Prion-binding activity in serum and proteins

Disclosed are methods and tools for the concentration and detection as well as quantification of pathological prion proteins as well as agents to be used in said detection and/or in the prevention or treatment of prion diseases. Said agents are factors with prion binding activities found in blood serum and blood plasma.

DETAILED DESCRIPTION F THE INVENTION As already mentioned above, there is a great need for a detection method for low concentrations of PrP Sc that can be used as a diagnostic test for transmissible spongiform encephalopathies (TSEs). There are basically three diagnostic principles for TSEs: histopathological detection of the typical spongiform changes in the CNS, detection of the scrapie-specific isoform of the prion protein, and the bioassay that detects infectivity. All these methods have limitations: histopathology is not useful for preclinical diagnosis since the structural changes appear late in the incubation period. Detection of the scrapie the Western specific is form of prion protein is more sensitive but still much less sensitive than the bioassay. The bioassay can, in principle, detect as little as 1 infectious unit but can last months or even years. The hitherto used Western blot technique is based on the partial protease resistance of PrP Sc that allows to distinguish between Prp C and PrP Sc . After protease treatment, PrP 27-30 —the protease resistant core of PrP Sc —can be detected but not PrP C which is completely digested. Although due to the “stickiness” of prions it was generally assumed that immuno affinity purification (IAP) cannot be applied, it has now been found that concentration can be achieved by applying magnetic beads (MB) carrying a prion binding site, preferably a factor with prion protein binding activity (PrPB). Thus, because the sensitivity of detection of absolute amounts of PrP 27-30 is a function of antibody affinity, and cannot be easily increased for each given antibody, in the scope of the present invention, despite of the hitherto assumed problems, first an “immuno affinity purification” (IAP) assay has been developed, using antibodies covalently crosslinked to solid phase material, e.g. magnetic beads. Because the monoclonal antibody (6H4 purchased at Prionics, Zurich, Switzerland, described in Korth et al., 1997), originally used for the development of the IAP, is not able to distinguish between PrP C and PrP Sc (it binds both undigested forms as well as digested PrP Sc , i.e. PrP 27-30 ), it is necessary to perform Proteinase K digestion prior to the IAP (see FIG. 1 ). For the development of the present IAP method, the following model system was used: Two tests were performed to determine the efficiency of the method. On the one hand, small amounts of a scrapie-infected mouse brain homogenate were diluted with water and then subjected to the PrP Sc concentration method. On the other hand, small amounts of a scrapie-infected brain homogenate was diluted with brain homogenate of non-infected mice in order to simulate a real situation in which a brain homogenate contains low amounts of PrP Sc (see FIG. 2 ). In FIG. 2 , lanes 1 to 6 and 10 represent usual Western Blots and lanes 7 to 9 and 11 to 13 represent immuno affinity purification (IAP). PrnP % is material from PrP deficient mice. MB are of course only used for IAP whereby 6H4 refers to MB coupled with 6H4 antibodies and − refers to uncoupled MBs. PRP C refers to brain homogenate of non-infected mice and PrP Sc refers to brain homogenate of scrapie-infected mice. PK refers to Proteinase K digestion whereby − refers to non digestion and &plus; to digested homogenate. The same abbreviations are used for the following figures. For prion analysis in homogenate, in particular of brain tissue, it is important to use in a first homogenation step low concentration of ionic detergent, followed by low speed centrifugation, preferably 500 g 30 minutes, 4° C. applied twice. For following steps high concentration of non-ionic detergent is used and a protein concentration of the homogenate of at most 5 mg/ml. Conditions for the proteinase K digestion are preferably 50 &mgr;g/ml PK, 37° C. and at least half an hour. Suitable incubation conditions for the beads with homogenate are e.g. about 1.5 hours at room temperature, whereby for low concentrations longer incubation times might be preferable. The concentration step in said first attempt was carried out by adding to digested homogenate magnetic beads (MB) carrying said 6H4. If a digestion step is needed, it has to be performed prior to the concentration step, whereby the digestion, usually by proteinase K, has to be stopped prior to the concentration step by deactivating the proteinase e.g. with phenyl methyl sulfonyl fluoride or another agent known to the skilled person. By applying the method of the present invention for e.g. brain tissue homogenate, PrP 27-30 can be concentrated up to amounts detectable by Western blot analysis from tissue comprising much less pathological prion protein than needed for the hitherto known tests. Using largely the same procedure, the above described method can also be applied as prion affinity assay (PAA) by exchanging the monoclonal antibody 6H4 by other substances to be examined, for example in order to find a binding partner for PrP Sc (see FIG. 3 ). As a positive control of this assay 6H4 (see FIG. 4 , lanes 1 - 3 ) is used and as a negative control mouse IgG or mouse albumin (see FIG. 4 , lanes 4 - 9 ). In order to investigate whether a given mouse serum containes IgG that specifically recognize PrP Sc magnetic beads that are already coated by the company DYNAL with sheep antibodies directed against mouse IgGs were used after preincubation with mouse serum. These beads —used without preincubation —were