WO2008122274A1

WO2008122274A1 - METHOD FOR ASSAYING PATHOLOGICALLY MODIFIED PRION PROTEIN (PrPSc)

Info

Publication number: WO2008122274A1
Application number: PCT/DE2008/000574
Authority: WO
Inventors: Claudia Engemann; Ulrike Krummrei; Christina König
Original assignee: Priontype Gmbh & Co. Kg
Priority date: 2007-04-04
Filing date: 2008-04-04
Publication date: 2008-10-16
Also published as: DE102007016324A1

Abstract

The invention relates to a method for assaying pathologically modified prion protein (PrPSc) in human body fluids, especially in human blood, plasma and serum samples.

Description

International patent application 4 April 2008

Applicant: Priontype GmbH Our sign: PTY-002 PCT

Method for the detection of pathologically altered prion protein

(PrP ^Sc )

The present invention relates to a method for the detection of pathologically altered prion protein (PrP ^Sc ) in human body fluids, in particular in human blood, plasma and serum samples.

Pathologically modified prion protein (PrP ^Sc ) is the causative agent of transmissible spongiform encephalopathies (TSE) in humans and in various animal species (eg BSE for bovine spongiform encephalopathy). These are infectious and always fatal degenerative diseases of the central nervous system. The resulting histopathological changes in the brain are associated with the accumulation of PrP ^Sc , a conformer of the naturally occurring cellular prion protein (PrP). The prion replication occurring in disease occurs by direct interaction between PrP and PrP, whereby PrP ^c adopts the conformation of the pathological PrP ^Sc . In contrast to PrP ^c, PrP ^Sc is characterized by an increased β-sheet fraction and a high resistance to proteases (eg proteinase K).

In humans, there are four types of TSE, Creutzfeldt-Jakob Disease (CJD), Gerstmann-Sträussler-Scheinker Syndrome (GSS), Familial Fatal Insomnia (FFI) and Kuru. The latter three diseases are extremely rare or geographically limited, while CJD is diagnosed at a rate of one million per year per year worldwide. In more than 90% of cases, CJD occurs sporadically (sporadic CJD, sCJD). In these cases, no source of infection can be detected. In addition, there are inherited or familial forms of CJD (fCJD) and iatrogenic CJD (iCJD), which are associated with external, mostly medical, e.g. through transplants.

However, since the end of 1995, the UK has been diagnosed with the first cases of unusual CJD, the so-called new variant of CJD or vCJD. According to the current state of knowledge, there is little doubt that the occurred cases of vCJD by the inclusion caused by the BSE agent. It is believed that this is via food intake, ie done orally. To date, 198 cases have been confirmed (as of January 2007, source: The National Creutzfeldt-Jakob Disease Surveillance Unit, Edinburgh, UK).

5 It is becoming increasingly apparent that infectious PrP ^Sc can be transmitted from animals to humans (BSE) and from human to human (Kuru). This goes hand in hand with the risk of transmission of PrP ^Sc through stored blood and transplants to an unmanageable number of blood and organ donation recipients. It is therefore essential to examine all stored blood for prions. Two criteria are to be considered: First, an easy to handle blood test for

.0 are available. Secondly, such a blood test must be highly sensitive, as only minimal amounts of PrP ^{c are} suspected in body fluids. The relatively long incubation period involves the risk that. Infected the pathogens, for example, by blood transfusions or organ transplants pass. This results in a high demand for the highly sensitive detection of prions in human blood.

_L 5 hi the literature various approaches for the highly sensitive detection of prions are described. One very sensitive approach is protein misfolding cyclic amplification (PMCA). Least undetectable levels of PrP ^Sc are stimulated to convert large amounts of PrP ^c, similar to the polymerase chain reaction. This method allows for the first time

JO shows the biochemical detection of PrP ^Sc in the blood of scrapie-infested hamsters (Castilla et al, 2005). It was also detected with this method PrP ^Sc from brain homogenate of a cow, which was 32 months previously orally experimentally infected with BSE-positive brain homogenate. The animal showed no clinical symptoms and was negative in standard Western Blot procedures. From brain samples from control animals no PrP ^{Sc could be} amplified

.5 (Soto et al, 2005). Furthermore, PrP ^{Sc was} detected in the blood of scrapie-infected hamsters in the presymptomatic phase (Saa et al, 2006). The serious disadvantage of this method, however, is the infectivity that is generated by the amplification. The accumulation of PrP ^Sc is dangerous; for obvious reasons, the blood processing industry will want to avoid any infectiousness. PrP ^Sc must be handled in S3 * 0 level safety labs, which is not practical in the context of blood bank screening.

