Patent Application: US-62009003-A

Abstract:
this invention relates to the field of the detection of prion diseases . the invention provides a binding molecule or antibody specifically reactive with an epitope which is exposed on a part of an aberrant conformer of a prion protein after treatment of said conformer with a protease wherein said epitope is not or only partly exposed on a prion protein which has not been treated with a protease .

Description:
here we describe the exposure of neo - epitopes by prp sc upon cleavage by pk . these neo - epitopes can be used to identify a novel class of binding molecules for the detection of prions in tissues containing high concentrations of prp c . this class is exemplified by the isolation and characterisation of a new monoclonal antibody ( 1e4 ) that has a low affinity for the prp c and undigested prp sc as compared to its affinity to pk - digested prp sc the so - called prp 27 - 30 ( the 27 - 30kd fragment of prp sc obtained after pk - digestion ). the remarkable specificity of this antibody was found upon analysis of a series of antibodies generated against prp - derived peptides mapped very close to the n - terminal end of prp 27 - 30 . the new class of binding proteins will improve the detection of prion in several species including human , bovine , ovine , and murine , as is indicated by showing examples of application of this class of binding molecules in various test systems , including western blotting , and immuno - histochemistry . furthermore , we will show examples of using these molecules for strain typing within and between species . as an example of this approach we will describe the selection , isolation and characterisation of mab 1e4 . monoclonal antibody 1e4 was isolated from fusion of spleen cells of a prnp 0 / 0 mouse , immunised with the peptide gqwnkpskpktn # ( corresponding to the bovine prp aa sequence 108 - 119 ;#= amidated carboxy - terminus ) coupled to klh at its n - terminal end via a cg - aa linker . sera from 6 out of 10 mice immunised with this klh - coupled peptide reacted positive in scrapie / bse dot - blot assay . moreover sera from two mice contained high titers of antibodies as detected in anti - peptide elisa and radio immunoassay ( ria ), detecting immobilised and fluid phase interaction , respectively . one of these mice was selected for fusion . four days after a final boost with the klh - coupled peptide , the spleen was collected , and spleen cells were isolated and fused with sp2 / 0 - ag14 myeloma cells . the fused cells were plated in 96 - well plates at a density of 10 5 cells / well . after 7 and 14 days of selection with ha ( hypoxantine and azaserine ), culture supernatants were screened for specific antibodies with the anti - peptide ria , resulting in 23 positive wells . limiting dilution of these clones revealed that only 10 clones were stable regarding igg production as measured with the anti - peptide ria . the cells from these 10 wells were subsequently subcloned three times by limiting dilution . each subcloning step was monitored with the anti - peptide ria , ultimately resulting in five stable clones . culture supernatant of clone 1e4 was tested with western blot demonstrating a strong binding reaction to bse brain homogenates treated with pk , whereas virtually no binding was found to brain homogenates from healthy controls . this observation showed the unique binding characteristic on the 1e4 monoclonal antibody that was subsequently analysed in detail . the binding properties of mab 1e4 were compared with those of generally used mab 6h4 ( prionics ag , zürich , switserland ) using western blot . bse samples were treated with pk and subsequently three - fold serial dilutions of the samples were prepared . mab 1e4 still yielded a positive reaction with a 243 - fold dilution of the pk - treated sample , whereas the 81 - fold dilution of the untreated sample was found negative ( fig1 ). using mab 6h4 the 81 - fold dilutions of both pk - treated and untreated bse samples was found positive . therefore this western blot indicates that 1e4 has at least a nine - fold higher affinity for the pk - digested bse sample as compared to untreated sample , assuming that 6h4 has a similar affinity for both types of antigens . this pivotal observation suggests that 1e4 has a higher affinity for the cleaved prp 27 - 30 than for the