Patent Application: US-97708907-A

Abstract:
the invention is related to diagnostic methods for detecting transmissible spongiform encephalopathies such as bse and scrapie and related disease in humans . the invention provides use of guanidine thiocyanate , or a functional equivalent thereof , for treating at least one sample derived from a mammal , including humans , for reducing the risk of scoring a false - positive test result in testing the sample for the presence or absence of aberrant prion protein .

Description:
phosphate buffered saline ( pbs ), ph 7 . 2 contained 136 . 89 mm nacl , 2 . 68 mm kcl , 8 . 10 mm na 2 hpo 4 and 2 . 79 mm kh 2 po 4 in water . ( a ) 10 mm phosphate buffer , ph 7 . 0 , 0 . 15 m nacl and 0 . 25 m sucrose , used by pan et al . ( 1992 ) to prepare microsomal fractions ; ( b ) lysis buffer ( collinge et al ., 1996 ) consisted of 0 . 5 % ( w / v ) tergitol ( type np - 40 , nonylphenoxy polyethoxy ethanol , sigma np - 40 ) and 0 . 5 % ( w / v ) deoxycholic acid , na - salt ( merck ) in pbs , ph 7 . 2 . guanidine thiocyanate ( gdnscn , purity & gt ; 99 %; sigma g 9277 ) solutions of 4 m were made up in water ( ph 5 . 8 ). alkaline phosphatase - conjugated goat anti - rabbit igg ( gar / ap ) was from southern biotechnology as . ( itk , diagnostics b . v ., uithoorn ). substrate for alkaline phosphatase was 5 - bromo - 4 - chloro - 3 - indolyl phosphate / nitro blue tetrazolium ( bcip / nbt ; tablets ; sigma b5655 ). usually , after prp extraction , protease inhibitors were added to the extracts . ( complete , protease inhibitor cocktail tablets ; boehringer nr . 1697498 , mannheim , germany .) proteinase k ( ec 3 . 4 . 21 . 14 , 20 units / mg lyophilisate nr . 745723 ) and pefabloc sc ( 4 -( 2 - aminoethyl )- benzenesulfonyl fluoride , hydrochloride nr . 1585916 ) were also from boehringer . incubation conditions for prp extracts with pk were 50 μg / ml enzyme for 30 minutes at 37 ° c . in order to stop this enzymatic reaction , the incubation mixture was made 1 mm in pefabloc added from a 100 mm stock solution of the inhibitor in water . as a blocking agent , nonfat dry milk ( protifar , nutricia ) was used . a number of hydrophilic ( 14 ) and hydrophobic ( 5 ) membranes were tested as carrier matrix . the most successful representatives , polysulphone or nitrocellulose membrane types , were selected . three membrane types were routinely used : nitrocellulose ( nc ) membrane with a 3 mm screen ( protran ba 85 / 21 ; 0 . 45 mm nr . 405891 ) was from schleicher and schuell gmbh ( dassel , germany ), immobilon - p ( polyvinylidenedifluoride , pvdf ) from millipore b . v . ( etten leur ) and zeta - probe ( quaternary amine - nylon membrane ) was from biorad . an ultra - turrax t25 mixer with a 10 mm shaft ( ika labortechnik gmbh , staufen , germany ) was used to homogenize brain tissue . the shaft was decontaminated in 1 m naoh . these were intentionally designed for scrapie diagnosis . antisera were induced in rabbits using synthetic peptides with sequences based on the sequence of ovine prp protein . the sequences have such differences with the rabbit prp sequence that they induce not only antibodies that recognize these peptides but also the authentic prp protein . other animal species like mouse , which have sequence differences , could be suitable as well . the sequences used for immunization were selected from the protease k - resistant domain of prp sc . the selected 12 - mer sequences ( seq id nos : 11 , 12 , 30 ) represent domains that have a low tendency to form secondary structure ( α - helix or β - sheet ). the antisera are reactive in diagnostic dot blotting but also in western blotting of both prp c and prp sc , in elisas with , as coated antigens , the above peptides or prp protein , and in immunohistochemical detection . with the peptide derived from the ovine prion protein sequence 94 - 105 ( seq id no : 11 ), antisera r521 and r522 were produced in rabbits . likewise , sequence 100 - 111 ( seq id no : 12 ) yielded antisera r504 , r505 , r593 , r594 , r595 , and r596 , and sequence 145 - 177 ( seq id no : 26 ) antiserum r532 . the sequence 126 - 143 ( ovine and bovine ) ( seq id no : 20 ) gave rise to antiserum r568 , while sequence 223 - 234 ( ovine and bovine ) ( seq id no : 30 ) yielded antisera r523 and r524 . peptides were synthesized and used to raise antipeptide antisera in rabbits following previously published procedures ( van keulen et al ., 1995 ). antisera were confirmed to be specific for sheep prp ( both undigested and after proteinase k treatment ) on western blots of partially purified prion protein from scrapie - affected sheep brain . sheep samples ( brain stem , cervical spinal cord ) were from scrapie - affected sheep , diagnosed by histopathological and immunohistochemical examination of the brain and from normal healthy sheep ( van keulen et al ., 1995 ). samples from bse - diagnosed cattle ( histopathology , immunohistochemical examination and western blotting ) were from the cervical spinal cord or brain stem . procedure for immuno - dot blotting : 0 . 