Patent Application: US-84436504-A

Abstract:
provided is a safe novel method for detecting transmissible spongiform encephalopathies . the method comprises : selecting a sample from a subject to determine whether the subject has transmissible spongiform encephalopathy ; and detecting abnormal prion protein in the sample . the method detects prp res without proteinase - k treatment by disrupting the sample in guanidine thiocyanate lysis solution followed by phenol purification of proteins , and demonstration of the abnormal prion isoform by western blotting using monoclonal antibodies against prion protein structure . guanidine salts effectively kill tse infectivity providing a laboratory safe environment and stabilize biomolecules so tse samples can be procured in the field and transported to the laboratory in guanidine lysis solution for processing at a later date . this method provides for rapid detection of the abnormal prion isoform diagnostic of tse and results are easily interpretable based upon very different western blot patterns for abnormal prion isoform versus the normal prion .

Description:
we have devised a safe method for purification and identification of abnormal prion protein isoform in tse - infected samples . although guanidine lyses is a component of many methods , especially for extraction of ribonucleic acids , this invention is the first demonstration of purification of abnormal prion protein combining guanidine lyses of the sample followed by phenol precipitation of the prion proteins . in this invention , a sample ( 100 mg of tissue for example ) is placed in a 1 . 5 ml eppendorf tube to which is added 800 μl guanidine thiocyanate lyses buffer ( 4m guscn , 0 . 5 % laurylsarcosine , 1 mm dithiothreitol , 0 . 3m nacl in 0 . 1m tris buffer [ ph 7 . 4 ]). the sample is homogenized with a sterile pestle and the guanidine lysed homogenate is incubated for 30 minutes at 37 ° c . followed by a 65 ° c . incubation for another 30 minutes . the preparation is then centrifuged for 20 min at 14 , 000 g , the pellet discarded , and the supernatant is placed in a new tube . 350 μl phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) is admixed with the supernatant , and the preparation is centrifuged for 5 min at 14 , 000 g at 4 ° c . the result is separation of an upper aqueous layer , an interphase layer and a lower phenol layer . the aqueous layer , which contains water soluble carbohydrates and nucleic acids is saved since it is suitable for future dna or rna extraction . the interphase , which contains denatured proteins , is discarded . the phenol layer is collected and proteins are precipitated from the phenol phase with 6 volumes isopropanol ( pusztai , 1966 ). the preparation is centrifuged for 5 min at 14 , 000 g at 4 ° c ., the supernatant is discarded , and the alcohol is allowed to evaporate . the protein pellet is then dissolved in t % sds buffer and electrophoresed on sds - page ( 4 - 12 % bis - tris acrylamide gel )( invitrogen ). the proteins are transferred to immuno - blot pvdf membrane ( biorad ) and immunoblotted with mab 3f4 ( dako corporation ) using invitrogen &# 39 ; s western breeze system which produces a finished blot in 4 - 5 hours . proteins precipitated from the phenol fraction when stained by coomassie blue revealed multiple protein bands which appear to represent most of the sample proteins ( fig1 ). no significant protein bands are seen at 38 and 28 kda on coomassie - stained preparations of cjd and normal brains as shown in fig1 . western blot of the normal brain protein preparation revealed the characteristic prp c pattern typically produced by monoclonal prion antibodies ( baron & amp ; biacabe , 2001 ) with bands at 38 kda , 33 - 34 kda , and 28 kda ( fig2 b ). the cjd - infected brains immunoblotted with mab 3f4 revealed a markedly different prion protein electrophoretic pattern with the major band at 28 kda and lower bands at 18 kda and 21 kda ( fig2 a ). protein preparations from cjd - infected brains shown in fig2 a revealed both prion isoforms , and the overall immunoblot patterns seen in the cjd patients were dramatically different as compared to the normal controls seen in fig2 b . therefore , this invention produces prion isoform patterns that are diagnostic of this disease , which is clearly evident when matched with clinical history and neuropathology of the individual cases . we found that treatment of the preparations from both normal brain and cjd brain with a small amount of proteinase - k ( 5 μg / ml ) for 2 minutes essentially removed both prion isoforms ( data not shown ) indicating that guanidine salts enhance activity of proteinase - k digestion . others have previously described extraction methods for detection of prion proteins without the need of proteolysis , but the results were ambiguous ( kang et al ., 2003 ; meyer et al ., 1999 ). gdhcl and gdscn solubilize prpby disaggregation and may reveal previously inaccessible epitopes ( kang et al ., 2003 ). we found that western blot of protein extracted by the method revealed both prion isoforms without the need of proteinase k treatment . cjd cases showed distinct pattern differences compared to the normal prion immunoblot pattern . mab 3f4 recognizes sequence 109 - 112 of human prp so there may be unique changes due to the guanidine treatment that may expose epitopes at this site which allows differentiation of the abnormal prion isoform from the normal host prion immunoblot pattern . we have been successful in purifying both prion isoforms whether using guanidine thiocyanate or guanidine hydrochloride for lyses of the sample ( data not shown for latter ). using the 4b4 monoclonal antibody specific for sheep prion protein , we found similar differences in immunoblots of prion proteins purified from normal sheep brain and scrapie - infected sheep brain ( fig5 ) wherein the scrapie abnormal prion could be easily identified in the scrapie - infected brains , but not seen in the normal sheep brains . the selection