Other publications describe prion detection in the blood of scrapie-infected sheep and red deer with chronic wasting disease in capillary electrophoresis (Schmerr et al, 1999) and the detection of femtomolar Concentrations of PrP ^Sc in cerebrospinal fluid of CJD patients with a prototype of two-color fluorescence correlation spectroscopy (Zeiss, Bieschke et al, 2000). These methods are supported by complex devices and are therefore not suitable for high throughput.

Another very sensitive test is the so-called improved CDI assay (Bellon et al, 2003); However, the test takes about 24 hours and is therefore not mature for the practice.

The currently most sensitive method of enzymatic signal amplification is the polymerase chain reaction (PCR) developed by K. Mullis in 1985. In addition to the

0 Amplification and modification of nucleic acid molecules for preparative purposes enables PCR to detect extremely sensitive target nucleic acids and has become widely used as an elementary tool in research and medical diagnostics. The cyclic amplification of the template ideally allows the detection of a single molecule of DNA. In contrast, the detection limit of a conventional

5 ELISAs typically at about 10 ^"1 - ^{10" 17} moles of antigen, and also by means of a radioimmunoassay (RIA) no less than 10 ^"18 moles individual approaches to development of highly sensitive enzymatic immunoassays chemiluminescent with a detection limit can be detected at. Although a zeptomol (about 600 molecules) has been published, it is not practicable as a routine procedure

'.0 Ultrasensitiver protein analysis are mainly in the extreme sensitivity of the detection method against nonspecific background signals, the development and adaptation of a methodology for an application example that can be transferred only with great effort to other applications and increased equipment, methodology and time , Therefore, the combination of the established PCR methodology for

Signal amplification with the specific antibody-antigen recognition of an ELISA, as described in 1992 by Sano et al. first published as Lnmuno-PCR (IPCR), particularly interesting. By combining the two standard methods of nucleic acid and protein analysis, a novel, extremely sensitive detection method for non-nucleic acid molecules could be presented in a simple modular design. The IPCR already allowed in a first

50 model attempt to improve the detection limit of a conventional ELISA by about five orders of magnitude. After PCR amplification of the marker DNA, the detection of the antigen is achieved indirectly via the quantitative analysis of the PCR products. Sano et al. Were able to detect 500 reference molecules of the model antigen bovine serum albumin (bovine serum albumin, BSA), which represents an over 1000-fold increase in sensitivity compared to conventional methods for protein analysis. While this approach allows for antigen-DNA coupling in one incubation step by using a protein chimera of streptavidin (STV) and protein A, the method is limited to detection of antigens from samples that do not contain immunoglobulin G (IgG). protein A binds to the Fc part of IgG molecules. An alternative approach that allows for the transfer of IPCR to commercially available reagents was published in 1993 by Zhou et al. Biotinylated antibodies are linked to biotinylated DNA via the STV-biotin interaction in two incubation steps, thus achieving coupling of the antigen with the marker DNA. This methodology is characterized by its enormous flexibility in addition to the easy commercial accessibility of biotinylated antibodies. Virtually any antibody and DNA fragment can be biotinylated. In addition, the sequence diversity of the DNA provides an almost inexhaustible reservoir of specific "marker molecules" for multiplexing approaches for the simultaneous detection of multiple antigens.The IPCR has hitherto been used to detect a wide variety of antigens, for example for quantifying bacterial antigens, as a comparison system for correlation with DNA. and mRNA analyzes or for the quantitation of human enzymes or messenger substances and in the research of prion diseases.

Thus, the technique of immuno-PCR was already used in the context of prion detection, but with worthy of discussion results. Gofflot et ah (2005) describe an immuno-quantitative PCR (iqPCR) for PrP detection in sporadic CJD, which is 10-fold more sensitive than a corresponding ELISA. This sensitivity is not sufficient for blood bank screening, especially when using pool samples.

