U.S. patent application number 12/965697 was filed with the patent office on 2011-04-14 for prion disinfection.
This patent application is currently assigned to NOVAPHARM RESEARCH (AUSTRALIA) PTY LTD.. Invention is credited to Steven Kritzler, Alex Sava, Michael Zalunardo.
Application Number | 20110086414 12/965697 |
Document ID | / |
Family ID | 3826963 |
Filed Date | 2011-04-14 |
United States Patent
Application |
20110086414 |
Kind Code |
A1 |
Kritzler; Steven ; et
al. |
April 14, 2011 |
Prion Disinfection
Abstract
The invention relates to a methods and compositions for treating
a surface, suspension or solution contaminated with a PrP.sup.Sc
prion protein or a surrogate thereof. The methods and compositions
employ a combination of one or more enzymes effective to cleave a
prion protein to fragments having a non-infective molecular weight,
and one or more agents selected to favour conformational unfolding
of the PrP.sup.Sc prion protein while not denaturing the one or
more enzymes.
Inventors: |
Kritzler; Steven; (Cronulla,
AU) ; Sava; Alex; (Paddington, AU) ;
Zalunardo; Michael; (Koonawarra, AU) |
Assignee: |
NOVAPHARM RESEARCH (AUSTRALIA) PTY
LTD.
Rosebery
AU
|
Family ID: |
3826963 |
Appl. No.: |
12/965697 |
Filed: |
December 10, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10467591 |
Dec 22, 2003 |
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PCT/AU02/00092 |
Jan 31, 2002 |
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12965697 |
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Current U.S.
Class: |
435/219 ;
435/262; 435/264 |
Current CPC
Class: |
A61L 2/12 20130101; A61L
2/025 20130101; A61L 2/0082 20130101; A01N 63/00 20130101; A61L
2/16 20130101; A61L 2202/24 20130101; A61L 2/085 20130101 |
Class at
Publication: |
435/219 ;
435/262; 435/264 |
International
Class: |
C12S 9/00 20060101
C12S009/00; C12S 99/00 20100101 C12S099/00; C12N 9/50 20060101
C12N009/50 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 7, 2001 |
AU |
PR 2938 |
Claims
1-21. (canceled)
22. A method of disinfection comprising the steps of treating a
surface, suspension, or solution contaminated with a PrP.sup.Sc
prion protein simultaneously 5 with a combination comprising: (1)
one or more enzymes effective to cleave a PrP.sup.Sc prion protein
to fragments having a non-infective molecular weight; (2) an
anionic surfactant selected to favor conformational unfolding of
the PrP.sup.Sc prion protein while not denaturing the one or more
enzymes; and (3) one or more agents selected to promote or protect
folding of the one or more enzymes, without preventing cleavage of
the PrP.sup.Sc prion protein, and (4) wherein the conditions are
selected to favor unfolding over refolding and wherein the
combination of the enzyme, the anionic surfactant and the one or
more agents is selected so as to effectively cleave a prion
surrogate which is a protein with a high degree of sheeting.
23. A method according to claim 22 wherein the treatment is
selected so as to result after cleavage in a predetermined
percentage of the protein fragments having a molecular weight of
less than a predetermined molecular weight.
23. A method according to claim 22 wherein at least 90% of the
protein fragments after cleavage have a molecular weight of less
than 27 kDa
25. A method according to claim 22 wherein at least 90% of the
protein fragments after cleavage have a molecular weight of less
than 25 kDa.
26. A method according to claim 22 wherein at least 90% of the
protein fragments after cleavage have a molecular weight of less
than 23 kDa.
27. A method according to claim 23 or 26, wherein the conditions
are selected to protect or refold the one or more enzymes while
irreversibly unfolding or at least opening the prion sufficiently
for access by the enzyme.
28. A method according to claim 22 further including in the
combination one or more agents selected from the group consisting
of irradiation, electric field, magnetic field, energetic vibration
and combinations thereof.
29. A method according to claim 28 wherein the energetic vibration
is one or more of ultrasound, electromagnetic or mechanical
vibration.
30. A method according to claim 22 wherein the one or more enzymes
are selected from serine proteinases, aspartic acid proteinases,
metalloproteinases, keratinises, collegenases and enzymes
possessing proteolytic activity.
