WO2006065897A2

WO2006065897A2 - Device for the detection of prions

Info

Publication number: WO2006065897A2
Application number: PCT/US2005/045229
Authority: WO
Inventors: Chester F. King
Original assignee: King Chester F
Priority date: 2004-12-14
Filing date: 2005-12-14
Publication date: 2006-06-22
Also published as: WO2006065897A3

Abstract

A device capable of obtaining a sample from an animal or human and processing the sample to detect a molecule of interest. The device comprises a two-sided needle, a sample collection tube that interconnects with a second tube containing processing agents via a compatible threaded coupling present on each tube. A compatible detection tube is provided. The present invention also relates to methods detecting defective prions in a sample obtained from an animal or a human by obtaining a sample, contacting the sample with proteinase K/cell lysing solution to form a mixture that will digest extraneous proteins in the sample but leave defective prions that may be in the sample unaffected, adding a monoclonal antibody specific for defective prions to the mixture, and detecting defective prions. The present invention also relates to method of detecting BSE in an animal prior to destruction of the animal comprising detecting defective prions as described above where the presence of defective prion proteins indicates the presence of BSE.

Description

TITLE: DEVICE FOR THE DETECTION OF PRIONS

INVENTORS: Chester F. King

7 Scotch Trail Fairfield PA 17320

USA

SPECIFICATION

TO ALL WHOM IT MAY CONCERN:

BE IT KNOWN THAT Chester F. King, a citizen of the United States of America, invented new and useful improvements for a device useful the detection of prions and other biological molecules as described in this specification:

We have designed a device useful for analysis of samples. The device is modular in design, and its discrete components make it versatile in the analysis of any type of sample. The device is useful for analysis of blood, urine, lymph, spinal fluid, other fluid or tissue from humans or animals. The device is useful for the detection of biological material, such as proteins, nucleic acids or prions.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a device for use in connection with analysis of samples. The device has particular utility in connection with analysis of biological materials. More particularly, the device has particular utility in connection with detection of defective prions.

Description of the Prior Art

The term prion describes an atypical infectious particle that is smaller than viruses and viroids. The currently held belief is that they are actually defective protein particles that accumulate in the brain due to their resistance to the normal protein regulatory pathways. Prions have been known to cause transmissible spongiform encephalopathies (TSE), characterized by a group of infectious neurodegenerative diseases such as scrapie in sheep, Creutzfeldt- Jakob disease (CJD) and kuru in humans, chronic wasting disease in cervids, bovine spongiform encephalopathy (BSE) or "Mad Cow" in cattle. The prion protein (PrP) can exist in two forms. One form, PrPc, is a normal, cellular structure that can be readily digested by proteases. A second form of the protein is commonly referred to as PrPsc or scrapie prion protein and is resistant to digestion by proteases. Other than the difference in structure between the two proteins, they seem identical chemically.

Defective prions are believed to be the primary reason for BSE. BSE is particularly relevant to humans because the disease is transmissible to humans. People potentially exposed to the disease cannot give blood or be an organ donor or receiver.

There are no rapid screening tests presently available for BSE or defective prions and most require a brain biopsy, extended turn-around times, and specialized laboratory analysis. These factors are the primary reasons there is not a test available to test feed supplied to the beef industry.

In addition to the possibility of transmission of TSEs to humans from the food chain as described above, there is particular interest in prions as the potential causative agent behind several other disease states for humans. For example, the molecular event of protein folding is central to both prion associated disorders and Alzheimer's disease. Understanding, detecting and eventually preventing abnormal folding of proteins is of prime importance for humans in prion associated disorders and neurodegenerative diseases such as Alzheimer's disease and Huntington's disease.

Therefore, a need exists for a new and unproved device, which can be used for the analysis of minimally invasive patient samples, particularly for detecting alternately folded proteins which may lead to disease. In this regard, the present invention substantially fulfills this need. The test of the present invention provides a low cost, easy to use blood-screening test for defective proteins in cattle, the causative agent for mad cow disease. The present invention allows for more cattle to be tested, testing prior to transport of animal, and early detection. The tests of the present invention do not require destruction of the animal, required hours not days for the results and can be used in the field. In this respect, the device according to the present invention substantially departs from the conventional concepts and designs of the prior art, and in doing so provides an apparatus primarily developed for the purpose of analysis of samples. Preferably the device according to the present invention is useful for providing a blood screening tests for prions in cattle but can be used to detect any other biologically relevant material.