the first negative control (see FIG. 5 , lanes 1 - 2 ). As a second negative control these beads preincubated with normal mouse serum were used in order to show that IgGs from normal mouse serum do not bind to any form of PrP (see FIG. 5 , lanes 3 - 4 ). Surprisingly the beads alone showed an affinity to PrP Sc but not to PrP 27-30 . Upon preincubation with normal mouse serum also PrP 27-30 is bound. Therefore it was hypothised that the sheep antibodies from DYNAL recognize a molecule that is associated with PrP Sc but digested away after PK-treatment. As PrP 27-30 is bound upon preincubation with normal mouse serum, this serum might contain the molecule with affinity to PrP Sc . The beads coupled to total mouse serum proteins did not show any affinity to any form of PrP. However, if the coupling of the total serum was performed in the presence of an excess of protein the beads showed the same binding to PrP 27-30 as the monoclonal antibody 6H4 (see FIG. 6 , lanes 4 - 6 ) whereas the beads that were coupled in the presence of an excess of albumine still did not show any affinity to any form of PrP (see FIG. 6 , lanes 1 - 3 ). Though it was not possible to measure any difference of the coupling efficency of the two conditions it might be that offering an excess of proteins causes a sponge on the surface of the beads that binds PrP 27-30 . We also checkled whether PK-treated brain homogenate might enhance the binding as in the case of bound PrP 27-30 total PK-digested brain homogenate is present: the addition of PK-digested brain homogenate from wild-type C57BL/6 mice or Prnp % mice allowed to bind PrP Sc in addition to PrP 27-30 (see FIG. 7 , lanes 1 - 3 ); the addition of inactive PK had no influence on the binding activity (see FIG. 7 , lanes 7 - 9 ). If coupled in the presence of an excess the activity of binding PrP 27-30 was also found in the serum of man, sheep, cow and in the serum of terminally scrapie-sick C57BL/6 mice (data not shown). Apart from an artefact it might well be that serum of several species contains activities (collectively termed PrP B ) that interact specifically with the pathogenic isoform of the prion protein and that are kinetically favoured in binding to the beads. The affinity to PrP 27-30 could then be understood assuming that native PrP Sc present in sick mice is saturated with PrP B which might be released upon proteolytic digest. Alternatively, partial proteolysis may expose PrP B binding sites on PrP Sc . However, the fact that the addition of PK-treated brain homogenate allows to bind PrP Sc indicates that there might be several different interactions leading to our observations. The template-directed refolding hypothesis predicts that PrP C and PrP Sc form heterodimers during the conversion process. Therefore we investigated whether PrP B is identical with PrP c . However, when coupling in excess PrP B activity was present in the serum of Prnp % mice at levels similar to those of wild-type mice, implying that PrP c does not contribute to the binding activity (see FIG. 8 ). If PrP B activity is not only caused by the special coupling conditions, it should be possible to “purify” it by fractionating mouse serum by differential ammonium sulfate precipitation. Indeed, it was possible to precipitate PrP B at an ammonium sulfate saturation below 50% whereby coupling of each fraction was performed in the presence of an excess of protein (see FIG. 9 ). While purified rabbit immunoglobulins against total mouse serum did not contain PrP B (data not shown), they efficiently bound PrP 27-30 upon preincubation with full mouse serum (see FIG. 10 , lanes 1 - 3 ) or with proteins precipitating between 25% and 50% ammonium sulfate saturation (see FIG. 10 , lanes 4 - 6 ). Preincubation with proteins precipitating between 75% and 100% ammonium sulfate saturation did not lead to PrP B activity (see FIG. 10 , lanes 7 - 9 ). This finding is important as it shows that the PrP B activity is a property of one or more serum proteins independent of the covalent crosslink to the surface of the beads. As the ammonium sulfate fractionation worked with human serum as well (data not shown), 58 fractions of human plasma were obtained by chromatography and differential precipitation and tested for binding activity to form an idea of the identity of PrP B . All fractions were not coupled in the presence of an excess of proteins. Therefore the results can directly be compared with 6H4 or mouse IgG. 20 fractions tested positive: Plasminogen, fibrinogen, antithrombin III, antithrombin III heparin complex, C1 esterase inhibitor, factor IX and several fractions containing protein mixtures (see FIG. 11 ). Purified plasminogen and also purified fibrinogen bound PrP Sc in addition to PrP 27-30 (see FIG. 12 ). Out of the 38 fractions that tested negative, 6 contained purified proteins: Prothrombin complex concentrate, albumin, activated prothrombin complex concentrate, factor XIII and thrombin. As mentioned, there are some hints that the binding of PrP 27-30 is caused by different effects. The activity that binds PrP 27-30 is termed spPrP B (s&equals;serum and p&equals;plasma) as it is present in serum and in plasma. Said activity is comparable to the activity found for plasminogen and fibrinogen. Plasminogen and fibrinogen were furthermore characterized as they both bind also PrP Sc . As calcium is an important cofactor in the coagulation cascade it was investigated whether PrP B activity is still intact if coagulation is inhibited by complexing calcium. In the presence of 10 mM EDTA the pathogenic PrP Sc and PrP 27-30 were still bound by plasminogen (see FIG. 13 , lanes 1 - 3 ) but only PrP 27-30 by fibrinogen (see FIG. 13 , lanes 4 - 6 ). At least in the case of plasminogen this finding speaks against the possibility that the PrP B activity is