31. A method according to claim 22 further including in the
combination one or more agents selected to promote unfolding from
the group consisting of heat, pH, organic solvents of the kind
which tend to denature proteins, chaotropic agents, surfactants
tending to bind proteins, inorganic salts which are strong
denaturants of proteins, agents which cause S--S bond scission,
substances having a strong affinity for hydrophilic residues of
amino acids, substances having a strong affinity for hydrophobic
residues of amino acids, substances promoting adsorption on
surfaces, anionic surfactants and combinations of the
foregoing.
32. A method according to claim 22 wherein the one or more agents
selected to promote or protect folding of the one or more enzymes
is selected from the group consisting of nucleophilic solvents,
weakly protic stabilizing solvents, non ionic surfactants, ionic
surfactants, zwitterionic and amphoteric surfactants, buffers,
surface active homo-co- or block copolymers, sulphated compounds,
deoxycholate, glycosaminoglycans and combinations of the
foregoing.
33. A composition for treating a surface contaminated with a
PrP.sup.Sc prion protein or a surrogate thereof comprising: (1) one
or more enzymes effective to cleave a PrP.sup.Sc prion protein to
fragments having a non-infective molecular weight; (2) an anionic
surfactant selected to favor conformational unfolding of the
PrP.sup.Sc prion protein while not denaturing the one or more
enzymes; and (3) one or more agents selected to promote or protect
folding of the one or more enzymes, without preventing cleavage of
the PrP.sup.Sc prion protein, and (4) wherein the conditions are
selected to favor unfolding over refolding and wherein the
combination of the enzyme, the anionic surfactant and the one or
more agents is selected so as to effectively cleave a prion
surrogate which is a protein with a high degree of sheeting.
34. A composition according to claim 33 wherein the anionic
surfactant and one or more agents are selected so as to result
after cleavage in a predetermined percentage of the protein
fragments having a molecular weight of less than a predetermined
molecular weight.
35. A composition according to claim 33 wherein the anionic
surfactant and one or more agents is selected so as to result after
cleavage in at least 90% of the protein fragments after cleavage
having a molecular weight of less than 27 kDa.
36. A composition according to claim 34 wherein the anionic
surfactant and the one or more agents is selected so as to result
after cleavage in at least 90% of the protein fragments having a
molecular weight of less than 25 kDa.
37. A composition according to claim 33 wherein the anionic
surfactant and the one or more agents is selected so as to result
after cleavage in at least 90% of the protein fragments having a
molecular weight of less than 23 kDa.
38. A method of treating a surface contaminated with a PrPSc prion
protein or a surrogate thereof wherein the surface is treated with
a composition according to any one of claims 33-37.
39. A method according to claim 38 wherein the surface is the
surface of a medical or surgical instrument.
40. A method according to claim 38 wherein the surface is a food
preparation or hospital work surface.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a 371 of PCT/AU02/00092, filed Jan. 31,
2002, which claims priority to Australian Patent Application No.
PR2938, filed Feb. 7, 2001, both of which are incorporated by
reference herein in their entirety.
FEDERALLY SPONSORED RESEARCH STATEMENT
[0002] Not applicable.
REFERENCE TO MICROFICHE APPENDIX
[0003] Not applicable.
TECHNICAL FIELD
[0004] This invention relates to compositions and methods for
inactivating prions and to means for disinfecting materials
contaminated by prions or by similar conformationally altered
proteins.
BACKGROUND OF THE INVENTION
[0005] Historically, infectious agents such as bacteria, fungi,
parasites, and viroids have well established methods of control
that involve various forms of disinfection and sterilization (e.g.
steam sterilization, dry sterilization, pasteurization, sterile
filtration, treatment with ethylene oxide, glutaraldehyde, phenols
or other disinfecting chemicals, radiation, etc.). With viruses,
there are also established methods for example lowering the pH to
4.0 or below, heating at 60.degree. C. for extended periods, or use
of organic solvents in high concentrations. In addition, UV
treatment, formaldehyde and specific antiviral agents have been
employed.
[0006] For some years now, new and previously unknown species of
pathogenic agents have appeared and have been reported in
scientific publications. These have been referred to as prions and
present one of the greatest challenges facing the health care
industry today. Prions are infectious particles that differ from
bacteria and other previously known infectious agents. While there
is no firm evidence on the exact structure of prions, a number of
diseases have been identified recently both in humans and animals,
that appear to be attributable to prions. As detailed in
PCT/US00/14353 (the content of which is incorporated herein by
reference), human diseases attributed to prions include Kuru,
Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker
disease (GSS), and Fatal Familial Insomnia. (FFI).
