MXPA06008396A - Cjd prion testing - Google Patents

Abstract

The present invention provides an assay method for detecting infectious prion protein in a sample from a mammalian subject, said method comprisi

Description

PROOF IN PRESS OF DISEASE OF CREUTZFELDT-JAKOB.

Description of the invention This invention relates to improvements in and related to test methods for testing transmissible spongiform encephalopathy (TSE) in mammalian subjects. Spongiform encephalopathies are a group of degenerative neurological diseases. Examples have been found in a number of mammalian species including sheep (where it is known as scrapie), cows (BSE) and humans (Creutzfeldt-Jakob disease (CJD), new variant CJD (nv CJD) and kuru). It has been reported that TSE of one species can be transmitted under laboratory conditions to mammals of another species. These species-crossing barriers by the infectious agent have led to a widespread concern that transfer to humans may occur as a result of ingestion of material from an infected edible animal, in particular bovine material. TSEs are characterized by a slow incubation period after which the clinical symptoms of progressive degeneration of the mental state appear, including aggression and lack of coordination. Post mortem studies reveal a characteristic pattern of vacuolation in brain tissue due to the destruction of neural cells, REF. : 174664 and to the deposition of unusual protein fibers. Although the form of the disease found in sheep has been known for many years, spongiform encephalopathies have become more important after the appearance of BSE in cattle and nvCJD in humans. It is believed that the causative agent of TSE is a so-called "prion", that is, an infectious agent that comprises only proteins and without nucleic acid. In TSE, a particular protein (called prion protein, PrP) has been identified as the infectious agent. PrP is a naturally occurring cellular protein which exists in two isoforms that differ in their tertiary structure and as a result can be distinguished by their response to enzymatic degradation, for example by proteinase K. In this way, the non-infectious isoform niPrP is completely digested by proteinase K while the infectious isoform iPrP is degraded to leave a detectable polypeptide residue PrP27-30. The amino acid sequences for many PrP of mammals are known and accessible, for example in SwissProt. The amino acid sequences for the PrP27-30 residue are also known. There is a high degree of homology between the different PrP sequences of mammals. Several companies have developed post mortem diagnostic tests for TSE, generally based on the use of antibodies that bind to PrP27-30 that is derived from a sample of proteolytically digested brain tissue. One of these trials available from Enfer, Dublin, Ireland, uses the technology described in EP-B-616613. More particularly, the Enfer assay uses two polyclonal antibodies developed against immunogenic conjugates of polypeptide sequences corresponding (i) to a section of PrP27-30 and (ii) to a section of PrP outside the PrP27-30 section. In EP-B-616613, these sections are called Vc and Va, respectively. Although commercial trials have been somewhat successful, there is a continuing need for trials for improved TSEs and in particular trials that can be carried out ante mortem or which do not require brain tissue samples. The present invention provides just that improved assay. Thus, viewed from one aspect, the present invention provides a test method for detecting an infectious prion protein in a sample from a mammalian subject, the method comprising: obtaining a sample containing prion protein from the subject; contacting the sample with an agent that serves to digest non-infectious prion protein and to partially digest infected prion protein, to produce a prion protein polypeptide residue; contacting the digested sample with an antibody capable of binding to a polypeptide having the amino acid sequence Vc (Gly-Gly-Gly-Trp) -Gly-Gln-Gly-Gly-R1-R2-His-R3-Gln-Trp-Asn-Lys-Pro-R4-Lys-Pro-Lys-Thr-R5-R6- Lys (-His-R7-Ala-Gly) (Vc) (where Ri is either Gly or absent, R2 is either Thr or Ser, R3 is an amino acid residue selected from Gly, Being and Asn; R and R5 are each independently either Asn or Ser; R6 is an amino acid residue selected from Met, Leu and Phe; R7 is either Val or Met; and wherein one or more of the residues in parentheses may be present or absent, with the proviso that if present they are attached to the rest of the peptide in the sequence); and detecting conjugates of the antibody and the prion protein polypeptide residue; further characterized in that the detection of the conjugates comprises the chemical, biological or biochemical amplification, in a particularly preferable biochemical or biological amplification, of a detectable species, and the detection of the amplified species.