5 g portions of brain tissue were cut down with a scalpel and homogenized with an ultra - turrax mixer ( 20 , 000 rpm / 15 seconds ) in 4 . 5 ml of ice - cold lysis buffer . the homogenates were centrifuged at 1000 × g for ten minutes or used without centrifugation as crude homogenate . if appropriate , an aliquot of the homogenate was incubated with pk at 37 ° c . for 30 minutes , after which the reaction was stopped with pefabloc ( 1 mm ). otherwise , a cocktail of protease inhibitors was immediately added to the homogenate . suitable dilutions of the turbid supernatants or crude homogenates in lysis buffer were spotted in 1 - 3 μl amounts onto two blotting membranes and left for 15 minutes . one membrane was incubated in 4 m gdnscn for ten minutes and the other membrane was left untreated . washing of the treated membranes was for ten minutes in pbs on a rocking platform . membranes were blocked with 5 % ( w / v ) protifar in pbs for one hour at 20 ° c . and washed in pbts with 1 % ( w / v ) protifar for five minutes at 20 ° c . a one to two - hour incubation with the primary antibody ( 1 / 1000 diluted in pbts ) at 20 ° c . was followed by three washing steps in pbts for five minutes each . next , the membranes were incubated with ap - conjugated goat anti - rabbit igg ( 1 / 1000 diluted in pbts ) for one to two hours at 20 ° c . and washed in pbts three times for five minutes . substrate solution was added and the reaction was stopped with water . after homogenizing brain stem tissue of a scrapie - affected sheep in extraction buffer ( a ) or in ( b ) (= lysis buffer ) and low - speed centrifugation that yielded supernatant 1 , aliquots of this supernatant were again centrifuged at a higher speed ( 11 , 000 × g , ten minutes : “ high speed ” supernatant 1 ). the loose pellets left from the first centrifugation step were adjusted with buffer to the original volume , re - extracted and centrifuged at 1000 × g , which yielded a supernatant 2 and a loose pellet . in addition , aliquots of all fractions were treated with pk . one μl extracts ( diluted 1 , 1 / 10 and 1 / 100 × in their respective buffers ) were spotted onto nc and immunodetection was with r522 - 7 , an antiserum that has proven to detect ovine prp ( van keulen et al ., 1995 ). for lysis buffer , the highest signal intensity was obtained for the supernatant 1 . compared to the results for lysis buffer , the signals for extraction buffer ( a ) were lower for all fractions , except for the pellet . for fractions of the lysis buffer , decreased intensities were observed after pretreatment with proteinase k , especially for supernatant 2 , which indicates that this fraction is relatively enriched with prp c . we observed dramatically intensified signals for the lysis buffer extracts when these were diluted in 4 m gdnscn . for supernatant 1 , even after a 100 - fold dilution , the signal was clearly visible , which means that in these scrapie brain stems , prp can be made visible in a tissue equivalent of 1 μg . investigation of brain stem extracts of a scrapie - negative sheep in lysis buffer revealed , even in an eighty - fold dilution , clear signals of prp c . however , after pretreatment with pk , a signal could no longer be observed . surprisingly , instead of applying this pk treatment , dilution of tissue extract in 4 m gdnscn also led to a dramatic decrease of signal intensity for prp c . next , instead of diluting lysis buffer - extracted samples in 4 m gdnscn , we applied serial dilutions of brain extracts of scrapie - positive and - negative sheep in duplicate on nc membranes and incubated one membrane in 4 m gdnscn for ten minutes while the other one was left untreated . immunodetection revealed that we could easily discriminate between scrapie - positive ( prp sc and prp c ) and scrapie - negative ( prp c ) samples : a higher intensity with 4 m gdnscn compared to an untreated sample means scrapie positive , while a lower intensity with gdnscn means scrapie negative . this finding is the basis for a rapid and simple diagnostic test for tses . in this test , there is , in general , no need for a preceding removal of prp c from the negative sample . as an alternative for gdnscn , we investigated the effects of other chaotropic agents . after dot blotting , 3 μl dilutions of extracts of scrapie - positive and - negative brain stems , separate nc membranes were incubated for ten minutes in chaotropic agents . the solutions used were : 4 m gdnscn , 7 . 