[0007] In addition to prion diseases of humans, disorders of
animals are included in the group of known prion diseases. Scrapie
of sheep and goats is perhaps the most studied animal prion
disease. Several lines of inquiry have suggested a link between
variant CJD and a preceding epidemic of bovine spongiform
encephalopathy (BSE). No successful therapeutic treatments have
been developed and as a result these diseases are always fatal.
Adding to the problem is the fact that the incubation period can be
up to 30 years in humans and this factor presents a major challenge
to the scientists involved, with some predicting an epidemic "in
the pipeline".
[0008] Groups possibly at risk of infection include patients who
may come into contact with infected medical instruments during
surgery, medical staff dissecting infected material, and healthcare
workers responsible for cleaning and sterilizing instruments. There
are also concerns that groups at risk may be broadened to include
veterinarians, abattoir workers, butchers in contact with cows or
beef primarily in Europe and more recently persons receiving blood
transfusions or organs from donors incubating a prion disease.
[0009] The structure of prions has been the subject of intense
investigation and different points of view have been expressed.
Some scientists believe they are extremely small viruses, while
most experts now believe that prions are actually infectious
proteins without a DNA or RNA core. More particularly the consensus
now is that the PrP gene of mammals expresses a protein which can
be the soluble, non-disease, cellular form PrP.sup.c or can be an
insoluble disease form PrP.sup.sc. Many lines of evidence indicate
that prion diseases result from the transformation of the normal
cellular form into the abnormal PrP.sup.Sc form. There is no
detectable difference in the amino acid sequence of the two forms.
The PrP.sup.c form is composed of a highly membrane associated
33-35 kDa protein which degrades on digestion with protease K.
However the PrP.sup.Sc form has an altered conformational form, in
particular having a high level of .beta.-sheet conformation.
Properties of PrP.sup.Sc useful in diagnosing the infective altered
conformational form are a protease resistant core of 27-30kDa.
Another distinctive feature of the altered conformational infective
form is that it acquires a hydrophobic core.
[0010] Conventional disinfection and sterilizing agents have no
significant effect on prions in an acceptable time. Attempts to
deactivate prions and/or to disinfect surfaces on which they may be
transmitted have shown an extraordinary resistance. The conditions
required are generally too severe to be practical for routine
disinfection, not only in terms of time and cost, but also in terms
of damage to materials and occupational health hazards involved.
For example in one study infectious PrP.sup.Sc particles have been
detected in a sample after 5-15 mins/600.degree. C. dry heat
although total destruction could be achieved at 1000.degree. C. in
15 mins and in from 1-10 hrs at >200.degree. C. It has been
proposed to treat with I M. caustic soda (pH14) for 2 hrs but that
treatment is extremely corrosive, dangerous to staff, and
aggressive to materials. U.S. Pat. No. 5,633,349 describes a
procedure for treating a biological material involving treatment
with 6-8 molar urea or 1-2 molar sodium thiocyanate for a minimum
of 12 hrs (preferably 18 hrs) which suffers from similar
disadvantages.
[0011] Because of the difficulties in decontamination it has been
proposed as preferably that surgical instruments used in brain
surgery should be used only once, but this implies a disposal risk
in addition to being expensive and for some instruments
impractical. PCT/US00/14353 describes a method of rendering prions
non-infectious by use of a polycationic dendrimer but it is not
clear whether that process is reversible or permanent or
commercially viable for disinfecting surfaces.
[0012] Although attention has been focused on the PrP.sup.c form
and the PrP.sup.Sc form it has also been suggested that the protein
can exist in an intermediate form which has a .beta.-sheet content
intermediate between the predominantly alpha helix structure of the
PrP.sup.c form and the predominantly .beta.-sheet conformation of
the PrP.sup.Sc form and which retains solubility in the absence of
a denaturant.
[0013] The assembly or misassembly of normally soluble proteins
into conformationally altered insoluble proteins is thought to be
causative of, or implicated in, a variety of other diseases.
Although the invention will be herein described in relation to
prions, it will be understood to be applicable to other insoluble
or enzyme resistant conformationally altered proteins implicated in
disease.
[0014] The above discussion of prior art is not to be construed as
an admission with regard to the common general knowledge in
Australia.
