Patent Application: US-94988004-A

Abstract:
the invention provides methods for the detection of prion diseases , such as scrapie of sheep , bovine spongiform encephalopathy of cattle , creutzfeld - jacob disease of man , whereby aberrant proteins or prion proteins are detected in tissues which can be sampled from live animals .

Description:
immunohistochemical detection of prion protein in lymphoid tissues of sheep with clinical cases of natural scrapie sheep . sixty seven sheep with nervous disorders resembling those of a scrapie infection were purchased . fifty - five sheep were diagnosed with scrapie by histopathological and immunohistochemical examination of the brain . ( van keulen et al ., 1995 ). one animal suffered from both a scrapie infection and a concurrent meningo - encephalitis probably caused by listeria monocytogenes . scrapie - positive sheep originated from 30 different flocks . the group consisted of 54 females and one male ranging in age from 2 to 5 years and comprised eight different breeds and cross - breeds . twelve sheep did not show any histopathological signs of a scrapie infection nor did they display any prp sc immunostaining in the brain . five of these sheep were diagnosed with meningo - encephalitis , one had intramyelinic edema of unknown cause , and 6 sheep showed no histopathological abnormalities . scrapie negative sheep were all females from 10 different flocks and two different breeds and crossbreeds , ranging in age from 1 to 5 years . necropsy . necropsy was performed within 36 hours after natural death or immediately after killing the animal by intravenous injection of sodium pentobarbital and exsanguination . the brain was removed from each sheep for scrapie diagnosis as described previously ( van keulen et al ., 1995 ). samples were taken from several lymphoid tissues including spleen , palatine tonsil , superficial cervical lymph node ( prescapular lymph node ), subiliac lymph node ( prefemoral lymph node ), medial retropharyngeal lymph node , tracheobronchial lymph node , mesenteric lymph node , and ileum . histological and immunohistochemical procedures . tissue samples were immediately immersed for 24 hours in periodate - lysine - paraformaldehyde fixative ( plp ) containing 2 % paraformaldehyde ( merck , darmstadt , germany ). samples were then trimmed to a maximum thickness of 2 mm and fixed for another 24 hours in freshly prepared plp . after fixation , tissue samples were washed in water , routinely dehydrated and embedded in paraffin . three sections of 5 μm were cut , mounted on 3 - aminoalkyltriethoxysilane - coated glass slides ( sigma , st . louis mo ., usa ), dried for at least 48 hours at 60 ° c . and deparaffinized . the first section was stained with hematoxylin - eosin ( he ). second and third sections were immunostained with anti - peptide serum directed against the ovine prion protein and pre - immune serum respectively according to the following procedure ; after 30 minutes immersion in 98 % formic acid ( merck ), sections were washed and autoclaved immersed in water for another 30 minutes at 121 ° c . in a pressure cooker . endogenous peroxidase was blocked with 0 . 3 % hydrogen peroxide in methanol ( merck ). incubation at room temperature for 1 hour with anti - peptide antiserum or pre - immune serum , diluted 1 : 1500 in phosphate - buffered saline ( ph 7 . 2 ) containing 1 % bovine serum albumin ( sigma ), was followed by incubation , first with biotin - conjugated goat - anti - rabbit igg and then with streptavidin - peroxidase for 10 and 5 minutes respectively ( dakopatts , glostrup , denmark ). as substrate we used aminoethylcarbazole ( zymed laboratories inc ., san francisco calif ., usa ) because its red color could easily be differentiated from the yellow - brownish ceroid / lipofuscin and hemosiderin pigment which was often present in the lymphoid tissues . between the various steps , sections were thoroughly rinsed in phosphate - buffered saline containing 0 . 05 % tween - 20 ( merck ). sections were counterstained with mayers hematoxylin for 30 seconds and mounted in glycergel ( dakopatts ). with every immunohistochemical staining , a section of the medulla oblongata of a confirmed scrapie - affected sheep was simultaneously stained for prp to check correct immunostaining procedures . peptide synthesis and anti - peptide antisera . five peptides with sequences derived from the ovine prion protein ( prp 94 - 105 , 100 - 111 , 126 - 143 , 145 - 177 , 223 - 234 ) were synthesized and used to raise anti - peptide antisera in rabbits following previously published procedures ( van keulen et al ., 1995 ). antisera were confirmed to be specific for prp ( both undigested and after proteinase k treatment ) on western blots of partially purified prion protein from scrapie - affected sheep brain according to established procedures ( hilmert and diringer , 1984 ). pre - immune sera were collected before immunization and served as negative control sera . the sera used have advantages which are based on a mixture of empirical , theoretical and analytical values the combination of which makes them invaluable in the diagnostic application . the preparation of the sera has been described in a publication of van keulen et al . 