2 m urea , 4 m kscn , 1 m thiourea , naoh ( ph 11 ) in water and 98 % formic acid ; besides one membrane was left untreated as a blank . results for immunodetection after kscn and thiourea did not differ from the blank . urea induced a slight increase for the scrapie - positive material and formic acid enhanced the intensity to the level of gdnscn , although this acid caused considerable shrinking of the nc membrane . optimum enhancement with pvdf as a carrier was achieved by using 50 % formic acid ; no membrane shrinkage was then observed . naoh ( ph 11 ), on the other hand , increased the signal for scrapie - negative material . treatment with 4 m gdnscn turned out to be the best discrimination between scrapie - positive and - negative tissue samples . moreover , this effect appeared to be ph - invariant since solutions of 4 m gdnscn at ph 4 and 7 ( in 50 mm phosphate buffer ), ph 6 ( in water ) and ph 9 ( in 50 mm carbonate buffer ) gave identical results . five classes of antipeptide antisera to linear epitopes of sheep prp sequences ( 94 - 105 ( seq id no : 11 ), 100 - 111 ( seq id no : 12 ), 126 - 143 ( seq id no : 20 ), 145 - 177 ( seq id no : 26 ) and 223 - 234 ( seq id no : 30 )) were examined . for comparative reasons , all sera were used in a 1 / 500 dilution in pbts . antisera to the 94 - 105 sequence ( seq id no : 11 ) ( r521 , r522 ) and to the 100 - 111 sequence ( seq id no : 12 ) ( r505 ) proved to have the best differentiating power . on the other hand , with the antisera r568 and r532 to the sequences 126 - 143 ( seq id no : 20 ) and 145 - 177 ( seq id no : 26 ), respectively , no immuno - enhancing effect of 4 m gdnscn on prp sc could be detected . comparison of results on nc membrane with those on zeta - probe showed , for the latter , a strong aspecific coloring of the entire membrane and consequently quaternary amine - nylon as a carrier was unsuitable . on the other hand , compared to nitrocellulose , a stronger adsorption for prp was shown for the pvdf membrane ( immobilon - p ). from brains of bse - positive cattle , obtained from the netherlands , the uk , ireland , belgium and switzerland , and of dutch bse - negative cattle ( diagnosed by histopathology and immunohistochemical examination ), brain stems were extracted with lysis buffer in the same manner as for sheep , and the low - speed supernatant 1 was used for further examination . brain stem extracts from confirmed scrapie - negative and - positive sheep were used for comparison . aliquots of extracts were also treated with proteinase k and 3 μl amounts of dilutions in lysis buffer of pk - treated and untreated extracts were spotted onto nc membranes . immunodetection was with 1 / 1000 dilutions of antisera to the 12 - mer sequences 94 - 105 ( seq id no : 11 ) ( antiserum r521 ), 100 - 111 ( seq id no : 12 ) ( r505 , r595 , r596 ), 223 - 234 ( seq id no : 30 ) ( r523 , r524 ) and to the longer sequences 126 - 143 ( seq id no : 20 ) ( r568 ) and 145 - 177 ( seq id no : 26 ) ( r532 ). highest immunoreactivity was shown with antisera r505 and r595 . after incubation with 4 m gdnscn , signal intensity of bse - negative samples diminished ; however , the immuno - enhancing effect of 4 m gdnscn on prp sc in bse - positive samples did not reach a comparable high level as for sheep prp sc in scrapie . surprisingly , antisera r523 , r524 and especially r532 showed stronger immunoreactivity with bovine prp sc than with prp c . immunoreactivity of antisera r521 and r568 with bovine prp was very poor . no signal was obtained with the pk - treated material of bse - and scrapie - negative animals . pvdf showed a higher adsorption than nc membranes since immunostaining could be observed at higher dilutions on pvdf . the detection limit of the test with sheep recombinant prp spotted on pvdf and using antiserum r521 or r595 is about 50 pg . using other detection methods , however , will , of course , result in even lower detection levels . thus far , 29 cases of bse and 131 negative controls were examined . the performance was 100 % ( table 1 ). the design of one of our tests is that of a dot blot immunoassay that has an intrinsically higher sensitivity than an analogous elisa assay in a microtiter plate , due to miniaturization within the blot and the higher binding capacity of the matrix material ( nitrocellulose , pvdf ) than of a smooth polysterene microtiter plate surface . because of the divergent immunoreactivity of sheep prp c and prp sc during denaturation , the discriminatory power for false - positive samples of our test is much higher than that of the assay of safar and coworkers ; in our assay , the signal for prp c during denaturation in 4 m gdnscn diminishes , whereas immuno - 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