SUMMARY OF THE INVENTION

The present invention provides an improved device, and overcomes the disadvantages and drawbacks of the prior art. As such, the general purpose of the present invention, which will be described subsequently in greater detail, is to provide a new and improved device and method of detecting prions. This device has all the advantages of the prior art and many novel features that result in a device and method of detecting prions which is not anticipated, rendered obvious, suggested, or even implied by the prior art, either alone or in any combination thereof. The present invention essentially comprises a device capable of obtaining a sample from an animal or human and processing the sample to detect an agent of interest that may be present in the sample. The device itself generally comprises a two-sided needle, where one side pierces the vein of an animal or human and the other side pierces the sterile septum of a tube which contains a vacuum thus permitting a sample to be collected hi a sample collection tube. The sample collection tube may interconnect with a second tube via a compatible threaded coupling present on each tube. The second tube may contain agents to process the sample. The device additionally includes a detection tube with a threaded coupling similar to that of the sample collection tube so that the detection tube may be connected to the second tube. Because the detection tube contains the means necessary, a molecule of interest that may be present in the sample may be detected. The present invention can not only detect, but quantitate the agent to provide a quantitative and/or a qualitative assay. The present invention also relates to methods detecting defective prions in a sample obtained from an animal. More specifically, comprising the steps of obtaining a sample from the animal, contacting the sample with proteinase K/cell lysing solution to form a mixture that will digest extraneous proteins in the sample leaving prions intact, contacting the mixture with a inactivating agent such as diisopropyl fluorophosphate (DFP) or phenyl methane sulphonyl fluoride (PMSF) to stop digestion, adding a monoclonal antibody specific for defective prions to the mixture, and finally detecting the presence of defective prions in the animal by detecting the presence of a monoclonal antibody/defective prion complex.

The present invention also relates to a method of detecting BSE in an animal prior to destruction of the animal comprising detecting defective prions in a sample as described above where the presence of defective prion proteins indicates the presence of BSE in the animal. The present invention also relates to methods detecting defective prions in a sample obtained from a human comprising the steps of obtaining a sample from the human, contacting the sample with proteinase K/cell lysing solution to form a mixture that will digest extraneous proteins hi the sample but leave defective prions that may be in the sample unaffected, contacting the mixture with a inactivating agent such as diisopropyl fluorophosphates (DFP) or phenyl methane sulphonyl fluoride (PMSF), adding a monoclonal antibody specific for defective prions to the mixture, and finally detecting the presence of defective prions hi the human by detecting the presence of a monoclonal antibody/defective prion complex. There has thus been outlined, rather broadly, the more important features of the invention 85 in order that the detailed description thereof that follows may be better understood and in order that the present contribution to the art may be better appreciated.

The invention may also include numerous variations in that the device of the present invention is capable of detection of any type of molecule for which a detection system is available. The methods of the present invention can detect the protein of interest in both it's 90 normal and defective (prion) form. Numerous types of detection methods can be used with the device because different types of detection tubes may be provided. The methods of the present invention can be used to detect defective prions in cattle, sheep, goats, deer, elk, cats, mink, zoologic bovids, primates, mammals or other species know to be susceptible to TSEs. There are, of course, additional features of the invention that will be described hereinafter and which will 95 form the subject matter of the claims attached.

Numerous objects, features and advantages of the present invention will be readily apparent to those of ordinary skill in the art upon a reading of the following detailed description of presently preferred, but nonetheless illustrative, embodiments of the present invention when taken in conjunction with the accompanying drawings. In this respect, before explaining the current 100 embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of descriptions and 105 should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions 110 insofar as they do not depart from the spirit and scope of the present invention.

It is another object of the present invention to provide a new and improved device that may be easily and efficiently manufactured and marketed.

An even further object of the present invention is to provide a new and improved device that has a low cost of manufacture with regard to both materials and labor, and which 115 accordingly is then susceptible of low prices of sale to the consuming public, thereby making such device economically available to the buying public.

Even still another object of the present invention is to provide a method of detecting BSE in cattle that involved a simple blood test to be used in the field prior to sale or slaughter.

Even still another object of the present invention is to meet the unmet need for an easy to 120 use, low cost screening test for defective prions in animals and humans.

Still yet another object of the present invention is to provide a device for a wide variety of different diagnostic systems. Each module of the device acts like a small, self- contained lab that processes the blood, fluid or tissue with specific reagent or reagents. The device therefore limits the exposure of the technician to the sample and greatly reduces the 125 possibility of technician reagent error as well.