due to unspecific coagulation. Because PrP B selectively interacts with the pathogenic PrP but not with PrP c , interaction may be conformation-specific. When the assay was carried out in the presence of 6 M urea the fraction containing purified plasminogen didn't bind PrP Sc nor PrP 27-30 (see FIG. 14 , lanes 8 - 9 ) under these conditions PrP Sc becomes protease-sensitive (see FIG. 14 , lanes 14 - 15 ). As the conformation of PrP Sc is thought to be responsible for the PK resistancy we conclude from this experiment that the interaction of plasminogen and PrP Sc is conformation-dependant. Furthermore it could be shown that PrP B activity of plasminogen is not dependent on the covalent crosslink to the beads by using magnetic beads coated with antibodies directed against plasminogen and preincubated with plasminogen (see FIG. 15 , lanes 3 - 4 ). There are two negative controls: 1. If beads coated with antibodies against plasminogen are not at all preincubated (see FIG. 15 , lanes 1 - 2 ) or preicubated with albumin (see FIG. 15 , lanes 5 - 6 ), the pathogenic isoform of PrP is not bound. 2. If beads coated with albumin are preincubated with plasminogen there is also no binding to the pathogenic isoform of PrP (see FIG. 15 , lanes 7 - 8 ). Furthermore it could be shown that at least spPrP B does not only bind the pathogenic PrP but also infectivity. For this purpose we inoculated indicator tga20 mice i.c. with 0.2% of the paramagnetic beads before eluting the other 99% of the beads and performing a western blot (see FIG. 16 ). The animals that were inoculated with beads that bind the pathogenic PrP did all develop the disease (see FIG. 17 , lanes 4 , 5 and 7 ). It was also determined whether the interaction between plasminogen and disease-associated prion protein represents a universal feature of spongiform encephalopathies. Human plasminogen (100 &mgr;g) was linked to tosyl-activated paramagnetic Dynabeads M-280 (Dynal, Oslo, 1 ml). Brain tissues from a healthy mouse ( FIG. 18 , lane 1 ), a scrapie-affected mouse (lanes 2 - 3 ), pooled brains of Swiss non-affected cows (lane 4 ) and brains of BSE-affected cows of various breeds (lanes 5 - 10 ) were homogenized as described and tested for the presence of PrP Sc . For this, 50 &mgr;g (mouse) or 1 mg (cow) homogenate were incubated with paramagnetic beads coupled to anti-PrP monoclonal antibody 6H4 (data not shown), BSA (negative control; data not shown), or plasminogen. Bead eluates (24 &mgr;l) were run on SDS-PAGE (5% stacking -12% resolving) and blotted on nitrocellulose membranes (Schleicher & Schuell, Dassel). For detection of disease-associated PrP, membranes were incubated with 6H4 (Prionics, Zurich)as primary antibody and rabbit-&agr;-mouse IgG 1 -HRP (Zymed, San Francisco) as secondary antibody. Membranes were then developed using ECL detection reagents. Signals were recorded on film and/or quantified using a Kodak Image Station. In all cases, plasminogen immobilized to magnetic beads captured PrP Sc from each species when subjected to the precipitation assay. It has been reported that various breeds of sheep are variably susceptible to scrapie. Susceptibility was mapped to polymorphisms at codons 136 , 154 , 171 within the sheep Prnp gene. Because these polymorphisms occur at the carboxy terminus of the protein and affect basic amino acids, and indirect evidence implies that the carboxy terminus of PrP Sc may participate to the binding to plasminogen, we have investigated whether genetic susceptibility to scrapie in sheep might correlate with the ability of PrP Sc to bind plasminogen. Brain tissue from non-affected and scrapie-affected sheep with the Prnp genotypes at codons 136 , 154 and 171 of VHQ/ARQ ( FIG. 18 , lanes 11 - 13 ), VRQ/ARQ (lanes 14 - 16 ), and VRQ/ARR (lane 17 - 19 ) were homogenized and subjected to the prion affinity assay. Plasminogen precipitated PrP Sc from all sheep genotypes investigated. FIG. 18 eluates from plasminogen beads incubated with brain homogenates were subjected to Western blot analysis. Species and breeds are indicated over the respective lanes. Infection with scrapie or with BSE, and digestion of samples with proteinase K, are marked with “&plus;” and “−” signs. Numbers listed underneath each lane indicate individual cows and sheep of various breeds and Prnp genotypes. Plasminogen beads immobilized PrP Sc in all samples tested. In addition, we tested brain tissues (500 &mgr;g) from several patients who died of sporadic Creutzfeldt-Jakob disease, Alzheimer's disease ( FIG. 19 ) and Binswanger's disease (data not shown) with the prion affinity assay. In all assays performed with homogenates of CJD patients, plasminogen was able to precipitate PrP Sc , while no signal was detectable with homogenates of non-CJD patients. Unambiguous positive signals were obtained from cases with plaque-like, patchy-perivacuolar and synaptic pattern of PrP depositions. The intensity of the Prp CJD signals in the precipitation assays correlated closely with histopathological findings ( FIG. 19 ). In FIG. 19 plasminogen precipitated PrP CJD from brain homogenate of three Swiss sCJD patients (a, b, c) exhibiting extensive plaque-like (a) or scant synaptic accumulation (b, c) of PrP CJD . For control we used brain homogenate from a patient suffering from Alzheimer's disease (d). Proteinase K digestion was carried out as indicated with “&plus;” signs over the corresponding lanes. Corresponding brain sections immunostained with antibody 3F4 (available from Dr. Richard Kascksak, Albert Einstein College, The Bronx, N.Y., USA or Draco, Denmark, Botrup) to PrP are displaced on the right side. In each case, the plasminogen-based assay and the Western blot show congruent results. In Alzheimer's disease, PrP C was detectable (−), but not PrP Sc (&plus;). Scale bars are 50 &mgr;m. 