non - cleaved prp sc . moreover the western blot also included a brain homogenate sample derived from a normal cow . mab 1e4 reacted only weakly with the latter sample , whereas 6h4 reacted very strongly with the sample suggesting that 1e4 has at least a nine - fold lower affinity for prp c than 6h4 has . so compared to 6h4 , 1e4 has a lower affinity for prp c and prp sc , but a higher affinity for prp 27 - 30 . these differences in affinity for prp c , prp sc , and prp 27 - 30 were more striking using a spot - blot assay ( fig2 ) using mab 1e4 the 270 - fold dilution of the pk - treated bse sample was found positive , whereas the 10 - fold dilution of the untreated sample was found negative . using mab 6h4 however the 90 - fold dilution of the pk - treated bse sample was positive , whereas the 810 - fold dilution of the untreated sample was found positive . furthermore mab 1e4 did not react with brain homogenate derived from a normal cow , whereas mab 6h4 reacted with the 2430 - fold dilution of the sample . these findings confirm that 1e4 has a lower affinity for bovine prp c and prp sc , but a higher affinity for prp 27 - 30 as compared to 6h4 . mab 1e4 also reacted with prion from 301v - infected mice , scrapie - infected sheep and scjd - and vcjd - infected human using western blots ( fig3 ). however the striking difference between the affinity for cleaved and non - cleaved prp sc observed for bse in cattle is not seen in these samples . the broad species reactivity in the western blot is also seen on the pepscan analysis of mab 1e4 . as already indicated the antibody originated from a mouse immunised with klh - cg - bovine prp 108 - 119 : the aa - sequence was gqwnskpktn # and pepscan analysis showed a linear epitope for mab 1e4 present in this aa region in bovine , sheep , human , mouse and hamster with qwnkps as core aa sequence ( fig4 ). furthermore a mimic of the epitope was found on mouse and hamster prp 217 - 222 ( aa sequence qyqkes ) implying the aa q109 , k112 and s114 to be essential residues for antibody binding in the original epitope for mab 1e4 . replacement analysis of prp 220 aa k by r , as it is present by bovine , sheep and human , resulted in absence of binding activity to mouse and hamster in the pepscan in this region . when we compared 1e4 with other prp mabs in pepscan analysis , 1e4 appeared to be the only antibody having high affinities for a broad range of species ( fig5 ). therefore the antibody would be suitable as a general antibody for detection prions in general and for bse in particular . the broad species reactivity of mab 1e4 is also found when the antibody is used for immuno - histochemistry of sections from brainstem of bse - infected cattle , brainstem of bse - and scrapie - infected sheep , and total brain of bse - and scrapie - infected mice . to further characterise the immunoglobutin that is produced by clone 1e4 , we rt - pcr - cloned the v h region of the ig expressed by clone 1e4 . the amplified cdna was sequenced and the hypervariable cdr3 region was identified . the cdr3 aa sequence of clone 1e4 is agdndaedy . the lower affinity for prp c , prp sc , and higher affinity for prp 27 - 30 was particularly found for bse - infected cattle , but not e . g . for scrapie infected sheep . this property of mab 1e4 might be exploited to facilitate discrimination between bse and scrapie in sheep and human cjd cases . hereby we assume that the position of pk - cleavage for prion in sheep with scrapie might differ from that in sheep with bse . thus , biochemical differences found between tse strains ( scrapie strains of hamster , scrapie of sheep , and bse of cattle ) have been reported . 6 , 13 p4 , 2d6 , 3f4 , kg9 , 6h4 ( prionics ag ). 9 , 10 , 11 , 14 , 15 we selected the bovine prp 108 - 119 aa sequence . the selected peptide cggqwnkpskpktn , corresponding to the bovine prp aa - sequence 108 - 119 plus the additional cg for flexibility and coupling , was synthesised from fmoc aa . coupling of klh to the n - terminal side of the peptide was done using a bifunctional linker mbs . the selected peptide was synthesised and coupled to klh before immunising a group of five prnp 0 / 0 mice ( charles weissmann ( zürich , switserland )). mice were immunised intraperitoneally using 30 μg of peptide suspended into freunds incomplete adjuvans / pbs . mice were boosted at 19 , 59 , and 136 days