Lastly, it is an object of the present invention to provide a new and improved device and method of use that permits testing of an infectious agent while limiting the contact of the user with the sample suspected of containing an infectious agent.

These together with other objects of the invention, along with the various features of 130 novelty that characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there is illustrated preferred embodiments of the invention. 135

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings wherein:

140 Figure 1 is a perspective view of the preferred embodiment of the device constructed in accordance with the principles of the present invention.

Figure 2 is a perspective view of the additional sections compatible with the device of the present invention.

Figure 3 A is a cut-away view of the device of the present invention. 145 Figure 3B is a cut-away view of a detection tube compatible with the device of the present invention.

The same reference numerals refer to the same parts throughout the various figures. DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now to the drawings, and particularly to FIGS. 1 -3, a preferred embodiment of the

150 device of the present invention is shown.

As shown in Figure 1 and the cut away view of Figure 3, a new and improved device of the present invention for detection of molecules in a sample is illustrated and will be described. The device comprises a two-sided needle, where one side pierces the vein of the subject and the other side pierces the sterile septum of a tube, which contains a vacuum, thus allowing a sample

155 to be taken. The device also comprises a sample collection tube having a sterile septum at one end of the tube, and at the other end of the tube a threaded coupling to open only when twisted. The sample collection tube should also have a mark to confirm that the volume of sample taken is sufficient for analysis. A vacutainer style housing for the needle, sterile septum, and sample collection tube is provided. The sample collection tube may be connected through the threaded

160 coupling to a second tube with a compatible threaded coupling. Preferably the contents of each tube will only mix when the threaded coupling is twisted to open a conduit between the two tubes. The second tube may contain materials need to process the collected sample. The second tube may also contain, within the interior walls of the tube a separate container. The separate container may hold molecules, a solution or an agent needed at some point in the sample

165 processing. The separate container may be broken and the device shaken to allow the contents of the second tube and the separate container to mix. In a preferred embodiment, the second section contains proteinase K, and the separate container within the second tube contains an inactivation agent such as diisopropyl fluorophosphate (DFP) or phenyl methane sulphonyl fluoride (PMSF). As shown in Figure 2, numerous additional cartridges, or tubes, may be

170 provided. Each cartridge may contain additional reactant or reagent materials, buffers, filters, or beads, detection means, stop solutions, or other solutions as needed to process and analyze the collected sample. Each cartridge could contain at each end, threaded couplings compatible with the other tube sections of the device as shown in Figuresl-2. Alternatively, the cartridges may be threaded at only one end as shown in Figure 3B. The additional cartridges could be connected

175 to the second tube after the device is mixed and turned to that the sample mixture is located in the second tube and the sample collection tube and needle can be removed without sample loss. Alternatively, the cartridges could be connected in series. The second tube may contain threaded couplings at each end so that additional cartridges could be added to each end of the second tube or the second tube may contain threaded coupling at one end and different cartridges, or tubes,

180 could be removed and connected to the second tube as necessary to carry out a desired method.

The device of the present invention maybe coupled with any sample collection system that permits sterile sample collection in place of the needle described above. The needle arrangement of the device may be modified for optimal collection of a different type of sample. The sections of the device may be made of any appropriate material. If biological materials are

185 to be analyzed, preferably the sections of the device are made of material to which those biological materials will not generally adhere. The dimensions of the device are any suitable to optimal performance of the device. For example, the detection tube of the device may contain a window or be made of a material suitable to be read by a spectrometer if a spectrometer appropriate detection means is used to detect a molecule in the sample after processing.

190 The present invention comprises a modular testing device in which each stage is either contained in a breakable container or cartridge within the device. Alternatively, the sample can be connected to the reagents in the next stage of processing through a threaded or vacuum coupling, which is sealed prior to use.

In use, the device of the present invention as described above could be used to test blood

195 or CSF from an animal for the detection of prion proteins associated with BSE.

Firstly, blood or cerebral spinal fluid (CSF) sample is drawn into a tube such as a vacutainer format: a two sided needle is used to pierce the vein of the subject animal while the other side is used to pierce the sterile septum of a tube which contains a vacuum, and potentially, a solution of 0.85% ammonium chloride or weak citric acid solution depending on the format of

200 the test (blood vs. CSF) and which reagent yields better test results. In this manner, the tube should filled to an approximately defined volume. The ammonium chloride will lyse all of the red blood cells in the sample leaving the target white blood cells available to continue the test. Once the sample has been drawn, the needle assembly can be safely discarded.