SUMMARY OF THE INVENTION
[0015] An object of the invention is to provide improved, or at
least alternative, means of disinfecting a surface infected with
prions. In certain preferred embodiments, the invention renders
prions inactive more efficiently, that is to say more effectively
in a given time, or as effectively in a shorter time, than prior
art methods. Certain highly preferred embodiments of the invention
achieve better than a 4 log reduction in less than 60 mins at below
60.degree. C. In some embodiments the invention is also applicable
to prions in situations other than on surfaces for example in
suspension in a solid, liquid or gaseous medium or in biological
systems and may have other in vitro or in vivo uses. It is an
object of some embodiments of the invention to provide improved
diagnostic tools and of other embodiments to provide novel epimers
for preparation of antibodies.
[0016] The term "prion protein" as herein used includes variants,
fragments, fusions, and analogues that have other interactions or
activities that are substantially the same as those of a full
length prion protein sequence, but which may be more convenient to
use and includes all forms of secondary structure The term is also
herein used to include prion surrogates, that is to say proteins
which are not themselves prions but which have similar structure or
exhibit similar behaviour to prions and can be used to model or
predict how a prion would perform under specified conditions. The
term "PrP.sup.Sc prion protein" is intended to have a similarly
broad meaning but is limited to prion proteins which by virtue of
their secondary or tertiary structure are enzyme resistant and
includes conformations which are similarly enzyme resistant
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Not applicable.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0018] According to a first aspect the invention provides a method
of disinfection comprising the steps of treating a surface
contaminated with a PrP.sup.Sc prion protein or a surrogate thereof
simultaneously with a combination of (1) one or more enzymes
effective to cleave a prion protein to fragments having a
non-infective molecular weight, and (2) one or more agents selected
to favour conformational unfolding of the PrP.sup.Sc prion protein
while not denaturing the one or more enzymes.
[0019] According to a second aspect the invention provides a method
according to the first aspect, further including (3) one or more
agents selected to promote or protect folding of the one or more
enzymes, without preventing cleavage of the prion protein.
Preferably, the conditions are selected to favour unfolding over
refolding.
[0020] It is presently accepted that proteins having a molecular
weight of less than 27 kDa are non-infective and safe, and
accordingly the method of the invention envisages digestion or
cleavage of the prion to fragments of which at least 90% and
preferably at least 98% are less than 27 kDa, and preferably less
than 25 kDa or more preferably less than 23 kDa. However, if in the
future a protein of less than 27 kDa should be found to be
infective, the method of the invention could be utilized to
fragment the protein to fragments of any safe size.
[0021] The term "prion surrogate" as used herein is according to
the FDA definition, that is to say, proteins having a similar
resistance to proteases due to the presence of .beta.-folding. The
term "agent" is herein used to include both chemical reagents for
example anionic surfactants, reagents to modify pH, and also
non-chemical agents which effect physical and/or thermodynamic
conditions such as pressure, temperature, irradiation and other
energetic influences which promote folding, or unfolding, as the
context requires. Folding agents are sometimes referred to as
"refolding" agents. Unfolding agents are sometimes referred to as
"denaturing" agents
[0022] According to a third aspect the invention provides a method
according to the first or second aspect wherein said one or more
agents selected to favour conformational unfolding includes one or
more agents selected from the group consisting of irradiation,
electric field, magnetic field, energetic vibration and
combinations thereof.
[0023] In highly preferred embodiments of the invention a
combination of chemical and physical agents is employed, for
example the agents of step (2) include an anionic surfactant in
combination with sonication by ultrasound.
[0024] For preference the prion is subjected to sound waves in the
ultrasonic range during the treatment. However the unfolding may be
induced or aided by other forms of radiation such as microwave
radiation, radiation in the radiofrequency, infra red, visible or
U.V spectrum, sound at audible or lower frequency, energetic
vibration from mechanical means such as magnetic or vortex
stirring. Other forms of energetic input may include from electron
beam irradiation, laser irradiation, or electrolysis.
[0025] According to other aspects, the invention extends to include
compositions for use in conducting the method, to novel prion
fragments produced by the method and to novel antibodies produced
from said fragments
[0026] According to the invention a contaminated surface, for
example a surgical instrument contaminated with a PrP.sup.Sc
protein, is treated with a combination of (1) one or more enzymes
effective to cleave the prion protein into fragments of a non
infective molecular weight, (currently, less than 27 kDa), and (2)
one or more agents selected to favour conformational unfolding of
the prion protein
[0027] PrP.sup.Sc protein is characteristically resistant to attack
by enzymes including proteolytic enzymes. Without wishing to be
bound by theory, the present inventors supposed that the resistance
of PrP.sup.Sc protein to attack by enzymes is a consequence of the
folded conformation (having a high ratio of .beta.-sheet secondary
structure relative to alpha helix structure). The invention
involves the conception that it is possible to select one or more
agents so as to promote, under selected conditions, unfolding of
the PrP.sup.Sc protein sufficiently for an enzyme to gain access
and cleave PrP.sup.Sc protein.