 EXAMPLES 
 Example 1: IAP method The IAP protocol is the following: Bring the brain tissue in a 15 ml FALCON tube, put it on ice and leave it there for all steps. Add Homogenate Buffer (0.5% DOC/0.5% NP-40 in PBS) to get 10% (w/v) homogenate. Pass the tissue through a 18 gauge needle and a 22 gauge needle by sucking up and down for 15 times each. Centrifuge the homogenate for 30 minutes at 500 g and 4° C. Keep the supernatant. Determine the protein concentration. Centrifuge the homogenate for 30 minutes at 500 g and 4° C. Keep the supernatant. If the protein concentration is higher than 10 mg/ml then bring the homogenate to a protein concentration of 10 mg/ml using the homogenate buffer. Bring the homogenate to a protein concentration of 5 mg/ml and 3% Tween 20/3% NP-40 all in PBS. Add to the tissue homogenate Proteinase K to get a final concentration of 50 &mgr;g/ml. Incubate for 60 minutes at 37° C. Add PMSF to get a final concentration of 5 mM. Add 0.25 volumes of IAP buffer (3% Tween 20/3% NP-40 in PBS). Resuspend the magnetic beads (covered with 6H4) according to the protocol described below) thoroughly. Pipette out 100 &mgr;l. Remove buffer. Add the homogenate to the beads and incubate the bead-sample mixture with continous mixing for 1.5 hours at room temperature. Collect the beads using the MPC (strong magnet). Wash three times with 1 ml Washing Buffer (2% Tween 20/2% NP-40 in PBS) and once with 1 ml PBS by vortexing for 15 seconds at room temperature and by using the MPC. Spin down the beads, discard the remaining supernatant using again the MPC. Add 24 &mgr;l ×Loading Buffer (50 mM Tris pH 6,8; 2% SDS; 0.01% bromphenol blue; 10% glycerol) . Heat to 95° C. for 5 minutes. If the samples are stored at −20° C. then heat them again for 30 seconds at 95° C. before performing SDS-PAGE followed by western Blot: Assemble the glass plates according to the manufacturer's instructions. Prepare in a Falcon tube the appropriate volume of the Resolving Gel (2.1 ml H 2 O, 1.5 ml 40% Acrylamid, 1.3 ml 1.5 M Tris pH 8.8, 50 &mgr;l 10% SDS, 50 &mgr;l 10% Ammoniumpersulfat, 2 &mgr;l TEMED). Mix the components in the order shown. Polymerization will begin as soon as the TEMED has been added. Pour the acrylamide solution into the gap between the glass plates. Leave sufficient space for the stacking gel (the length of the comb plus 1 cm). Using a pasteur pipette carefully overlay the acrylamide with water. Place the gel in a vertical position at room temperature. After polymerization is complete (30 minutes), pour off the overlay and wash the top of the gel several times with deionized water to remove any unpolymerized acrylamide. Prepare in a Falcon tube the appropriate volume of the Stacking Gel (1.48 ml H20, 0.25 ml 40% Acrylamid, 0.25 ml 1.0 M Tris pH 6.8, 20 &mgr;l 10% SDS, 20 &mgr;l 10% Ammoniumpersulfat, 2 &mgr;l TEMED). Mix the components in the order shown. Polymerization will begin as soon as the TEMED has been added. Pour the stacking gel solution directly onto the surface of the polymerized resolving gel. Immediately insert a clean Teflon comb into the stacking gel solution, being careful to avoid trapping air bubbles. Place the gel in a vertical position at room temperature. After polymerization is complete (30 minutes), remove the Teflon comb carefully. Mount the gel in the electrophoresis apparatus. Add Running buffer to the top and bottom reservoirs. Remove any (25 mM Tris, 250 mM glycine, 0.1% SDS) bubbles that become trapped at the bottom of the gel between the glass plates. Load 24 &mgr;l of each of the samples in a predetermined order into the bottom of the wells (1. well: Low -range marker). Load an equal volume of 1×Gel-loading Buffer into any wells that are unused. Attach the electrophoresis apparatus to an electric power supply (the positive electrode should be connected to the bottom reservoir). Apply 10 V/cm to the gel. After the dye front has moved into the resolving gel (30 minutes), increase the voltage to 14 V (cm and run the gel until the bromophenol blue reaches the bottom of the resolving gel (1 hour). Then turn off the power supply. Cut six sheets of absorbent paper (Whatman 3 MM or equivalent) and one sheet of nitrocellulose to the size of the gel (6 cm×8 cm). If the paper overlaps the edge of the gel, the current will short-circuit the transfer and bypass the gel, preventing efficient transfer. Wet the absorbent paper, the nitrocellulose and the gel by soaking in Transfer (39 mM glycine, 48 mM Tris, 0.037% SDS, 20% methanol) Buffer. On the bottom plate of the apparatus (the anode), assemble the gel, nitrocellulose, and paper in this order: bottom electrode, three layers absorbent paper soaked in transfer buffer, one nitrocellulose membrane soaked in transfer buffer, polyacrylamide gel slightly wetted with transfer buffer, three layers absorbent paper soaked in transfer buffer. Check carefully for air bubbles and gently remove