A similar test was described by Barletta et al. (2005). However, this test appears to be less robust: Two PCR amplifications are performed, interrupted by over-pipetting into other reaction vessels and addition of fresh PCR reagents. The PCR expert understands that these conditions provide unreliable results. In addition, the authors themselves discuss that "variables", such as the ratio of biotin to streptavidin in the conjugate, are not consistent and cause variations in the test results to lead. The proteinase K digestion involves several steps that complicate the workup. Overall, this test is very sensitive, but in a developmental stage it seems far removed from the prototype stage. This does not seem like a procedure that allows reliable blood bank screening.

The invention is therefore based on the object to provide a more sensitive, faster and cheaper method for the detection of PrP ^Sc in human body fluids.

The object is achieved by the subject matter defined in the claims.

The following figures illustrate the invention.

Figure 1 shows a schematic representation of an immuno-PCR (IPCR) approach in sandwich format. The steps are analogous to an ELISA in sandwich format, but the detection antibody is not coupled with an enzyme but with DNA (marker DNA). Furthermore, the marker DNA is subjected to a PCR amplification. The quantification of the PCR product allows for indirect quantification of the antigen by comparison with calibration series of known antigen concentrations (external standards).

FIG. 2 shows in a graph the comparison of the titration of recPrP and the detection in ELIS A method and the IPCR method according to the invention.

Figure 3 shows the result of an anti-PrP western blot. Bovine brain homogenate PrP is spiked in serum with and without proteinase K treatment.

FIG. 4 shows in a graph the result of the detection of spiked recPrP in serum with the method according to the invention.

Figures 5 A and B show the result of an anti-PrP western blot. Bovine brain homogenate PrP was treated with various proteinase K concentrations. Figure 5 A: mAb with epitope in proteinase K resistant region of PrP; FIG. 5B: mAb with epitope in proteinase K-labile region of PrP. Figure 6 is a graph showing the result of comparing the signal strength of vCJD positive brain homogenate spiked in plasma with and without accumulation by IP. Without IP, a dilution of 1: 1000 is detected, while with IP, even the 1: 100000 dilution is clearly determined to be positive. The ff can therefore increase the proven amount of PrP 5 by a factor of 100.

The present invention relates to a method for the detection of pathologically altered prion proteins (PrP ^Sc ) in human body fluids, comprising the steps of

0 Incubating an isolated sample of the body fluid to be examined with a protease, and then

Immunoprecipitation to enrich PrP ^Sc, and then the detection of PrP ^Sc from the thus treated sample by Immuno-PCR.

With the aid of the method according to the invention, the pathologically altered prion protein (PrP ^Sc ) contained in body fluids is prepared. Furthermore, the naturally occurring PrP is removed from the sample. From a larger sample volume PrP is then enriched by immunoprecipitation. By the sample preparation according to the invention PrP ^Sc in body fluids, for example, with an immunological detection using

0 anti-PrP antibodies are detected. The method allows detection with high sensitivity, so that even the lowest, previously undetectable concentrations of PrP ^Sc in body fluids can be detected.

The method according to the invention is suitable for all body fluids, e.g. Blood, serum, plasma, tears, ocular fluid, cerebrospinal fluid, urine, saliva, lymph, peritoneal fluid or milk. Preferably, the method is used for plasma, blood or serum samples, the samples being in particular as preserves to be used for administration to other persons or patients.

In a first step, the sample is subjected to a protease treatment to unmask the PrP ^Sc epitopes. For this purpose, the sample material is mixed with a protease, eg proteinase K. For the protease treatment, a detergent or a detergent-containing solution can be added to the sample. The detergent may be an ionic, nonionic or zwitterionic detergent such as sodium dodecyl sulfate (SDS), N-lauroyl sarcosinate, Sodium desoxycholate, octylglucoside, Nonidet P-40, Triton X-100, Tween 20 or 80, CHAPS, Zwittergent 3-14. Preferred is the addition of SDS or sodium deoxycholate in a final concentration ranging from about 0.1% to about 10% in the sample, especially at a final concentration of about 1% SDS or sodium deoxycholate in the sample. The protease

5 Digestion may be carried out at standard conditions for the particular protease or according to the manufacturer, preferably at 37 ° C for 10 min. The enzyme concentration used may range from about 10 μg / ml to about 1000 μg / ml depending on the sample material, preferably about 800 μg / ml enzyme is used. The proteinase K digestion may preferably be performed at 900 rpm in a thermomixer.