[0028] Many proteins are prone to loose their natural three
dimensional folding pattern ("secondary and tertiary structure")
and to become "denatured". The denaturation includes breakdown of
the intramolecular interaction, especially hydrogen and disulphide
bonds, and thus the loss of the secondary structure which virtually
all native proteins have in at least parts of the molecule, and
which generally is decisively responsible for the activity of the
protein.
[0029] Those skilled in the art appreciate that enzymes are
themselves proteins and tend to be readily denatured by agents
which promote protein unfolding. It is not clear whether that is
because the unfolding agent binds to the enzyme, preventing the
enzyme from binding to a target substrate, or more likely because
the unfolding agent promotes unfolding of the enzymes
conformational structure, rendering it inactive or "denatured" or a
combination of those effects. PrP.sup.Sc protein on the other hand
is highly resistant to unfolding. It has hitherto been considered
impossible to formulate a system in which an enzyme retains
activity in the presence of an unfolding agent effective to
influence such an intractable protein as PrP.sup.Sc. Surprisingly
the present inventors have found that either (i) certain unfolding
agents selectively unfold or relax the PrP.sup.Sc protein while not
unfolding (denaturing) a selected enzyme or (ii) that folding and
unfolding agents can be combined in such a way that the folding
agent selectively promotes or retains enzyme activity, while the
unfolding agent selectively and sufficiently unfolds the prion to
provide access to the enzyme for prion scission.
[0030] These intrinsically conflicting desiderata are met in the
present invention by classifying agents as promoting "folding" or
"unfolding" and then determining their relative effect on enzymes
and on PrP.sup.Sc protein or a surrogate thereof
[0031] The surface may be first treated with the one or more agents
and the one or more enzymes may be added subsequently but in
preferred embodiments the surface undergoing treatment is subjected
to both simultaneously.
[0032] The enzyme is preferably a proteolytic enzyme. Suitable
enzymes are: [0033] non-specific proteinases:--e.g. serine-,
aspartic-, metalloproteinases [0034] more specific
proteinases--e.g. keratinases, collagenases etc [0035] any other
enzyme(s) that posses proteolytic activity
[0036] The one or more agents selected to favour conformational
unfolding of the prion protein are chosen to be effective to
provider access by the enzyme to the prion protein. In general the
folding-unfolding of a polypeptide chain may be a thermodynamically
reversible equilibrium process or may be irreversible.
[0037] By way of example only, agents which promote unfolding
(denaturation) include: [0038] (1) heat--increases in temperature
up to about 150.degree. C. [0039] (2) pH--values below 3 and above
9 (global effect resulting from ionization of many side chain
residues accessible to the solvent) or in some molecules may be
attributable to local effects due to ionization of specific groups
(e.g. serine proteases due ionization of N-terminal amino group of
the carboxylate) [0040] (3) Selected organic solvents of a kind
which tend to denature, dissolve or swell proteins. Generally the
products are not completely unfolded and possess an ordered
conformation which differs from the native state. Solvents which
favour helical conformations (i.e. unfolding) are exemplified by
N-dimethylformamide, formamide, m-cresol, dioxan, CHCl.sub.3,
pyridine, dichlorethylene, and 2-chloroethanol. This group also
includes solvents which have a weak tendency to form hydrogen bonds
such as the alcohols, ethanol, n-propanol, methanol (especially in
mixture with 0.01% HCl), Also, solvents which tend to disorganize
the structure e.g. dimethylsulphoxide (DMSO) at high
concentrations, dichloroacetic acid and trifluoroacetic acid, and
other electrophillic solvents [0041] (4) Certain organic solutes
and chaotropic agents.--Such as urea, guanidine hydrochloride
(GuHCl). The transition to randomly coiled polypeptide is complete
for 6-8M GuHCl at room temp except for some exceptionally stable
proteins. These agents may be markedly influenced by temperature,
pH other reagents and to conditions. [0042] (5) Certain
surfactants--Ionic surfactants tend to bind to proteins and
initiate unfolding of tertiary structure. Anionic detergents are
very strong denaturants. E.g. Sodium dodecyl sulfate (SDS) is able
to completely unfold many (but not all) proteins at concentrations
close to the critical micelle concentration. Dodecyl benzene
sulfonate is also a denaturant. The detergents do not necessarily
result in a complete unfolding since in some cases it appears that
the hydrophobic part of the detergent might interact with the
ordered structure of the protein to form micellar regions. Cationic
surfactants are usually less effective unfolding agents than