them either by using a gloved hand or by rolling a pipet over the sandwich. Dry any buffer that may surround the gel-paper sandwich. Carefully place the upper electrode (the cathode) on top of the stack. Put a weight on it. Connect the electrodes and commence transfer. Running time is 1 hour with a current of 1 mA/cm 2 . After transfer, disconnect the power source. Carefully disassemble the apparatus. Mark membrane to follow orientation (usually by snipping off lower left-hand corner, the number one lane). Rinse the membrane three times with TBS-T. Add Blocking Buffer (5% (w/v) nonfat dry milk in TBS-T). Incubate at room temperature with agitation for 30 minutes. Rinse the membrane three times with TBS-T. Add to 2.5 &mgr;l of mAB 6H4 (2 mg/ml) 12.5 ml of 1% (w/v) nonfat dry milk in TBS-T. Incubate at room temperature with agitation for 1 hour or overnight at 4° C. Remove the membrane from the antibody solution and wash three times for 10 minutes each in TBS-T. Add to 1.25 &mgr;l of relativ anti mouce IgG1-HRP 12.5 ml of 1% (w/v) nonfat dry milk in TBS-T. Incubate at room temperature with agitation for 1 hour. Remove the membrane from the antibody solution and wash three times for 15 minutes each in TBS-T. Mix 1 ml of detection solution 1 with 1 ml of detection solution 2 from the ECL Western blotting detection reagents (Amersham Pharmacia Biotech). Incubate for precisely 1 minute at room temperature without agitation. Drain off excess detection reagent by putting the membrane on a absorbent paper. Gently place the membrane, protein side down, on a SaranWrap. Close SaranWrap to form a envelope avoiding pressure on the membrane. Place the membrane, protein side up, in the film cassette. Work as quickly as possible. Switch off the lights and carefully place a sheet of autoradiography film such as (Hyperfilm ECL) on top of the membrane, close the cassette and expose for some seconds (15″, 30″). 
 Example 2: PAA method Couple the protein of interest to magnetic beads: Bring 100 &mgr;g of protein into approx. 1 ml of Coupling Buffer (0.1 M borate buffer pH 9.5: dissolve 6.183 g H3BO3 in 800 ml distilled water, Adjust pH to 9.5 using 5 M NaOH and adjust volume to 1000 ml with distilled water; if necessary, change buffer by dialysis). (If coupling was performed in the presence of an excess, 1 mg was used for 1 ml of coupling buffer.) Make a homogeneous suspension of the Dynabeads M-280 Tosylactivated by Dynal using a pipette and by vortexing for approximately 1 min. Pipette out 1 ml of Dynabeads and wash as follows: Place the tube in the DYNAL MPC. Leave to separate for 2 minutes. Remove the supernatant taking care not to disturb the Dynabeads. Remove the tube from the Dynal MPC and resuspend the Dynabeads in PBS. Repeat these steps and resuspend the Dynabeads in the coupling buffer containing the antibodies. Incubate for 24 h at 37° C. with tilt rotation. Place the tube in the magnet for 3 minutes and remove the supernatant. Wash the coated Dynabeads six times: 2 × in PBS/BSA (add 0.1% (w/v) bovine serum albumin (final concentration) to PBS), pH 7.4 for 5 minutes at room temperature; 1 × in Blocking Buffer (0.2 M Tris pH 8.5 with 0.1% (w/v) BSA: dissolve 2.42 g Tris in 80 ml distilled water. Adjust pH to 8.5 using 1 M HCl, add 0.1% BSA and adjust volume to 100 ml with distilled water) for 4 h at 37° C.; 1 × in PBS/BSA, pH 7.4 for 5 minutes at room temperature; 1 × in 1% Tween 20 for 10 minutes; 1 × in PBS/BSA, pH 7.4 for 5 minutes at room temperature. Store the coated Dynabeads in PBS/BSA pH 7.4, 0.02% sodium azide. Then prepare Sample I: Add 1 ml of PAA Buffer (3% NP-40/3% Tween 20 in PBS) to 10 &mgr;l of not infected brain homogenate (Protein concentration 5 mg/ml; 0.5% DOC/0.5 NP-40). Then prepare Sample II and III: Add 1 ml of PAA Buffer (3% NP-40/3% Tween 20 in PBS) to 10 &mgr;l of infected brain homogenate (Protein concentration 5 mg/ml; 0.5% DOC/0.5 NP-40). Incubate Sample I and Sample II for 30 minutes at 37° C. without PK. Incubate Sample III for 30 minutes at 37° C. with PK at final concentration of 50 &mgr;g/ml (add 50 &mgr;l of PK 1 mg/ml). Add PMSF to all samples to get a final concentration of 5 mM (add 50 &mgr;l of 100 mM PMSF). Resuspend the Magnetic Beads thoroughly. Pipette out 100 &mgr;l. Add the beads to the Samples and incubate the bead-sample mixture with continuous mixing for 1.5 hours at room temperature. Collect the beads using the MPC. Wash three times with 1 ml Washing Buffer and once with 1 ml PBS by vortexing for 15 seconds at room temperature and by using the MPC. Spin down the beads, discard the remaining supernatant using again the MPC. Add 24 &mgr;l 1 × Loading Buffer. Heat to 95° C. for 5 minutes. If the samples are stored at −20° C. then heat them again for 30 seconds at 95° C. before loading on the gel. As a positive control of this assay 6H4 is used and as a negative control mouse IgG or mouse albumin (see FIG. 4 ). 