Secondly, once the draw volume has been confirmed to exceed the minimum volume

205 required to proceed with the test, it will be mixed with the next reagent in the test by the tester piercing another septum to a tube of the device that contains a proteinase K / cell Iy sing solution. Although the basic format may vary, in one variation the tester will twist the device so as to connect the two tubes through a sealable threaded connection or piercing a separate septum within the device. The second tube will contain a sample processing agent such as proteinase K.

210 Also contained in this tube will be a separate container which contains a measured amount of either diisopropyl fluorophosphate (DFP), phenyl methane sulphonyl fluoride (PMSF), or other proteinase K inactivating agent. Once the digestion has run to completion, the reagents may be mixed by breaking the internal container and shaking as in a fluorescent glow stick.

Thirdly, once the proteinase K has digested the extraneous proteins sufficiently and been

215 inactivated, the protein of interest otherwise known as the defective prion form, should remain relatively unaffected and available for detection using a monoclonal antibody (mAb) format.

Finally, monoclonal antibodies can be introduced as either part of the next test solution, or they may be attached to a solid phase such as a bead or the wall of the tube itself that can be inserted into the digested solution. In any case, the detection format cartridge will be introduced

220 to the sample through the threaded coupling. The sample collection and second tubes will be separated at the threaded coupling and the detection cartridge threaded into place. There are several detection formats available to be chosen based on performance and ease of use. This format will also allow a certain amount of flexibility in the detection method based on available equipment, sensitivity desired, or other parameters. In either case, specific mAbs will bind to the

225 protein of interest.

The device of the present invention offers a wide variety of various tubes, or cartridges, for use with different technologies that can be selected to detect the monoclonal antibodies. One example of a detection technology is fluorescent detection, wherein a tube is coated with specific protein and fluor-labeled antibodies are in solution. In use the tube is coated with a

230 protein known to bind to the defective prion. Monoclonal antibodies with fluorescent tags are used to detect if the prion is bound. After the sample is added and allowed to equilibrate, the fluid is poured off and a buffer added (a buffer cartridge which threads into place is possible if necessary), finally the sample is read in a standard test appropriate for the fluor used. This method offers the advantages of being relatively easy and straight forward and requiring no

235 decanting.

Another example of a detection technology is magnetic detection, wherein a tube contains magnetic beads coated with a protein known to bind to the defective prion. In use the defective prion proteins bind to magnetic beads, the magnetic beads are pulled from the solution using a magnetic field while solution is removed and replaced with detection buffer and the

240 beads are detected using fluorescently labeled antibodies as described above for the fluorescent detection method. This method offers the advantages of allowing concentration of sample for increased sensitivity because the replacement volume of detection buffer can be reduced to increase signal per volume of fluid.

Another example of a detection technology is a bead test in which a tube contains beads

245 coated with a specific protein known to bind to the defective prion and one half of a two part detection system such as alpha screen donor beads (Packard). The other part of the detection system, or the acceptor beads, would be conjugated to the antibody to the prion. The signal would only be generated when the two are in close proximity. In use the test solution is added to the tube containing the conjugated beads and antibody. The protein would bind to both the beads

250 and the antibody causing them to come together. Positive results would be determined by the amount of emissions after the sample is hit with a specific wavelength laser. This method offers the advantages of being very sensitive.

Another example of a detection technology is a solid phase test or modified ELISA format. A sample tube has a membrane on the top that has been coated with protein known to

255 bind to the target prion. This protein will capture prions as they flow across the membrane which can be detected using the Ab conjugate - (HRP, Alkaline Phosphatase, fluor, etc.). In use, the detection cartridge containing membrane is threaded onto PK cartridge either gravity or vacuum assist will cause fluid to flow across membrane allowing proteins to bind fluid can be discarded and proteins captured on membrane detected using appropriate means. Alternatively,

260 membrane may be small enough to be suspended in tube. This method offers the advantages of being very simple, that the membrane can be removed and washed with standard wash buffer using "squirt bottle", that a second threaded cartridge containing the detection substrate can be used if desired and colormetric results rather then fluorescent results with the level of color being proportional to level of prions detected.