anionic. Dodecyl ethoxy sulfates tendency to denature bovine serum
albumin (BSA) decreases with increasing ethoxy groups and
disappears for ethoxy greater than six [0043] (6) Inorganic salts
can induce conformational transitions in proteins. For example
LiBr, CaCl.sub.2, KSCN, NaI, NaBr, sodium azide are strong
denaturants. Although these salts do not necessarily lead to
completely unfolded protein, the residual ordered structure may be
disrupted by energy input e.g. increasing temperature. Anions such
as
CNS.sup.->I.sup.->Br.sup.->NO.sub.3.sup.->Cl.sup.->CH.sub.-
3COO.sup.->SO.sub.4.sup.- exhibit similar behaviour as do
guanidinium salts and tetraalkyl ammonium salts However
(GuH).sub.2SO.sub.4 has been observed to protect certain proteins
against denaturation. [0044] (7) agents which cause scission of the
S--S bond such as thioglycols [0045] (8) Other substances with
strong affinity to either hydrophilic or hydrophobic residues of
amino acids [0046] (9) Adsorption on certain surfaces and
interfaces including zeolites, including air/liquid interfaces.
These include, for example, but are not limited to: finely divided
alumina, silicas and other chromatographic and stationary phases.
[0047] (10) Ultrasonic energy, Infra red and microwave radiation,
high pressure, and subjecting protons to the action of electric
and/or magnetic fields may be able to promote unfolding
(refolding), and even shaking or stirring may be influential.
[0048] In preferred forms of the invention a combination of agents
is used for example a surfactant and/or a suitable solvent with
ultrasound is employed. It is unclear whether the input of energy,
such as from ultrasound, assists in driving the folding/unfolding
equilibrium in favour of unfolding of the PrP.sup.Sc protein at a
greater rate than it does in denaturing the enzyme, or whether it
merely assists in providing access of the reagents or enzymes to
the prion, or whether it is effective in activating the enzyme.
Other methods of applying energy include application of sound waves
in the sub-sonic range. However energetic vibration may be induced
by other forms of electromechanical radiation or energetic
vibration from mechanical means such as magnetic or vortex
stirring. Other forms of energetic input may include from electron
beam irradiation, laser, or electrolysis.
[0049] As indicated above, most of the discussed unfolding agents
would be expected effectively to denature the enzyme. Either the
unfolding agent and its conditions of use must be carefully
selected so as to permit digestion of the PrP.sup.Sc protein or its
surrogate without denaturing the enzyme, or alternatively the
unfolding agent must be combined with a folding agent.
[0050] Suitable folding agents include: [0051] (1) Nucleophillic
solvents and highly hydrogen bonded organic solvents. There is
competition between the energy of the peptide hydrogen bonds and
the strength of hydrogen bonds between solvent molecules. When
solvent molecules are linked by strong hydrogen bonds the
equilibrium is shifted to towards stabilization of peptide hydrogen
bonds. Solvents such as dioxan, acetonitrile, dimethylformamide,
pyridine, and, at low concentrations, dimethylsulphoxide(DMSO)
which are good proton acceptors but weak proton donors have a very
weak tendency to disrupt peptide hydrogen bonds and tend to induce
ordered conformation, especially in globular proteins. [0052] (2)
Stabilizing solvents such as polyhydric alcohols (e.g. glycerol,
ethylene glycol, and propylene glycol, sucrose and the like) which
are weakly protic and in the presence of which proteins tend to
remain conformationally stable and may be used as stabilizing
agents. [0053] (3) Non-ionic surfactants such as alkyl, phenyl or
alkyl ethoxylates, propoxylates or copolymers thereof,
alkylpolyglucosides etc, sarcosinates (e.g. sodium-
(N-lauroyl)sarcosinate) do not alter the tertiary structure of
protein and any unfolding occurs in the region of the isotherm
where a significant increase in surfactant binding by non-specific
cooperative interaction begins The effects of SDS can be reduced by
addition of non-ionic or amphoteric surfactants [0054] (4)
Zwitterionic and amphoteric surfactants, [0055] (5) High
concentrations of buffers, (e.g. phosphates, acetates, citrates,
borates) [0056] (6) Surface active homo-, co-, or block-polymers
which contain weakly hydrophobic and weakly hydrophilic zones in
alternating arrangement, [0057] (7) Protective agents such as
sulphated compounds, deoxycholate, glycosaminoglycans
[0058] If a folding agent is combined with an unfolding
(denaturing) agent then the agents and conditions must be selected
to protect or refold the enzyme while irreversibly unfolding or at
least opening the prion sufficiently for access by the enzyme. For
example, the pH and/or temperature may be selected so that the
folding agent acts selectively on the enzyme while the unfolding
agent acts selectively on the PrP.sup.Sc protein.
