Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-10-13
2004-08-24
Spector, Lorraine (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S413000, C530S427000, C435S007100, C435S962000, C435S975000
Reexamination Certificate
active
06780979
ABSTRACT:
The present invention relates to a novel method of purifying PrPres from a biological sample in order to use it for the qualitative and/or quantitative detection of PrPres in said sample.
Transmissible subacute spongiform encephalopathies are caused by non-conventional transmissible agents (NCTA), also called prions, the precise nature of which is still unknown at the present time. TSSE comprise essentially Creutzfeldt-Jakob disease (CID) in humans, scrapie in sheep and goats and bovine spongiform encephalopathy (BSE) in cattle; other encephalopathies have been revealed in mink or certain wild animals such as stag and elk.
The outcome of these diseases is inevitably fatal and no effective treatment is currently available.
In transmissible subacute spongiform encephalopathies, there is an accumulation of a host protein, PrP (or prion protein), in an abnormal form (PrPres), mainly in the central nervous system; PrPres copurifies with the infectiousness and its accumulation precedes the appearance of histological lesions. In vitro it is toxic to neuron cultures.
Two biochemical properties usually make it possible to distinguish between PrPres and normal PrP: PrPres is partially resistant to proteases and is insoluble in anionic surfactants.
To be able to detect the PrPres present in a sample, it is necessary to subject said sample to different operations in order to enrich it in PrPres, while eliminating the normal PrP, so that the PrPres can then be detected by any appropriate specific method without causing:
false positives due to the presence of normal PrP or other contaminants, or
false negatives due to an insufficient concentration of PrPres in the final biological sample.
A number of methods of isolating and/or purifying PrPres have been proposed for this purpose. They are essentially based on the method developed by Hilmert and Diringer (Nature, 1983, 306, 476-478) and generally involve an extraction with a detergent, differential ultracentriugations and a treatment with proteolytic enzymes (Multhaup G. et al., EMBO J., 1985, 4, 6, 1495-1501; Takahashi K et al., Microbiol. Immunol., 1986, 30, 2, 123-131; Hope J. et al., EMBO J., 1986, 5, 10, 2591-2597; Grathwohl K. U. D. et al., Arch. Virol., 1996, 141, 1863-1874; Kascsak R. J. et al., Immunol. Investig., 1997, 26, 259-268; R. E. Race et al., J. Gen. Virol., 1992, 73, 3319-3323; Doi et al., J. Gen. Virol., 1988, 69, 955-960; T. Muramoto et al., Am. J. Pathol., 1993, 143, 5, 1470-1479; Farquhar C. F. et al., Gen. Virol., 1994, 75, 495-504 and J. Gen. Virol., 1996, 77, 1941-1946). They have the disadvantage of comprising a large number of steps including several ultracentrifugations, which are cumbersome to carry out and result in cumulative losses of PrPres; these in turn lead to an insufficient sensitivity to obtain a high-quality detection threshold and quantification of the PrPres.
These various methods require research laboratory equipment and implementation times which are incompatible with use in the field, particularly in abattoirs.
Now, there is a need for rapid verification of the absence or presence of a transmissible subacute spongiform encephalopathy at the time when the animal is slaughtered.
Consequently the inventor set out to provide a method of purifying a biological sample in order to use it for a rapid and reliable detection of PrPres, said method being sufficiently simple to carry out that it can be used in the field, especially in abattoirs, and thereby meeting practical needs better than the methods of the prior art. In fact, the method according to the invention is:
simple to carry out,
reliable and
easy to interpret it increases the detection sensitivity threshold of PrPres by eliminating the false positives (normal PrP and other contaminants), and it eliminates the false negatives because it enables a substantial amount of PrPres to be obtained, in absolute terms, since it is possible to treat large amounts of biological material with a purification yield in excess of 80%; this is of particular value in abattoirs and produces samples in which PrPres is readily detectable with customary diagnostic tests.
The present invention relates to a method of purifying PrPres from a biological sample, characterized in that it comprises essentially:
(1) the incubation, for 30 seconds to 2 hours, preferably for 30 seconds to 10 minutes, at a temperature below 80° C., of said biological sample with a buffer A comprising at least one surfactant in an amount of between a quarter and four times, preferably of between a quarter and one and a half times, the weight of the biological sample, and optionally prior, subsequent or simultaneous incubation with a protease, to form an opalescent to turbid micellar or lamellar suspension S
1
; under the temperature and quantity conditions mentioned above, whatever the surfactant or surfactant mixture may be, it does not solubilize most of the PrPres, which remains in suspension, whereas the normal PrP is solubilized, or even destroyed, if protease is added; said incubation is preferably carried out at a temperature below 50° C., in the presence of an amount of surfactant of between a quarter and one and a half times the weight of the biological sample; according to the invention, the protease can in fact be added either before, after or simultaneously with the surfactant;
(2) the addition, to said micellar or lamellar suspension S
1
obtained in (1), of a buffer B in an amount suitable for clarifying said suspension (for example by forming a microemulsion or a microsuspension), said buffer B consisting of a solvent or solvent mixture which does not solubilize the PrPres and has a dielectric constant of between 10 and 25; this gives a suspension S
2
which is limpid to the naked eye;
(3) the centrifugation of the suspension S
2
obtained in step (2); said centrifugation is carried out for example for 2 to 10 minutes at a speed below 20,000 g, preferably at a speed of between 3500 g and 17,500 g; the PrPres ends up in the centrifugation residue with a PrPres purification yield surprisingly of between 80 and 100%; advantageously the centrifugation time and speed can be adapted to give the same result, namely a PrPres purification yield of between 80 and 100%; and
(4) the solubilization of said residue in a buffer C comprising at least one surfactant, as defined in step (1), at a concentration of between 0.1% and 5%, preferably of between 0.25% and 1%, based on the volume of buffer C (w/v), and/or at least one chaotropic agent at a concentration of between 0.1 M and 8 M, at a temperature between room temperature and 100° C., preferably equal to or greater than 80° C.; under such temperature conditions, the above-mentioned surfactants, preferably ionic surfactants, and/or the chaotropic agents solubilize the PrPres.
Said steps (1) and (2) can be carried out simultaneously or successively; they are preferably carried out successively.
In one advantageous mode of carrying out said method, if the biological sample is a tissue or an organ, it is homogenized prior to step (1), for example by mechanical grinding in a homogenization buffer consisting of a neutral buffer such as water, or an isotonic buffer such as 5% glucose.
In another advantageous mode of carrying out said method, the temperature used in step (1) is between room temperature and 50° C.; it is preferably 37° C.
Buffer A preferably comprises a surfactant selected from the group consisting of:
anionic surfactants such as SDS (sodium dodecylsulfate), sarkosyl (lauroylsarcosine), sodium cholate, sodium deoxycholate or sodium taurocholate;
zwitterionic surfactants such as SB 3-10 (decyl sulfobetaine), SB 3-12 (dodecyl sulfobetaine), SB 3-14, SB 3-16 (hexadecyl sulfobetaine), CHAPS or deoxyCHAPS;
non-ionic surfactants such as C12E8 (dodecyl octaethylene glycol), Triton X100, Triton X114, Tween 20, Tween 80, MEGA 9 (nonanoylmethylglucamine), octylglucoside, LDAO (dodecyldirethylamine oxide) or NP40; or
surfactant mixtures such as a mixture of an ionic surfactant and a non-ionic surfactant, especially the mixture SDS/Tween 80 or
Commissariat a l'Energie Atomique
Morgan & Lewis & Bockius, LLP
Seharaseyon Jegatheesan
Spector Lorraine
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