Subsequently, the reaction is preferably stopped by immediate incubation at 95 ° C for 10 min.

In a second step, PrP ^{Sc is} enriched by means of immunoprecipitation (IP). For enrichment, a solid support is coupled with an antibody specific for PrP

5 is directed. The solid carriers used are preferably magnetic beads, but also, for example, beads consisting of agarose, sepharose or acrylic. Preferably, Bruker MB-IAC ProtG, Bruker MBCovAC Select, Pierce Magnabind Protein A, Pierce Magnabind Protein G or Dynabeads Protein A or G are used for immunoprecipitation. The antibodies coupled to the solid support may be commercially available anti-PrP antibodies.

0 such as B. WD3C7 from Biodesign International, F89 / 160.1.5 from Calbiochem, clones SAF from Cayman Chemical, C-C2 from Priontype, 3F4 from Chemicon or Dako, 6H4 and 34C9 from Prionics. Preferably, WD3C7 or 6H4 are covalently coupled through a crosslinker to the solid supports listed above.

For the method according to the invention, 500 μl to 5 ml of sample can be used, for example, with 10 μl to 1 ml of anti-PrP dynabeads (about 1.3 g / cm ³ , 2.8 μm ± 0.2 μm) for 1 to 24 h, preferably be incubated for about 2 h. Subsequently, the beads can be washed with PBS, in particular with 0.5 ml, wherein the washing process can be repeated about 6 times, in particular 4 times. The DynPeads bound PrP ^Sc can then with 10 ul to 100 ul, preferably with 30 ul iθ a detergent-containing buffered solution, preferably 1% Na-deoxycholate in PBS at 65 ° C to 100 ⁰ C, preferably at 95 ° C for 5 min to 15 min, in particular eluted for 10 min. In a third step, the eluted PrP ^{Sc is} detected by means of an immuno-PCR with an antibody directed against PrP ^Sc . The antibody is coupled to a DNA which is amplified in a subsequent polymerase chain reaction (PCR). The method of detecting an agent by means of immuno-PCR is known in the art and already described in US 5,665,539. The nucleic acid linked to the antibody can be chosen freely. Preferably, a nucleic acid is used that is either completely artificial or that is not naturally present in the sample in which the agent is to be detected. If, for example, the agent, here in particular PrP ^Sc , is to be detected in a human sample, no human DNA will of course be used as the DNA to be amplified. The nucleic acid can be connected to the antibody in a variety of ways. For example, DE 19941756 describes a suitable process. Both the antibodies and the nucleic acids are coupled with biotin and are linked to one another via a biotin-specific binding molecule, preferably streptavidin.

The immuno-PCR can be further performed by incubating the sample with a carrier coated with an antibody to the PrP ^{Sc to} be detected. PrP ^Sc then binds the antibody on the carrier. In a subsequent step, the second antibody labeled with the DNA is then added. This second antibody may be the same antibody that is bound to the carrier. This may be the case especially if PrP ^Sc has multiple epitopes for these antibodies. It can be used as a labeled antibody but also a different antibody from the first antibody. The antibodies may in particular be the antibodies directed against PrP described above, in particular WD3C7 being used as capture antibody and 6H4 being being labeled with the nucleic acid.

For a further increase in the sensitivity of the immuno-PCR, additional stabilizing components may be added to the buffers used in the immuno-PCR. The components may be DNA, proteins, detergents and chelating stabilizers.

Surprising here is the effect that the individual components complement each other supportive, so that a combination of these components leads to a significant increase in efficiency of the IPCR. The DNA used here are preferably mixtures of a Variety of DNA sequences, in particular to preparations of DNA mixtures for molecular biology, for example from fish, especially fish sperm DNA. Preferably, a mixture of different genomic DNAs is used for the DNA added to the buffers and solutions of the immuno-PCR. The DNA is preferably sheared.

For the proteins used, protein mixtures such as milk powder are also preferred in order to cover the widest possible range of interaction possibilities. As detergents, non-ionic detergents such as, for example, the molecules of the Tween family (Tween 20, Tween 80) are preferred, as chelating agent it is preferred to add EDTA to the buffers used, with the exception of the PCR buffer. In a special

0 preferred embodiment of the experimental conditions, therefore, the use of a uniform buffer system based on 20 mM Tris / HCl pH 7.4, 5 mM EDTA, 0.1% Tween 20, 2.5% skimmed milk powder, 0.5 mg / ml DNA as the basis., For. all used washing, blocking, dilution and storage buffers in the IPCR.