post immunisation ( dpi ). sera of the mice were collected at 73 dpi and analysed using various immuno - assays . two different assays were used for detecting the peptide specific responses — anti - peptide elisa and anti - peptide ria —, whereas also assays were used to detect responses against prp c ( prp c elisa ) and prp sc ( scrapie / bse dot - blot ) elisa plates were coated with bsa - coupled peptide and incubated with various dilutions of mice sera that were collected at − 7 and 73 dpi . bound antibodies were detected using a goat - anti - mouse - hrp conjugate and using tmb as a substrate . radiolabelled bsa - coupled peptide ( 125 i - peptide , iodogen method ) was incubated with mice sera . subsequently bound and free - labelled peptide was separated by addition of rat - anti - mouse - immunoglobulin coupled sepharose , incubation and centrifugation . gamma counter quantified radioactivity in the pellet . elisa plates were coated with normal bovine or ovine crude brain homogenate and incubated with various dilutions of mice sera that were collected at − 7 and 73 dpi . then the bound antibodies were detected using a goat - anti - mouse - immunoglobulin - hrp conjugate and using tmb as a substrate . strips of pvdf membrane were spotted with pk - treated crude brain homogenate derived from normal sheep , scrapie - infected sheep , normal cattle and bse - infected cattle . samples were diluted three - fold and nine - fold and 3 μl spots were applied in duplicate . subsequently the strips were incubated with various dilutions of mice sera that were is collected 7 days before immunisation and 73 dpi . bound antibodies were detected using a goat - anti - mouse - immunoglobulin - hrp conjugate , enhanced chemiluminescence as substrate , and visualised using nbt , bcip as substrate . four days after the final booster , spleen cells of a selected mouse were fused with sp2 / 0 - ag14 myeloma cells in a 3 : 1 ratio and were plated in 96 - well cluster plates ( density 10 5 cells / well ). hybridoma culture supernatants were screened for presence of specific antibodies by anti - peptide ria and scrapie / bse dot - blot assay one and two weeks later . clones giving positive results in both anti - peptide ria and scrapie / bse dot - blot assays were subcloned three times by limiting dilution . anti - peptide ria was used to monitor production of specific antibodies during the cloning steps . after three limiting dilution subcloning cycles , the finally selected clones were characterised using western blotting , spot - blotting , pepsean , immuno - histochemistry , and cdr3 . western blotting was carried out according to the prionics check kit specifications ( prionics ag ). briefly , proteins present in pk - treated or crude brain homogenates derived from normal sheep , scrapie infected sheep , normal cattle and bse infected cattle were first separated on sds - 12 . 5 % polyacrylamide gels and blotted on pvdf membranes . subsequently the membranes were incubated with selected mab supernatants . bound antibodies were detected using a goat - anti - mouse - hrp conjugate , enhanced chemiluminescence as substrate , and visualised by exposure to ecl hyperfilm . protease - k - treated or crude brain homogenates derived from normal sheep , scrapie infected sheep , normal cattle and bse infected cattle were diluted three - fold and nine - fold and 5 μl spots were applied to a nitrocellulose membrane . after drying and blocking the membranes were incubated with selected mab supernatants . bound antibodies were detected using a goat - anti - mouse - immunoglobulin hrp conjugate , enhanced chemiluminescence as substrate , and visualised by exposure to ecl hyperfilm . solid phase synthesis of peptides on polyethylene pins and immunoscreening by elisa were carried out according to established pepscan ™ procedures . 18 , 19 complete sets were synthesised of overlapping 15 - mer peptides in 3 μl polyethylene wells , covering the amino sequences of human , bovine , ovine , murine and hamster prp and binding of antibodies to these sets was determined . 20 , 21 , 22 , 23 , 24 tissues were collected and embedded in paraffin and sections were cut , and stained with ) 1e4 ( 1 : 10 dilution ). 25 the v h region of clone 1e4 was rt - pcr amplified using primers as described by dattamajumdar et al . 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