265 Another example of a detection technology is an agglutination test wherein a tube contains two bead populations - one coated with the protein known to bind to the defective prion and one coated with the antibody to the prion. In use, the test solution is added to the tube containing the beads, the protein would bind to both types of beads causing them to clump together. The test could be read fairly subjectively as a "clump" or not similar to blood typing

270 tests currently in use. This method offers the advantages of being very simple.

In yet another example of the technology, a metal such as copper or zinc may be used to tag the prion. An electrical field may be used to detect and/or separate the tagged prions.

The present invention also envisions methods of detecting BSE in an animal prior to destruction of the animal comprising detecting defective prions in a sample as described above

275 wherein the presence of defective prions is indicative of BSE in the animal. This method offers several advantages over existing methods of detecting BSE. The present method can be performed on an animal prior to slaughter using blood or other easily obtained sample. Existing tests require that animal be slaughtered to obtain a sample for testing. The present test can be performed by a technician in the field and does not require a laboratory. The results of the

280 present test are obtained very quickly. In other tests, days are needed to obtain results.

The present invention also relates to methods detecting defective prions in a sample obtained from a human comprising the steps of obtaining a sample from the human, contacting the sample with proteinase K/cell lysing solution to form a mixture that will digest extraneous proteins hi the sample but leave defective prions that may be in the sample unaffected, contacting

285 the mixture with a inactivating agent such as diisopropyl fluorophosphates (DFP) or phenyl methane sulphonyl fluoride (PMSF), adding a monoclonal antibody to defective prions to the mixture, and finally detecting the presence of defective prions in the human by detecting the presence of a monoclonal antibody/defective prion complex.

While a preferred embodiment of the device and method of detecting defective prions has

290 been described in detail, it should be apparent that modifications and variations thereto are possible, all of which fall within the true spirit and scope of the invention. With respect to the above description then, it is to be realized that the optimum dimensional relationships for the parts of the invention, to include variations in size, materials, shape, form, function and manner of operation, assembly and use, are deemed readily apparent and obvious to one skilled in the art,

295 and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention. And although the device has been described for detecting defective prions, it should be appreciated that the device herein described is also suitable for detecting any biological molecules. Therefore, the foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to Limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.

Claims

CLAIMS305 I/We claim:

1. A device for collection and analysis of a molecule of interest in a sample obtained from a subject, comprising: a two-sided needle, where one side pierces the vein of the subject and the other side pierces the sterile septum of a tube which contains a vacuum;

310 a sample collection tube containing a mark to confirm volume of sample collected, a sterile septum at one end of the tube, and at the other end of the tube a threaded coupling to open only when twisted or pierced; a housing for the needle, sterile septum and a sample collection tube; a second tube containing agents to process the sample, the second tube optionally 315 containing, within its interior, a separate container for another agent, the second tube having at one end a threaded coupling compatible with the sample collection tube thread; a detection tube containing at one end a threaded coupling similar to that of the sample collection tube so that the threaded coupling is compatible with the second tube and containing within the tube detection molecules for detecting a molecule of interest in the sample. 320 2. A method of detecting defective prions in a sample obtained from an animal comprising: obtaining a sample of blood or CSF from the animal; contacting the sample with proteinase K/cell lysing solution to form a mixture that will digest extraneous proteins in the sample but leave defective prions that may be in the sample 325 unaffected; contacting the mixture with a inactivating agent such as diisopropyl fluorophosphates (DFP) or phenyl methane sulphonyl fluoride (PMSF); adding a monoclonal antibody to defective prions to the mixture; detecting the presence of defective prions in the sample by detecting the presence 330 of a monoclonal antibody/defective prion complex, thereby detecting defective prions in a sample obtained from an animal.

3. A method of detecting BSE in an animal prior to destruction of the animal comprising: detecting defective prions in a sample obtained from the animal according to the 335 method of claim 2, wherein, the presence of defective prions in the sample obtained from an animal is indicative of the presence of BSE.

4. A method of detecting defective prions in a sample obtained from a human comprising:

340 obtaining a sample of blood or CSF from the human; contacting the sample with proteinase K/cell lysing solution to form a mixture that will digest extraneous proteins in the sample but leave defective prions that may be in the sample unaffected; contacting the mixture with a inactivating agent such as diisopropyl 345 fluorophosphates (DFP) or phenyl methane sulphonyl fluoride (PMSF); adding a monoclonal antibody to defective prions to the mixture; detecting the presence of defective prions in the sample by detecting the presence of a monoclonal antibody/defective prion complex, thereby detecting defective prions in a sample obtained from a human.