1995 . the immunochemical properties of these sera are partly published . the specific sera used in this example have been designed for scrapie diagnosis , however , guidance can be found in the below given indications for the development of sera that are applicable in diagnosis of the other ses , provided one selects the sequences as corresponding to the species specific sequence of the prion protein . when needed one may select other animals than rabbits to generate the specific sera . 1 : the sera have been induced with synthetic peptides with sequences based on the sequence of prp protein . 2 : the sera have been induced in rabbits . 3 : the peptides sequences have such differences with the rabbit prp sequence that they induced not only antibodies which recognized these peptides but also the authentic prp protein . 4 : the peptides used for immunization are kept short ( 12 mers ); this shortness is supposed to have a critical role in the high specificity for the scrapie forms of prp and thus in the binding in the tissue sections even after harsh denaturing and degradative treatments . 5 : the sequences used for immunization and yielding the specific scrapie prp staining were selected from the protease k resistant domain of the prp sc . 6 : the sera of use in the diagnostic ihc are also well reactive in other immunochemical tests such as : western blotting of both prpc and prp sc , elisa with prp protein , pepscan with 12 mer peptides with overlapping sequences of sheep prp . 7 : the peptides selected have properties ( hydrophilicity , flexibility , surface occurrence ) which are — when used for immunization — advantageous for eliciting antibodies with binding to the antigen on which the sequences have been based . 8 : the antisera elicited show the right specificity when analyzed in pepscan with 12 mer peptides . the addition of a foreign dimeric glycine at either the n - terminus or the c - terminus of these peptides does not decrease the specificity of the peptides but more probably does make the immunization more effective , supposedly because it makes the peptides stand out farther away from the carrier protein and makes them more flexible on the carrier protein properties which are important determinants in antigenicity . 9 : the sequences selected for peptide synthesis and immunization represent domains which have a low tendency to form secondary structure ( a - helix or β - sheet ) and are not part of the four regions described in the literature a being able to form β - sheet as synthetic peptides . an identical and distinct immunolabelling pattern was detected with all anti - peptide antisera in the lymphoid tissues of scrapie - affected sheep . because the five antisera were directed against different epitopes of the prp protein , cross - reactivity of the anti - peptide antisera with another protein can be excluded . we therefore classified the immunolabelled protein as prp . we further defined this prp as scrapie - associated prp ( prp sc ), because no prp immunoreactivity was seen in any of the lymphoid tissues of scrapie - negative sheep . replacing the anti - peptide antisera with pre - immune sera did not result in any immunolabelling . prp sc was located within the primary and secondary lymphoid follicles of the spleen , palatine tonsil , lymph nodes , and solitary follicles or peyer &# 39 ; s patches of the ileum . the prp sc immunolabelling pattern consisted of a reticular network in the center of the lymphoid follicle which varied in staining intensity . apart from this network , fine to coarse granules of prp sc were seen in the cytoplasm of non - lymphoid cells within the follicle . several of these cells were identified as macrophages because of the simultaneous presence of ceroid / lipofuscin pigment in their cytoplasm . no immunolabelling of the b lymphocytes in the lymphoid follicle was seen . occasionally , additional immunolabelling was found in specific cells and regions of the lymphoid tissues . in the spleen , individual cells in the periarterial lymphatic sheath ( pals ) and the marginal zone surrounding the splenic corpuscles contained granules of prp sc sometimes combined with ceroid / lipofuscin pigment within the cytoplasm . no prp sc was seen in the red pulp of the spleen . in the palatine tonsil and ileum , branches or granules of prp sc were found interspersed between the lymphocytes of the dome area between the follicles and the crypt epithelium . in the lymph nodes , granules of prp sc were seen between the lymphocytes of the paracortex . prp sc was detected in 54 ( 98 %) of the 55 scrapie - affected sheep in the spleen , tonsil , retropharyngeal lymph node and mesenteric lymph node . in the tracheobronchial , prefemoral and prescapular lymph node , prp sc was seen in a slightly lower percentage of the sheep ( table 1 ). prp sc was found in solitary lymphoid follicles or peyer &# 39 ; s patches of the ileum in 24 ( 89 %) of the 27 sheep in which lymphoid tissue was present in the sections of the ileum . in only 1 of the 55 scrapie - affected sheep , prp sc could not be detected in any of the lymphoid tissues . the percentage