of the prion antibody is not a limiting factor for this invention since , in our hands , all prion monoclonal antibodies directed to the prion protein specific for each species of animal have worked well . others have shown variable immunostaining with different monoclonal prion antibodies suggesting that one may have to be selective in choice of antibody to be used in our invention ( demart et al ., 1999 ). in the future , we will continually test newer prion antibodies as they become available to determine the best fit for our detection method . a major application of the invention was revealed when we found subtle changes in immunoblot pattern indicative of a hidden cases of tse in review of a series of brains diagnosed as alzheimer &# 39 ; s disease . it has been reported that 13 % of alzheimer &# 39 ; s disease brains have either associated cjd infection or are misdiagnosed and actually represent a case of cjd ( manuelidis , 1993 ). on study of twenty brains submitted from the lousiana state university medical center brain bank as alzheimer &# 39 ; s disease with our invention , we found several cases that had an immunoblot prion isoform pattern indicative of cjd . in fig3 a representative group of alzheimer cases from this series are shown wherein case number 2 has the immunoblot pattern of cjd . the diagnosis of cjd was confirmed on review of the neuropathology of that case which shows the reliability of the invention . two other suspicious cases in this series ( data not shown ) also revealed spongiform encephalopathy on review of the histopathology . in addition , we showed that in an established case of cjd , that there is significant variation in abnormal prion deposition in samples from different sites in the brain ( fig4 ). in that case , the frontal cortex revealed lack of abnormal prion isoform whereas the basal ganglia and cerebellum revealed abundant abnormal prion isoform ( fig4 ). this variability in distribution of abnormal prion distibution in brains of cjd patient has been noted by others ( bell et al ., 1997 ) and was the primary reason why we sampled several different sites on study of the alzheimer cases . therefore , our invention was not only able to detect cjd cases in a collection of brains misdiagnosed as alzheimer &# 39 ; s disease but also showed the importance of multiple sampling of such cases . this method provides significant advantages over currently used methods to extract prion proteins ( madec et al ., 1998 ; baron & amp ; biacabe , 2001 ). our guanidine method for extraction of prion protein from cjd brain tissues was developed primarily for laboratory safety reasons . guanidine salts have been shown to effectively kill the transmissible agent of tse ( manuelidis , 1997 ). the methodology of this invention differs markedly from other current methods where the sample is infectious at several stages of the purification procedure ( prusiner , 1984 ). 2 provides easy - to - do test without need for special equipment or expert training this method is substantially less labor intensive than current methods used to purify prion proteins . other currently used methods purify the prions by disrupting the tissues with proteases and detergents using repeated ultracentrifugations , using speeds requiring highly specialized expensive equipment available only in a research laboratory with a need for skilled technicians ( prusiner , 1984 ; bastian , 1991 ). the method outlined in this invention is safe , requires minimal technician time , no specialized training or expertise and no specialized equipment . the evaluation is made within 4 hours after receipt of the sample : briefly , the sample is incubated in the guanidine lyses buffer for one hour , treated with phenol from which the protein is extracted and run on western blot . as shown in fig1 , this method purifies abundant protein from the samples thereby allowing many experiments to be performed on the protein extracts . in our hands , this method provides a reliable way to screen proteins extracted from brains of suspect cjd cases for presence of abnormal prion protein . all eight of cjd samples submitted from the prion center showed abnormal pattern consistent with prion disease . we have examined an additional ten cjd cases with the same results ( data not shown ). we found by using this invention that there is some variability in distribution of abnormal prion protein in cjd cases . we have had the opportunity to screen 20 cases at the lsu dementia brain bank for abnormal prion proteins using this invention . screening of alzheimer cases in this fashion is important since 13 % of cases are really cases of cjd , either in combination with alzheimer &# 39 ; s disease or representing a misdiagnosis . we found several cases in the lsu alzheimer collection which show the immunoblot prion pattern consistent with cjd . in fig3 , case number 2 showed abnormal prion isoform pattern on western blot and on further histopathological review revealed spongiform encephalopathy consistent with cjd . the rapid diagnosis possible using this invention allows significant advantages to protecting the neurosurgeon , the pathologist or the mortician . in the current atmosphere , the neurosurgeon operating on a dementia case for biopsy must compromise his surgery by using disposable instruments or risk contaminating his usual set of instruments , which may end up being used on several patients before a final diagnosis is obtained . using our methodology , a diagnosis can be rendered within 4 hours after submission of the tissue to the laboratory which would allow the operating room to hold the surgeon &# 39 ; s instruments until a decision can be made whether to dispose of certain of them and to perform more extensive cleanup of the operating room . furthermore , the biopsy sample can be held for a few hours in formalin until a diagnosis is rendered . if a test based upon this invention is positive , the sample can then be processed in a separate histology laboratory so that