 Example 3 In order to investigate whether a given mouse serum containes IgG that specifically recognize PrP Sc magnetic beads that are already coated by the company DYNAL with sheep antibodies directed against mouse IgGs were used after preincubation with mouse serum. These beads were the first negative control. As a second negative control these beads preincubated with normal mouse serum were used in order to show that IgGs from normal mouse serum do not bind to any form of PrP. Surprisingly the beads alone showed an affinity to PrP Sc but not to PrP 27-30 . Upon preincubation with normal mouse serum also PrP 27-30 is bound (see FIG. 5 ). Therefore it was hypothised that the sheep antibodies from DYNAL recognize a molecule that is associated with PrP Sc but digested away after PK-treatment. As PrP 27-30 is bound upon preincubation with normal mouse serum, this serum might contain the molecule with affinity to PrP Sc . 
 Example 4 The beads coupled to total mouse serum proteins did not show any affinity to any form of PrP. However, if the coupling of the total serum was performed in the presence of an excess of protein the beads showed the same binding to PrP 27-30 as the monoclonal antibody 6H4 whereas the beads that were coupled in the presence of an excess of albumine still did not show any affinity to any form of PrP (see FIG. 6 ). Though it was not possible to measure any difference of the coupling efficency of the two conditions it might be that offering an excess of proteins causes a sponge on the surface of the beads that binds PrP 27-30 . 
 Example 5 We also checkled whether PK-treated brain homogenate might enhance the binding as in the case of bound PrP 27-30 total PK-digested brain homogenate is present: the addition of PK-digested brain homogenate from wild-type C57BL/6 mice or Prnp % mice allowed to bind PrP Sc in addition to PrP 27-30 ; the addition of inactive PK had no influence on the binding activity (see FIG. 7 ). 
 Example 6 If coupled in the presence of an excess the activity of binding PrP 27-30 was also found in the serum of man, sheep, cow and in the serum of terminally scrapie-sick C57BL/6 mice (data not shown). 
 Example 7 The template-directed refolding hypothesis predicts that PrP C and PrP Sc form heterodimers during the conversion process. Therefore we investigated whether PrP B is identical with PrP C . However, when coupling in excess PrP B activity was present in the serum of Prnp % mice at levels similar to those of wild-type mice, implying that PrP C does not contribute to the binding activity (se FIG. 8 ). 
 Example 8 If PrP B activity is not only caused by the special couspling conditions, it should be possible to “purify” it by fractionating mouse serum by differential ammonium sulfate precipitation. Indeed, it was possible to precipitate PrP B at an ammonium sulfate saturation below 50% whereby coupling of each fraction was performed in the presence of an excess of protein (see FIG. 9 ). While purified rabbit immunoglobulins against total mouse serum did not contain Prp B (data not shown), they efficiently bound PrP 27-30 upon preincubation with full mouse serum or with proteins precipitating between 25% and 50% ammonium sulfate saturation. Preincubation with proteins precipitating between 75% and 100% ammonium sulfate saturation did not lead to Prp B activity (see FIG. 10 ). This finding is important as it shows that the PrP B activity is a property of one or more serum proteins independent of the covalent crosslink to the surface of the beads. 
 Example 9 As the ammonium sulfate fractionation worked with human serum as well (data not shown), 58 fractions of human plasma were obtained by chromatography and differential precipitation and tested for binding activity to form an idea of the identity of PrP B . All fractions were not coupled in the presence of an excess of proteins. Therefore the results can directly be compared with 6H4 or mouse IgG. 20 fractions tested positive: Plasminogen, fibrinogen, antithrombin III, antithrombin III heparin complex, C1 esterase inhibitor, factor IX and several fractions containing protein mixtures (see FIG. 11 ). Purified plasminogen and also purified fibrinogen bound PrP Sc in addition to PrP 27-30 (see FIG. 12 ). Out of the 38 fractions that tested negative, 6 contained purified proteins: Prothrombin complex concentrate, albumin, activated prothrombin complex concentrate, factor XIII and thrombin. 
 Example 10 As calcium is an important cofactor in the coagulation cascade it was investigated whether PrP B activity is still intact if coagulation is inhibited by complexing calcium. In the presence of 10 mM EDTA the pathogenic PrP Sc and PrP 27-30 were still bound by plasminogen but only PrP 27-30 by fibrinogen ( FIG. 13 ). At least in the case of plasminogen this finding speaks against the possibility that the PrP B activity is due to unspecific coagulation. 
 Example 11 Because Prp B selectively interacts with the pathogenic PrP but not with PrP C , interaction may be conformation-specific. When the assay was carried out in the presence of 6 M urea the fraction containing purified plasminogen didn't bind PrP Sc nor PrP 27-30 ; under these conditions PrP Sc becomes protease-sensitive ( FIG. 14 ). As the conformation of PrP Sc is thought to be responsible for the PK resistancy we conclude from this experiment that the interaction of plasminogen and PrP Sc is conformation-dependant. 
 Example 12 Furthermore it could be shown that PrP B activity of plasminogen is not dependent on the covalent crosslink to the beads by using magnetic beads coated with antibodies directed against plasminogen and preincubated with plasminogen ( FIG. 15 ). 
 Example 13 Furthermore it could be shown that at least spPrP B does not only bind the pathogenic PrP but also infectivity. For this purpose we inoculated indicator tga20 mice i.c. with 0.2% of the paramagnetic beads before eluting and performing a western blot. The animals that were inoculated with beads that bind the pathogenic PrP did all develop the disease ( FIG. 16 , FIG. 17 ). 