of lymphoid follicles that contained prp sc was estimated for the sections of the spleen , tonsil and lymph nodes . in the palatine tonsil of 98 % of the scrapie - affected sheep , over 60 % of the lymphoid follicles contained prp sc . in the tonsils of 93 % of the sheep with scrapie the percentage of prp sc - positive lymphoid follicles even exceeded 80 %. in the spleen or lymph nodes , prp sc accumulation in more than 60 % of the lymphoid follicles was only present in less than 30 % of the sheep . immunohistochemical detection of prion protein in lymphoid tissues of sheep with pre - clinical cases of natural scrapie . we selected a group of 10 purposely bred lambs , six of them homozygous for the prp allele with valine ( v ) at position 136 and glutamine ( q ) at position 171 . in several breeds , this prp vq allele is significantly associated with an increased susceptibility for scrapie ( belt et al ., 1995 ). the remaining four lambs were heterozygous and possessed one prp vq allele and one prp ar allele ( alanine at position 136 and arginine at position 171 ). the prp ar allele is significantly associated with increased resistance of sheep for scrapie . in a flock with natural scrapie we observed that sheep with the genotype prp vq / vq died from scrapie at approximately 25 months of age and that the majority of the sheep with the genotype prp vq / ar were still healthy at 70 months of age . since we expected that the prp vq / vq sheep would almost certainly develop clinical signs of scrapie within approximately 25 months after birth and that the prp vq / ar sheep would stay healthy , we regarded these two groups of sheep as a suitable model to study changes at known stages of the incubation period . all 10 sheep - were born and raised on the same farm , in an environment where scrapie has been occurring for several years . they were kept here until six months old , when they were transferred to our institute , to a paddock where various scrapie positive animals had spent their last days . tonsil biopsies were collected under general anaesthesia , which was achieved by intravenous application of a combination of ketalar ( ketamine - hcl ) 4 mg / kg , xylazine ( rompun ) 0 . 05 mg / kg and atropine 0 . 1 mg / kg . we used a mouth gag , a laryngoscope , and a biopsy forceps with a head of approximately 4 mm in diameter . tonsils in sheep are not as readily accessible as in some other species , such as man , where they often protrude into the pharyngeal lumen . in sheep , they are hidden , surrounding a small cavity . it proved , however , sufficient to take a biopsy of the edge of the entrance to this cavity , the fossa tonsillaris , thereby collecting in general sufficient material ( follicles !) to allow examination . some experience in the technique was obtained by collecting , just before the animals were euthanized , tonsillar biopsies from 11 sheep , among them clinically affected scrapie sheep . histological procedures included immunostaining with specific ( anti - prp sc ) anti - peptide - sera , as described above and in van keulen et al ., 1995 . from the 11 sheep , eight proved to be scrapie positive while three turned out negative , as was confirmed histologically and by ihc of brain tissue during post mortem examination . the tonsillar biopsies of all eight positive animals showed a positive immune - staining in the ihc , whereas no immuno - staining could be detected in the three negative cases . in the actual experiment , we planned to take tonsillar biopsies sequentially , at regular intervals and starting at an age of six months . for logistic reasons this was delayed . we collected biopsies from both groups for the first time at approximately 10 months after birth , when none of the sheep showed clinical signs of scrapie . the youngest sheep were nine - and - half months , the oldest sheep was 10 months and one week . after ihc - staining we found clear , already extensive , prp sc staining in the tonsillar biopsies of all six susceptible prp vq / vq sheep , whereas no immune - staining was detectable in the tonsillar biopsies of any of the resistant prp vq / ar sheep . we have thus detected the scrapie associated prp sc in tonsils of 10 months old sheep , which is at less than half - way the incubation period as they are expected to develop scrapie when approximately 25 months old . in sheep that are expected to develop scrapie at a much later stage or stay healthy during their whole life span , we did not detect this prp sc protein . we conclude that ihc - staining and related methods provide the possibility for pre - clinical diagnosis in sheep scrapie as well as for other ses like bse - and cjd . belt , p . b . g . m ., i . h . muileman , b . e . c . schreuder , j . bos - de ruijter , a . l . j . gielkens , and m . a . smits . 1995 . identification of five allelic variants of the sheep prp gene and their association with natural scrapie . j . gen . virol . 76 : 509 - 517 . fraser , h . and a . g . dickinson . 1978 . studies of the lymphoreticular system in the pathogenesis of scrapie : the role of spleen and thymus . j . comp , pathol . 88 : 563 - 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