5 In practice, the carrier used for the immuno-PCR, such as. B. membranes of nitrocellulose or polyvinyl difluoride, nylon, beads, or PCR tubes, plastic polymers such as polypropylene, polycarbonate, polystyrene, z. For example, a microtiter plate is coated with a solution containing the specific antibodies. The binding of the capture molecules is carried out by adsorption to the surface of the solid phase or by chemical

0 Coupling methods to bind proteins to solid supports. Preferably, a PrP-specific antibody is bound in a concentration of 1-10 μg / ml in 50 mM carbonate buffer, pH 9.6, or 50 mM boric acid, pH 9.5 overnight by absorption to a plastic surface, preferably a microtiter plate. Subsequently, the antibody solution is removed by washing the microtiter plate several times, preferably 3 times; there will be a

.5 physiological saline, preferably PBS, with a detergent, preferably a nonionic detergent, preferably Tween-20, in a concentration of 0.01 to 1%, preferably 0.05%. Thereafter, the surface is saturated with a proteinaceous solution, commercial reagents such as Stabilcoat or Stabilguard from Surmodics, Superblock from Pierce or the above-mentioned uniform buffer system or iθ self-produced proteinaceous buffered solutions, such as BSA or casein (about 3-5%). ) in PBS or DNA preparations in buffered solutions, eg DNA fragments from fish sperm. The incubation time for the saturation is between 30 minutes and 5 hours, preferably 1 hour at room temperature. Thereafter, the solution is discarded. Subsequently, the sample is incubated with the solid phase between 30 minutes and 5 hours, preferably 1 hour at room temperature.

The present invention further relates to a kit for the treatment of PrP ^Sc from body fluids and for the detection of PrP ^Sc . According to the invention, the kit contains the required reagents, enzymes and antibody-coated plates. The kit contains in particular a detergent-containing buffer, a protease in solid form or in solution, one or more test plates coated with anti-PrP antibody, sample diluent (PBST), a

Negative control, a positive control, buffer A preferably as concentrate, buffer B preferably as concentrate, conjugate diluent, conjugate, PCR master mix.

The following exemplary embodiments illustrate the invention.

Example 1: Protease Treatment

It is thought that PrP is masked by prominent proteins in the blood (possibly chaperones, albumin, immunoglobulins) and thereby inhibits molecular interactions with PrP (Telling et al., 1995; DebBurman et al., 1997). First, it was shown that PrP may be masked in serum or plasma. In so-called spiking experiments, serum or plasma was supplemented with brain homogenate or recPrP to simulate body fluids containing PrP. For this purpose, 100 μl of serum were spiked with 10 μl of 10% brain homogenate from a BSE-positive bovine and the sample was divided. One half was not treated, one half was incubated with 100 ug / ml proteinase K for 37 ⁰ C for 10 minutes. To unmask the PrP epitopes, 500 μl serum was spiked with SDS so that the final concentration was 1% SDS. Both samples were separated by SDS-PAGE and analyzed by Western blot with the anti-PrP antibody WD3C7 (Biodesign International). The visualization was done by chemiluminescence. In Fig. 3 it is clear that PrP spiked in serum (even in the gel!) Is not accessible for molecular interactions: PrP is only after PK digestion with antibodies to mark and thereby visualize. For the detection of PrP it is therefore necessary to remove these dominant serum proteins.

Example 2: Sensitivity in serum / plasma

RecPrP was spiked at various concentrations in serum samples previously used with

Proteinase K were treated to the same conditions as described in Example 1 to obtain. These samples were analyzed by the following methods. In Fig. 4 it is shown that the detection limit of 10 pg / ml recPrP is confirmed in the serum.

Example 3: Elimination of prominent proteins

In the next step it was shown that under the chosen conditions for proteinase K digestion PrP ^{c is} removed and at the same time PrP ^{Sc is} not removed. This is considered essential because suboptimal PK digestion can lead to false positive or false negative results. For this purpose, BSE-positive brain homogenate with different PK concentrations (25-800 μg / ml) was treated for 10 min at 37 ° C. Here, too, demasking of the PrPΕpitope 500 μl serum with SDS was added so that the final concentration was 1% SDS. The samples were divided by SDS-PAGE. and analyzed in Western blot with two different anti-PrP antibodies (Figure 5).