 Example 14 The prior art offers a large number of options for determining and characterizing the binding characteristics of a given peptide or protein to a certain target. Binding assay for determining the selectivity of a PrPSc specific binding partner using solid state-bound technologies includes e.g. microtiter plate formats, paramagnetic beads, non-magnetic beads, plasmon surface resonance, interferometry, coincidence detection, mass spectrometry/mass spectroscopy, electrospray analysis, and combinations thereof. For use in the present invention the following two approaches are preferred. 1. The peptides or protein or fragments thereof to be tested are coupled to a solid phase material: a. Use micro particles such as magnetic beads as solid phase and perform immunopreciptiation. Couple the peptides to magnetic beads. Incubate the beads with PrPSc, PrP 27 - 30 or PrPC. Detect whether the prion protein has bound to the peptides either by Western blot analysis or by microparticle immunoassay. b. Use surface of a micro titer plate as solid phase and perform ELISA. Coat the surface of the wells of a micro titer plate with the peptides. Add PrPSc, PrP 27 - 30 or PrPC to the wells. Detect whether the prion protein has bound to the peptides by ELISA. 2. PrPSc (or PrP 27 - 30 ) and PrPC are coupled to a solid phase material: a. Use micro particles such as magnetic beads as solid phase and perform immunopreciptiation. Couple PrPSc (or PrP 27 - 30 ) and PrPC, respectively, to magnetic beads. Incubate the beads with the peptides or protein fragments to be tested. Detect whether the peptides have bound to the PrPSc but not to PrPC either by Western blot analysis or by microparticle immunoassay. b. Use surface of a micro titer plate as solid phase and perform ELISA. Coat the surface of the wells of a micro titer plate with PrPSc (or PrP 27 - 30 ) and PrPC, respectively. Add peptides or protein fragments to be tested to the wells. Detect whether the peptides have bound to PrPSc but not to PrPC by ELISA. 
 Example 15 The prior art offers many possibilities to determine and detect certain parts of a protein which are involved in specific binding of the protein to a certain target. Method to identify suitable fragments of plasminogen as PrP Sc specific binding partners includes e.g. forward genetic selection using phage display, ribosomal display, bacterial protein fragment affinity assay, and combinations or derivations thereof. Accordingly, those parts of plasminogen that are involved in the specific binding to PrP Sc can be determined as follows: 1. Produce peptide libraries of plasminogen fragments and/or mutants displayed on phage, expose them to a solid phase coated with the pathological prion protein and select for the clones with the maximal binding affinity to PrPSc but minimal affinity to PrPC. 2. Express fragments and/or mutants of plasminogen in a host cell such as bacteria, yeast, fungi or eukaryotic cells, purify the peptides, label them and test them for binding activity. 3. Express fusion proteins with plasminogen fragments and/or mutants in a host cell and test them for PrPSc affinity in a binding assay. References Aguzzi, A. (1997). Neuro-immune connection in spread of prions in the body&quest; The Lancet 349, 742-743. Aguzzi, A. (1998). Protein conformation dictates prion strain. Nat Med 4, 1125-6. Aguzzi, A., Blattler, T., Klein, M. A., Raber, A. J., Hegyi, I., Frigg, R., Brandner, S., and Weissmann, C. (1997). Tracking prions: the neurografting approach. Cell Mol Life Sci 53, 485-95. Aguzzi, A., and Collinge, J. (1997). Post-exposure prophylaxis after accidental prion inoculation. Lancet 350, 1519-20. Aguzzi, A., and Weissmann, C. (1997). Prion research: the next frontiers. Nature 389, 795-798. Aguzzi, A., and Weissmann, C. (1998). Spongiform encephalopathies. The prion's perplexing persistence. Nature 392, 763-4. Aguzzi, A., and Weissmann, C. (1996). Spongiform encephalopathies: a suspicious signature. Nature 383, 666-7. Bernoulli, C., Siegfried, J., Baumgartner, G., Regli, F., Rabinowicz, T., Gajdusek, D. C., and Gibbs, C. J. (1977). Danger of accidental person-to-person transmission of Creutzfeldt-Jakob disease by surgery &lsqb;letter&rsqb;. Lancet 1, 478-479. Blattler, T., Brandner, S., Raeber, A. J., Klein, M. A., Voigtlander, T., Weissmann, C., and Aguzzi, A. (1997). PrP-expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 389, 69-73. Brandner, S., Isenmann, S., Raeber, A., Fischer, M., Sailer, A., Kobayashi, Y., Marino, S., Weissmann, C., and Aguzzi, A. ( 1996 ). Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature 379, 339-43. Bruce, M. E., Will, R. G., Ironside, J. W., McConnell, I., Drummond, D., Suttie, A., McCardle, L., Chree, A., Hope, J., Birkett, C., Cousens, S., Fraser, H., and Bostock, C. J. (1997). Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent &lsqb;see comments&rsqb;. Nature 389, 498-501. Chazot, G., Broussolle, E., Lapras, C., Blattler, T., Aguzzi, A., and Kopp, N. (1996). New variant of Creutzfeldt-Jakob disease in a 26-year-old French man &lsqb;letter&rsqb;. Lancet 347, 1181. Duffy, P., Wolf, J., Collins, G., DeVoe, A. G., Streeten, B., and Cowen, D. (1974). Possible person-to-person transmission of Creutzfeldt-Jakob disease. N Engl J Med 290, 692-3. Fraser, H., Brown, K. L., Stewart, K., McConnell, I., McBride, P., and Williams, A. (1996). Replication of Scrapie in Spleens of Scid Mice Follows Reconstitution With Wild-Type Mouse Bone Marrow. Journal of General Virology 77, 1935-1940. Gibbs, C. J., Jr., Joy, A., Heffner, R., Franko, M., Miyazaki, M., Asher, D. M., Parisi, J. E., Brown, P. W., and Gajdusek, D. C. (1985). Clinical and pathological features and laboratory confirmation of Creutzfeldt-Jakob disease in a recipient of pituitary-derived human growth hormone. N Engl J Med 313, 734-8. Hill, A. F., Butterworth, R. J., Joiner, S., Jackson, G., Rossor, M. N., Thomas, D. J., Frosh, A., Tolley, N., Bell, J. E., Spencer, M., King, A., Al-Sarraj, S., Ironside, J. W., Lantos, P. L., and Collinge, J. (1999). Investigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples. Lancet 353, 183-9. Hill, A. F., Desbruslais, M., Joiner, S., Sidle, K. C., Gowland, I., Collinge, J., Doey, L. J., and Lantos, P. ( 1997 ). The same prion strain causes vCJD and BSE &lsqb;letter&rsqb; &lsqb;see comments&rsqb;. Nature 389, 448-50. Hill, A. F., Zeidler, M., Ironside, J., and Collinge, J. (1997). Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. Lancet 349, 99. Hilton, D. A., Fathers, E., Edwards, P., Ironside, J. W., and Zajicek, J. (1998). Prion immunoreactivity in appendix before clinical onset of variant Creutzfeldt-Jakob disease &lsqb;letter&rsqb;. Lancet 352, 703-4. Kitamoto, T., Muramoto, T., Mohri, S., Doh ura, K., and Tateishi, J. (1991). Abnormal isoform of prion protein accumulates in follicular dendritic cells in mice with Creutzfeldt-Jakob disease. J. Virol. 65, 6292-6295. Klein, M. A., Frigg, R., Flechsig, E., Raeber, A. J., Kalinke, U., Bluethmann, H., Bootz, F., Suter, M., Zinkernagel, R. M., and Aguzzi, A. (1997). A crucial role for B cells in neuroinvasive scrapie. Nature 390, 687-90. Klein, M. A., Frigg, R., Raeber, A. J., Flechsig, E., Hegyi, I., Zinkernagel, R. M., Weissmann, C., and Aguzzi, A. (1998). PrP expression in B lymphocytes is not required for prion neuroinvasion. Nat Med 4, 1429-33. Korth, C., Stierli, B., Streit, P., Moser, M., Schaller, O., Fischer, R., Schulz-Schaeffer, W., Kretzschmar, H., Raeber, A., Braun, U., Ehrensperger, F., Hornemann, S., Glockshuber, R., Riek, R., Billeter, M., Wuthrich, K., and Oesch, B. (1997). Prion (PrP Sc )-specific epitope defined by a monoclonal antibody. Nature 390, 74-7. Lasmezas, C. I., Cesbron, J. Y., Deslys, J. P., Demaimay, R., Adjou, K. T., Rioux, R., Lemaire, C., Locht, C., and Dormont, D. (1996). Immune system-dependent and -independent replication of the scrapie agent. J Virol 70, 1292-5. O'Rourke, K. I., Huff, T. P., Leathers, C. W., Robinson, M. M., and Gorham, J. R. (1994). SCID mouse spleen does not support scrapie agent replication. J. Gen. Virol. 75, 1511-1514. Raeber, A. J., Klein, M. A., Frigg, R., Flechsig, E., Aguzzi, A., and Weissmann, C. (1999). PrP-dependent association of prions with splenic but not circulating lymphocytes of scrapie-infected mice. EMBO J 18, 2702-2706. Schreuder, B. E., van Keulen, L. J., Smits, M. A., Langeveld, J. P., and Stegeman, J. A. (1997). Control of scrapie eventually possible&quest; Vet Q 19, 105-13. Schreuder, B. E., van Keulen, L. J., Vromans, M. E., Langeveld, J. P., and Smits, M. A. (1998). Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie. Vet Rec 142, 564-8. Vankeulen, L. J. M., Schreuder, B. E. C., Meloen, R. H., Mooijharkes, G., Vromans, M. E. W., and Langeveld, J. P. M. (1996). Immunohistochemical Detection of Prion Protein in Lymphoid Tissues of Sheep With Natural Scrapie. Journal of Clinical Microbiology 34, 1228-1231. Weber, T., and Aguzzi, A. (1997). The spectrum of transmissible spongiform encephalopathies. Intervirology 40, 198-212. Weissmann, C., and Aguzzi, A. (1997). Bovine spongiform encephalopathy and early onset variant Creutzfeldt-Jakob disease. Curr Opin Neurobiol 7, 695-700. Wells, G. A., Hawkins, S. A., Green, R. B., Austin, A. R., Dexter, I., Spencer, Y. I., Chaplin, M. J., Stack, M. J., and Dawson, M. (1998). Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Vet Rec 142, 103-6. Will, R., Cousens, S., Farrington, C., Smith, P., Knight, R., and Ironside, J. (1999). Deaths from variant Creutzfeldt-Jakob disease. Lancet 353, 9157-9158. Will, R., Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A, Poser S, Pocchiari M, Hofman A, and Smith (1996). A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347, 921-925.