[0059] Bovine albumin with high globulin content (Sigma product
A7906), beta-galactosidase (G7279) and rabbit muscle myosin
(M0163)) were used as models of proteins with low-solubility and
high beta-sheet content. The molecular weight of the above proteins
are significantly larger than that of the prions. The identifying
of molecular mass of peptide fragments after enzymatic digest is
easier, as most of proteases used in the experiment have molecular
mass (20-35 KDa) similar to prions.
[0060] SDS-PAGE was conducted using the method described by Laemmli
U.K., Nature, 227, pages 680-685,(1970). The protein solution was
boiled for 2 min in sample buffer containing 2% SDS. 1.5 mm
polyacrylamide slab gels (8-12%) were loaded with 10 microlitres of
the protein per lane, and subjected to non-reducing conditions (ie.
no beta-mercaptoethanol in sample buffer). Electrophoresis was
performed for 1 hour at 150 V, until the dye front was at the
bottom of the gel. The gel was then removed and the protein bands
visualized by staining either with Coomassie brilliant blue R-250
(Sigma) or silver stained (Bio-Rad). The molecular mass value for
proteins was determined by using calibrating curve obtained with
prestained low molecular weight markers (Bio-Rad marker
161-0318).
[0061] Cleaving the proteins was considered sufficient when no
fragments with molecular mass larger than 14.4 KDa (the molecular
weight of lysozyme) were detected after the combined action of
unfolding agents and enzymes. That indicates that the treatment was
effective on the surrogate.
[0062] When peptide fragments with molecular mass of larger than
14.4 KDa were present, the results was reported as positive
[0063] To prove that the method of the invention may be used to
deactivate prions, and not merely cleave the surrogate, the prion
detection test developed by Prionics AG was employed.
[0064] The "Prionics-Check" is an immunological test for the
detection of prions in animal tissues that use a novel antibody
developed by PRIONICS AG. The PrP.sup.Sc remaining in the reaction
mixture is bound by the antibody and detected using an enzyme
coupled to the antibody.
[0065] 100 mL aliquots of the solutions as described in table 1
were spiked with approx. 1 microgram of recombinant prion protein
and the Prionics Check was performed as per the procedure described
in Appendix 1.
[0066] When PrP.sup.Sc was detected after the enzymatic digest, the
results were reported as positive. Table 1 shows that in control
experiments 1-1 to 6-1 fragments having a mass greater than 14.4
KDa were detected as was PrP.sup.Sc. However in experiments 1-2 to
6-2 no fragments were detected with a mass greater than 14.4 KDa
and no PrP.sup.Sc was detected. 1-1 differs from 1-2 by inclusion
of an unfolding agent (3% DOBS) shown to be effective in 30 min at
70.degree. C.
[0067] 2-2 and 3-2 differ from 2-1 and 3-1 respectively by
inclusion of sonication. In 2-2 an unfolding agent 3% DOBS in
combination with 25% Terric 164 (a folding agent) was effective at
25.degree. C. with sonication at 40 kHz In 3-2 a similar result was
obtained with 10% SDS as unfolding agent and a zwitterionic
surfactant as folding agent at 2.6 mHz.
[0068] 4-2 differs from 4-1 by the combination of borax with SDS at
a more elevated temperature.
[0069] 5-2 differs from 5-1 by combining DOBS with Triton X-100 in
the absence of boron
[0070] 6-2 differs from 6-1 by increasing the concentration of DMSO
from reversibly unfolding (0.05%) to irreversibly unfolding
(0.5%)
[0071] As will be apparent to those skilled in the art from the
teaching hereof the invention may be performed using other
combinations of agents without departing from the inventive concept
herein taught.