Figure 5 A shows that PrP ^Sc remains detectable at all selected proteinase K concentrations. Here, the detection antibody used was an anti-PrP antibody with epitope in the proteinase K-resistant region of the prion protein (mAb WD3C7). PrP ^Sc is stable under the chosen conditions. Fig. 5 B shows that is fully digested in the same PK-treated samples PrP ^c. Here, an anti-PrP antibody (C-C2 from Priontype, Germany) with epitope in the proteinase K-labile region of the prion protein was used.

Example 4: Immunoprecipitation

Immunoprecipitation (IP) can enrich proteins with larger volumes. This concentration allows a hundred- to thousand-fold increase in sensitivity depending on the antibody used. First, the purified mAb WD3C7 (Biodesign International) was coupled to Dynabeads Protein G by a linker according to the manufacturer's instructions. Uncoupled free antibody was removed by several washes. Following the same protocol, two irrelevant mAbs for the production of control matrices for the control of IP were coupled to the Dynabeads. In no case was PrP precipitated here.

Following the PK digestion as described in Example 1, 500 μl of serum were mixed with 10 μl of WD3C7 beads and rotated for 2 hours at room temperature. It was then washed 4 times with PBS. Elution of bound PrP was performed with 30 μl of sodium deoxycholate (1% in PBS, 10 min, 95 ° C). This eluate was analyzed in the IPCR. Fig. 6 Figure 4 shows the comparison of signal strength of vCJD positive brain homogenate spiked in plasma with and without EP accumulation. Without EP, a dilution of 1: 1000 was detected, with EP, however, even the 1: 100000 dilution was clearly determined to be positive. The IP can therefore increase the proven amount of PrP by a factor of 100.

Example 5: Immuno-PCR

For carrying out the immuno-PCR Nunc ™ Top-Yield ™ strips coated overnight at 4 ° C (containing antibody solution 30 ul / well; 5 ug / ml 6H4 (Prionics) in 35mm NaHCO ₃ / 15mM Na ₂ CO _3, pH 9 6, or coated in 50 mM boric acid, pH 9.5, followed by washing the plates for 3 μl with 240 μl / well wash buffer A (Cat.No .: 23-004, Chimera, Germany) and then for 1 h at RT with 200 μl / Well blocking solution (150 mM NaCl, 20 mM Tris, 4.5% skimmed milk powder (Oxoid), 1 mg / ml DNA MB-grade, cat.-no. 114671490001, Roche, _Mannheim, Germany), 5 mM EDTA (Sigma, Germany) or 200 μl / well of Stabilguard (Surmodics, USA) according to the manufacturer's instructions. The blocking solution was filtered off with suction.

For the EPCR, controls (serial dilutions of recPrP and positive control, PBS / 0.05% Tween 20 as a negative control) and 30 μl each of prepared samples were incubated on the anti-PrP mAb-coated microtiter plate. This primary incubation was carried out for 1 h at room temperature and 600 rpm. This was followed by washing steps 2 × 30 sec and 2x4 min with 240 μl / well wash buffer B (cat. No. 23-005, Chimera, Germany). Subsequently, the incubation was carried out with DNA-coupled detection antibody, 45 min at RT and 600 rpm were 30 .mu.l DNA-coupled detection antibody in ^"a concentration of 1 pmol / ml in a 1:. 270 dilution in TBS-conjugate diluent (20 mM Tris / HCl pH 7.4, 5 mM EDTA, 0.09% Tween 20, 0.5% Skim milk powder (LP0031, Oxoid), 0.1 mg / ml DNA (MB grade, cat.-no. 114671490001, Roche, Mannheim, Germany) followed by washing steps 4χ30 sec, 4χ4 min and 3 x 1 min with 240 μl / well wash buffer B (cat No. 23-005, Chimera, Germany) At the start of the PCR, 30 μl / well PCR master mix were used (16mM (NH ₄ ) ₂ SO ₄ ; 67mM Tris-HCl (pH 8 at 25 ° C); 0.01% Tween 20; 1.5mM MgCl ₂ ; 0.5mg / ml BSA; 0.2mM dNTPs ( ea), 0.2 μM TaqMan or Scorpions probe, 1 pmol / μl primer (each), 0.05 U / μl Taq) pipetted The master mix used by Chimera Biotech already contained the primers required for amplification in the appropriate concentration and the usual for a PCR n buffer substances and nucleotides. The PCR is carried out as follows:

