 Aug 11; 2(8398): 345
PT letter
VT
SIR, - In the subacute spongiform virus encephalopathies (SSVE) there is no measurable immune reaction.[1] However, scrapie-associated fibrils (SAF) - reviewed in Dr Rohwer's letter (Ju1y 7, p 36) - can now be isolated in large amounts from the brains of hamsters with scrapie.[2] We have developed an efficient method of raising antibodies against SAF-protein that may be of general use in the study of SSVE.
Antibodies were prepared by immunising a rabbit with SAF-protein from fifteen hamster brains. The material was divided into three portions and injected, first with complete Freund's adjuvant and then twice with incomplete Freund's adjuvant with intervals of 20-30 days between injections. The injections were given subcutaneously into three areas of the back of the animal and intra-muscularly in both hind legs. Blood was withdrawn about 10 days after each injection. After the second booster antibody could be detected by enzyme-1inked immunosorbent assay at serum dilutions up to at least 1:6400. The specificity of the antibodies for SAF-protein was determined by Western blotting.
To prepare the immunogen SAF purified from twenty scrapie hamster brains[2,3] were sonicated and extracted at 100° three times with 1.5 ml of "tris" buffered saline containing 0.5% sodium dodecyl sulphate in two Eppendorf vials. Supernatants withdrawn after spinning in an Eppendorf centrifuge for 15 min were combined, and 4-5 mg of haemocyanin from Helix pomatia (Serva) and 100 µl of a 2.5% solution of glutardialdehyde were added. The solution was stirred at room temperature for 3 h to couple SAF-protein and haemocyanin[4] and then dried over phosphorus pentoxide in a desiccator. The residue was extracted three times with 1 ml of acetone:acetic acid:triethylamine:water (85:5:5:5) to remove detergent[5] and excess glutardialdehyde. The residue was then dispersed in 4 ml of distilled water by ultrasonication. Four portions of 1 ml were mixed with 1 ml of adjuvant and injected into four rabbits. After the second booster all four animals were positive for antibodies against SAF-protein.
To raise an immune response in one rabbit against the major protein of an SSVE-specific morphological structure (SAP), which itself could either be an infectious form of an unconventional slow virus or an abnormal protein of amyloid appearance, necessitates the preparation of SAF from only fifteen hamster brains. Research with antibodies raised by this easy method may lead to a better understanding of the pathogenesis of natural human and animal SSVE.
1. Kasper KC, Bowman K, Stites DP, Prusiner SB. Toward development of assays for scrapie-specific antibodies. Adv Exp Med Biol 1981; 134: 401-13.
2. Hilmert H, Diringer H. A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters. Biosc Rep 1984; 4: 165-70.
3. Diringer H, Hilmert H, Simon D, Werner E, Eblers B. Towards purification of the scrapie agent. Eur J Biochem 1983; 134: 555-60.
4. Schwoch G, Hamann A, Hilz H. Antiserum against the catalytic subunit of adenosine 3'-5'-cyclic monophosphate dependent proteinase kinase. Biochem J 1980; 192: 223-30.
5. Itenderson LE, Oroszlan S, Konigsberg W. A micromethod for complete remova1 of dodecyl sulfate from proteins by ion-pair extraction. Analyt Biochim 1979; 93: 153-57.
IN Die Autoren immunisierten 4 Kaninchen mit dem aus scrapieassoziierten Fibrillen infektiöser Hamsterhirne isolierten Prionprotein, welches in einer Glutardialdehyd-Lösung an Hämocyanin gebunden wurde. Je ein Drittel des Proteins wurde zunächst mit vollständigem und dann in Abständen von 20-30 Tagen noch zweimal mit unvollständigem Freundschem Adjuvans subkutan und intramuskulär injiziert. Jeweils 10 Tage nach jeder Injektion wurde der Antikörpertiter im Blut bestimmt. Nach der zweiten Injektion waren Antikörper gegen das Prionprotein bei allen 4 Kaninchen und selbst nach einer Verdünnung auf 1:6400 nachweisbar. Zur Immunisierung eines Kaninchen sollen 15 Hamstergehirne erforderlich sein.
MH Animal; *Antibody Formation; Brain/*immunology/ultrastructure; Hamsters; Nerve Tissue Proteins/*immunology; Rabbits; Scrapie/*immunology/pathology; Sheep
AD H.Diringer, H.C.Rahn, L.Bode, Robert-Koch-Institut des Bundesgesundheitsamtes, D-1000 Berlin 65, West Germany
SP englisch
PO England
OR Prion-Krankheiten D