TABLE-US-00001 TABLE 1 SDS- SDS- PAGE SDS- PAGE beta- PAGE Prionics
No. Test procedure/solutions Albumin galactosidase myosin Check
1-1. Distilled water + + + + Warm to 70 C. Keep in water bath at 70
C. for 30 min, 15 units protease activity per mL, pH 9 Cool down to
25 C. 1-2. 3% DOBS and Distilled water diluted - - - - 1:100 Warm
to 70 C. Keep in water bath at 70 C. for 30 min, 15 units protease
activity per mL, pH 9 Cool down to 25 C. 2-1. 3% DOBS, 25% Teric
164, + + + + diluted 1:100 15 units protease activity per mL pH 9
25 C. for 30 min 2-2 3% DOBS, 25% Teric 164 diluted - - - - 1:100
15 units protease activity per mL sonicated with 40 kHz ultrasound
25 C. for 30 min 3-1 10% SDS 10% Empigen + + + + BS/AU
(zwitterionic surfactant) diluted 1:100 15 units protease activity
per mL pH 9 25 C. for 30 min 3-2 10% SDS 10% Empigen - - - - BS/AU
(zwitterionic surfactant) diluted 1:100 15 units protease activity
per mL pH 9 25 C. sonicated 2.6 mHz for 30 min 4-1 10% SDS, 4%
borax diluted 1:100 + + + + 15 units protease activity per mL pH 9
25 C. for 30 min 4-2 10% SDS, 4% borax diluted 1:100 - - - - 15
units protease activity per mL pH 9 55 C. for 30 min 5-1 15% DOBS,
5% Triton X100 4% + + + + Borax diluted 1:100 15 units protease
activity per mL pH 9 25 C. for 30 min 5-2 15% DOBS, 5% Triton X100
diluted - - - - 1:100 15 units protease activity per mL pH 9 25 C.
for 30 min 6-1 .05% DMSO + + + + 15 units protease activity per mL
pH 9 25 C. for 30 min 6-2 .5% DMSO - - - - 15 units protease
activity per mL pH 9 25 C. for 30 min DOBS = dodecyl benzene
sulfonic acid (Sigma Product No. D2525) DMSO = dimethylsulfoxide
(Sigma Product No. D5879) Protease = Subtilisin Carlsberg (Sigma
Product No. P5380) Sonication at 40 kHz performed using ultrasonic
bath supplied by UNISONICS Pty Ltd. Sonication at 2.6 mHz performed
using Disonics Pty Ltd. ultrasonic nebulizer.
Appendix 1
Prionics Check Test Method
[0072] The protocol below outlines using the protease resistant
core of PrP.sup.Sc, from a recombinant source known to be
representative of the naturally occurring infectious agent. It has
been proven experimentally that the protease resistant core of the
prion is not infectious, but indicates the presence of infectious
agents [0073] 1. Weigh 1 microgram of PrP.sup.sc or B SE infected
animal brain homogenate that contains 1 mcg or PrP.sup.Sc and
reconstitute it in 1 ml of deionised water [0074] 2. Add to 10 ml
of test solution and subject to appropriate deactivation protocol
[0075] 3. Take 10 microlitre aliquot of the test solution and add
it to 10 microlitres of sample buffer [0076] 4. Perform SDS-PAGE of
[0077] untreated PrP.sup.sc solution used for spiking (positive
control) [0078] solution under study
[0079] All proteins or protein fragments are separated in an
electric field according to their size. The small proteins migrate
faster than the large proteins. After a period of time the smallest
fragments of the decomposed prion proteins migrate out of the gel
while the resistant PrP.sup.Sc fragments will be present in the
lower half of the gel. In the control sample where the prion
protein remains resistant to protease, non-cleaved PrP.sup.Sc
molecules will remain higher up in the gel. [0080] 1. Transfer
proteins from the gel to nitrocellulose membrane by Western
blotting. [0081] 2. Add monoclonal antibodies (Prionics Product No.
01-020). [0082] 3. Allow to bind to the proteins and then wash away
non-bound antibodies. [0083] 4. The horseradish-peroxidase
conjugated to primary antibodies is allowed to react with a
chemoluminescence substrate (ECL product No. RPN 2209 supplied by
AMERSHAM Life Science) [0084] 5. Expose the membrane to X-Ray film,
develop the film. [0085] 6. Assess whether prion protein are
present. Report results as positive when the antibodies are
retained at the position corresponding to molecular mass of prion
protein.
* * * * *