Temperature duration repetitions

95 ° C 5 mm Ix

Measurement at the end of this

50 ° C 30 s

step

72 ° C 30 s 4Ox

95 ⁰ C 20 s

For evaluation, the Ct value determined by the real-time PCR software is used. The Ct value corresponds to the cycle in which the fluorescence signal exceeds a certain threshold. Since the Ct value is inversely proportional to the amount of marker DNA and thus also to the amount of PrP, the ΔCt value is calculated for quantification as follows:

ACt = total number of cycles (40) - Ct

For the quantification ΔCt of the dilution series is plotted against the decadic logarithm of the concentration. The linear regression-determined equation is used to quantify the concentration of unknown samples.

Example 6: Sensitivity comparison of the classical ELISA with the developed method

To compare the sensitivities of the classical ELISA and the immuno-PCR, recPrP in PBST was diluted in several steps and analyzed in the classical ELISA. In parallel, this dilution series was investigated in the process. Nunc ™ Top-Yield ™ strips were coated overnight at 4 ° C with antibody solution as described in Example 5.

The detection of the bound recPrP takes place via the addition of the DNA-coupled antibody WD3C7 and the performance of the IPCR as indicated in Example 5. Fig. 2 illustrates the high sensitivity of the developed method based on comparative data with the classical ELISA (same antibody combination with colorimetric detection). The S / P ratios of both measurement methods are shown. In the conventional ELISA 100 ng / ml, in the method 10 pg / ml are detected. This means a 10000 times higher sensitivity of the procedure compared to the ELISA. Example 7: Validation J

The suitability of the method for detection of PrP ^Sc in body fluids was validated on blanks. For this purpose, blanks were examined by the CID Resource Center of the NIBSC (UK), which were decrypted only after testing and transmission of results by the authority. These 22 plasma samples were spiked by the NIBSC with varying amounts of brain homogenate from vCJD patients. The samples were digested with proteinase K, PrP by D? enriched and analyzed in the EPCR. Table 1 shows the results of the workup with and without IP. The 1: 100000 dilution is used in the sample preparation with D? recognized as correctly positive; this corresponds to a detection of 0.00024 nl 10 percent brain homogenate. All negative samples were correctly negative (n = 4, not shown). , - -

Tab. 1: Detection of vCJD-positive brain homogenate spiked in plasma in the process

Example 8:

The specificity of the established method was tested by testing plasma samples from healthy volunteers and Alzheimer's patients. All 375 samples were correctly diagnosed negative. The method has a specificity of 100%.

Tab. 2: Specificity of the procedure

Example 9: Overview of the Performance of Various Methods for the Detection of vCJD Brain Homogenate

Reference material from the WHO Working Group on International Reference Materials for the

Diagnosis and Study of Transmissible Spongiform Encephalopathies "was presented with various

Methods analyzed in different laboratories (Minor et al, 2004). The method detects the least amount of vCJD brain homogenate (0.00024 nl, Table 3).

Table 3: Overview of methods for highly sensitive detection of PrP

literature

Barletta JM, Edelman D. C, Highsmith WE, Constantine NT (2005) Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR. J. Virol. Methods. 127, 154 Bellon A., Seyfert-Brandt W., Lang W., Baron H., Groner A., Vey M. (2003) Improved conformation-dependent immunoassay: suitability for human prion detection with enhanced sensitivity. J. Gen. Virol. 84, 1921

Bieschke J., Giese A., Schulz-Schaeffer W., Zerr L, Poser S., Eigen M., Kretzschmar H. (2000) Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets. Proc. Natl. Acad. Be. U.S.A. 97, 5468

Castilla J., Saa P., Soto C. (2005) Detection of prions in blood. Nat. Med. U, 982

DebBurman S.K., Raymond G.J., Caughey B., Lindquist S. (1997) Chaperone-supervised conversion of prion protein to its protease-resistant form. Proc. Natl. Acad. Be. U.S.A. 94, 13938

Gofflot S., Deprez M., El Moualij B., Osman A., Thonnart J.F., Hougrand O., Heinen E., Zorzi W. (2005) Immunoquantitative PCR for prion protein detection in sporadic Creutzfeldt-Jakob disease. Clin Chem. 5_1, 1605