other tissue samples are not contaminated . there are many instances of iatrogenic transmission of cjd through transplantation of dura or corneas derived from cadavers . at the time of obtaining the tissue from the cadaver , a small piece of the tissue can be lysed in the guanidine salt solution and submitted to the laboratory for detection of abnormal prion using this invention . the test results can be obtained before the sample homograft is placed in the tissue bank for distribution , thereby protecting the future patient from contamination by cjd through transplants . similarly , if the pathologist must autopsy a dementia case , or a case without clinical history , a tissue sample can be taken from the brain and processed , while dissection of the organs can be delayed for one day . this will prevent contaminated tissues being processed along with routine specimens in the histology laboratory . similarly , the mortician is often presented with a corpse where the clinical diagnosis is unknown or there is a history of dementia of unknown cause . a small brain sample can be obtained by trocar and examined by this method which would allow the mortician to proceed as usual or defer to use of special embalming methods or incineration so as to protect his facility from cjd contamination . this method can also be easily applied to field studies where samples are obtained from suspect animals in a situation where tissues cannot be frozen and instead are preserved by fixation . fixation with formalin does not allow for western blotting or dna studies and samples can only be studied by immunohistochemistry . this has presented a significant problem for the usda since basing the entire assay procedures on immunohistochemical evaluation alone is known to give inconclusive results as well as show decreased sensitivity compared to other methods . using our method , tissues are minced in the guanidine thiocyanate lyses buffer at the location where the sample is taken , which effectively kills the transmissible agent ( manuelidis , 1997 ) and stabilizes biomolecules for days until analysis in a laboratory . western blotting could then give conclusive results in just a few hours following arrival to the laboratory . immunoblot studies of sheep scrapie reveal presence of abnormal prion isoform which are not present in the normal sheep brain samples ( fig5 ). this method can easily and rapidly test whether samples submitted to a taxidermist for trophy preservation are infected with tse . this would both protect the taxidermist as well as providing epidemiological evidence of spread of the disease into the local wild animal population . in the case of meat processing plants , a sample could be assayed by this invention while the meat is curing and long before meat is distributed to commercial outlets . such testing would prevent a situation as occurred recently in washington state where standard testing of a suspect case of bse took over a week so that much of the meat from the bse - infected canadian cow had been distributed to the market place , and much had been consumed . beef derived products are used in preparation of many commercial products including ice cream and lipstick . fetal calf serum is widely used as an essential component of growth media for cell cultures . samples of batches of serum or other beef products can be spot checked using this method to prevent distribution of a contaminated product . similarly , food supplements derived from cattle or other animals can be checked . of particular interest is the widespread use of elk velvet which should be screened since cwd is prevalent in the elk population . both prion isoforms can be compared by the same preparatory method . direct comparison between the two prion isoforms has not been possible in the past since treatment with proteinase - k necessary to extract prp res destroys the prp c isoform ( bolton et al ., 1987 )). current studies therefore are comparing prion protein isoforms each prepared very differently . peptide mapping of these preparations from our study could lead to significant increase in our understanding of the nature of the prion isoforms including the relationship of the normal prion to the abnormal isoform and the mechanism of transformation of one isoform to the other . since we have both prion isoforms in the same preparation using this invention , selective sequencing of the various fragments should allow us to better understand the nature of transformation of the normal prion isoform to the abnormal misfolded prion . 1 . bastian f o . 1991 . review of theories of the transmissible agent , p . 49 - 64 . in f o bastian ( ed ), creutzfeldt - jakob disease and other transmissible spongiform encephalopathies . mosby / year book publishers , st . louis , mo . 2 . spraker t r , miller m w , willams e s , getzy d m , adrian w j , schoonveld g g , spowart r a , o &# 39 ; rourke k i , miller j m , merz p a . spongiform encephalopathy in free - ranging mule deer ( odocoileus hemionus ), white - tailed deer ( odocoileus virginianus ) and rocky mountain elk ( cervus elaphus nelsoni ) in northcentral colorado . j wildlife dis 1997 ; 33 : 1 - 6 . 3 . will r g , ironside j w , zeidler m , cousens s n , estibeiro k , alperovitch a , poser s , pocchiari m , hofman a , smith p g . a new variant of creutzfeldt - jakob disease in the uk . lancet 1996 ; 347 : 921 - 925 . 4 . houston f , foster j d , chong a , hunter n , bostock c j . transmission of bse by blood transfusion in sheep . lancet 2000 ; 356 : 1771 - 1772 . 5 . vamvakas e c . risk of transmission of creutzfeldt - jakob disease by transfusion of blood , plasma , and plasma derivatives . j clin apheresis 1999 ; 14 : 135 - 143 . 6 . ironside j w . 1996 . neuropathological diagnosis of human prion disease : morphological studies , p . 35 - 38 . in h f baker and r m ridley ( eds . ), prion diseases . humana press , totowa , n . j . 7 . prusiner s b . 1984 . prions , p . 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