Minor P., Newham J., Jones N., Bergeron C, Gregori L., Asher D., van Engelenburg F., Stroebel T., Vey M., Barnard G., Head M .; WHO Working Group on International Reference Materials for the Diagnosis and Study of Transmissible Spongiform Encephalopathies. (2004) Standards for the assay of Creutzfeldt-Jakob disease speeimens. J. Gen. Virol. 85, 1777

Saa P., Castilla J., Soto C. (2006) Presymptomatic detection of prions in blood. Science 313, 92

Sano T., Smith C.L., CR. Cantor CR. (1992) Immuno-PCR: very sensitive antigen detection by means of speeifie antibody-DNA conjugates. Science, 258, 120

Schmerr MJ, Jenny AL, Bulgin MS, Miller JM, Ffamir AN, Cutlip R. C, Goodwin KR (1999) Use of capillary electrophoresis and labeled with the same spongiform encephalopathy. J. Chromatogr. A. 853_, 207 Soto C, Other L., Suardi S., Cardone F., Castilla J., Frossard MJ, Peano S., Saa P., Limido L., Carbonatto M., Ironside J., Torres JM, Pocchiari M., Tagliavini F. (2005) Pre-symptomatic detection of prions by cyclic amplification of protein misfolding. FEBS Lett. 579, 638

Telling G.C, Scott M., Mastrianni J., Gabizon R., Torchia M., Cohen FE, DeArmond SJ, Prusiner SB (1995) Prion propagation in mice expressing human and chimeric PrP transgenic implicates the interaction of cellular PrP with another protein. Cell 83, 79

Zhou, H., Fisher, R.J., Papas, T.S. (1993): Universal Immuno-PCR for Ultra-Sensitive Target Protein Detection, Nucleic Acids Res, 21, 6038-6039.

Claims

claims

A method for detecting pathologically altered prion proteins (PrP ^Sc ) in human body fluids comprising the steps of

a) incubating an isolated sample of the body fluid to be examined with a protease, and then b) immunoprecipitation to enrich PrP ^Sc _; and then c) detection of the PrP ^Sc obtained in step b) by means of immuno-PCR.

2. The method according to claim 1, wherein the protease in step a) is proteinase K and in a concentration in the range of about 10 μg / ml to about 1000 μg / ml, in particular with a

Concentration of 800 μg / ml in the sample.

3. The method according to any one of the preceding claims, wherein the incubation in step a) in the presence of a detergent, for example SDS, N-lauroyl sarcosinate, sodium deoxycholate, octylglucoside, Nonidet P-40, Triton X-100, Tween, CHAPS, Zwittergent 3- 14 is performed.

4. The method according to any one of the preceding claims, wherein the body fluid is CSF, blood, plasma or serum.

5. The method according to any one of the preceding claims, wherein in step b) the solid support for immunoprecipitation is Bruker MB-IAC ProtG, Bruker MBCovAC Select, Pierce Magnabind Protein A, Pierce Magnabind Protein G or Dynabeads Protein A or G.

6. The method according to any one of the preceding claims, wherein in step b) the capture antibody in the immunoprecipitation WD3C7 from Biodesign International, F89 / 160.1.5 from Calbiochem, clones SAF from Cayman Chemical, C-C2 from Priontype, 3F4 from Chemicon or Dako, or 6H4 or 34C9 from Prionics.

7. The method according to any one of the preceding claims, wherein in step c) the immuno-PCR is carried out in the presence of stabilizing components.

8. The method of claim 7, wherein the stabilizing components are DNA, proteins, detergents or chelating agents. ι

9. The method of claim 7, wherein the stabilizing components are a mixture of DNA, proteins, detergents or chelating agents.

10. The method according to claim 8 or 9, wherein the DNA is genomic DNA, in particular sheared genomic DNA.

A method according to any one of claims 8 to 10, wherein the DNA is fish sperm DNA.

12. Kit for detection of pathologically altered prion proteins (PrP ^Sc ) containing a detergent-containing buffer, protease, test plates coated with anti-PrP antibody, sample diluent (PBST), negative control, positive control, buffer A (2Ox concentrate), buffer B ( 20 x concentrate), conjugate diluent, conjugate, PCR master mix.