The antibody capable of binding to Vc is preferably an antibody developed against an immunogenic conjugate of a synthetic polypeptide with the amino acid sequence Vc (or more preferably Vc 'as described below), for example, by vaccination of a mammal with the same and the collection of serum or IGg that binds to Vc. The sample containing prion protein used in the method of the invention can be a sample of any tissue, fluid or body material containing prion proteins, for example, muscle, tonsils, brain, "blood, urine, feces, etc. The sample it may also be blood, serum or plasma from blood banks, blood products (eg, clotting factors), tissue products, culture media containing mammalian products (eg, BSA), pharmaceutical components derived from mammalian species ( for example, heparin), etc. For post-mortem tests, brain tissue will be desirably used because of its high prion content.For ante-mortem tests the sample is preferably a tissue sample of biopsy, blood, urine or feces. it is preferentially pretreated to lyse any cell therein, for example by homogenization or other methods of tissue disruption. with the prion protein digestion agent, for example proteinase K, under conditions and for a period sufficient for the digestion of the non-infectious prion protein. These treatments and conditions and durations of treatment are well known in the art. If the sample is desired, it can be treated before and / or after its digestion to separate the components of the sample and / or digestion products that are not undigested or partially digested prion protein, for example by centrifugation, chromatography, etc. . 0. After digestion, the sample is contacted with the Vc binding antibody. This antibody can be prepared as described in EP-B-616613. In a particularly preferred manner the antibody is prepared using a conjugate of a polypeptide of the sequence GQGGSHSQWNKPSKPKTNMKHVGC (Ve ') with an immunogenic vehicle, for example. tetanus toxoid, ovalbumin, etc. The conjugation can be carried out, either using a standard binding agent, for example, m-maleimido-benzoyl-N-hydroxy sulfosuccinimide ester (SMBS), etc. The antibody is preferably polyclonal. Standard techniques for the production of antibodies can be used. The Vc binding antibody can be immobilized on a substrate (eg, a flat surface, optionally superparamagnetic beads, cylinders, meshes, tubes, etc.) using conventional protein immobilization techniques. Alternatively, a non-immobilized Vc binding antibody can be used. The precise series of steps in the detection stage of the assay method will depend on whether an immobilized or non-immobilized Vc-binding antibody is used, and the technique selected for chemical amplification., biological or biochemical. . By chemical amplification is meant that a non-biochemical chemical reaction (for example, a reaction catalyzed by a chemical that is not normally found in a biological environment) is used to generate a detectable species, the presence or absence of which is an indicator of the presence of antibody conjugates. Residual prion protein polypeptide. By biological amplification it is tried to say that a microorganism is used to generate a detectable species (for example, chemical substance, microorganisms, etc.), the presence or absence of which is indicative of the presence of antibody conjugates: prion protein polypeptide residue. By biochemical amplification is meant that a biochemical reaction (eg, an enzymatic reaction or a nucleic acid amplification such as PCR) is used to generate the detectable species (eg, chemical substance), the presence or absence of which is Indicator of the presence of antibody conjugates: prion protein polypeptide residue. The material that is amplified or which causes the amplification to occur can be conjugated to the Vc binding antibody or to an additional agent capable of binding to the antibody: residue conjugates, for example a second antibody. When conjugated to the Vc binding antibody, its ability to function can be unaffected by conjugation to PrP27-30 or it can be activated or deactivated by this conjugation. As a result, an unreacted antibody may or may not have to be separated from the antibody: residue conjugates. This is conventional in immunoassay procedures and the person of ordinary ability will readily appreciate the steps that must be taken. When the material to be amplified or which causes the amplification is separated from the Vc binding antibody, it will generally be necessary to separate the unconjugated antibody from the antibody-residue conjugates. Again, this is conventional in immunoassay procedures and the person of ordinary ability will readily appreciate the steps that must be taken. The material that is amplified or that causes the amplification may comprise more than one component. In this case, one of the components can be conjugated to the Vc binding antibody and the other to a separate agent capable of binding to the antibody: residue conjugate. Again it will be clear to the person of ordinary ability if separation of the unconjugated Vc binding antibody from the antibody-residue conjugates is required. When using this system of two or more components, it is preferable that the different components together create an amplification effect different from that which can be achieved with the individual components on their own, for example, they can be catalysts (eg example, enzymes) that catalyze different stages of a multi-stage reaction, or they can be viral agents having different effects on the same or different target microorganisms. When a second binding agent is used in the assay method of the invention, it is also preferably an antibody. However, other bonding agents may be used if desired. The term "antibody" as used herein, unless the content dictates otherwise, may be an antibody as such or a functional fragment (e.g., a Fab fragment) thereof, a single chain antibody or an antibody construct. oligomeric These materials can be produced in conventional manner. To avoid unnecessary false negatives, the test method of the invention also preferably includes testing a portion of the original sample for PrP content. This can be done conventionally, for example, using PrP binding antibodies as for example in the commercial BSE tests. In this case, the "PrP binding antibody must be" capable of binding to iPrP and / or niPrP or fragments thereof exposed by denaturation or partial digestion. These antibodies are described in the literature. However, in a preferred embodiment, a Va-binding antibody is used in undigested sample with PK, ie analogously to the Vc-binding antibodies with the omission of the niPrP digestion step. Particularly preferably, the antibody used is a Va-binding antibody as defined below. Especially preferably, this antibody is one developed against an immunogenic conjugate of a synthetic polypeptide with the amino acid sequence Va (or most preferably Va7 as described below), for example by vaccinating a mammal therewith and collecting serum or IgG that binds to Va. By a Va binding antibody is meant one capable of binding to a polypeptide with the sequence (Pro-Gly-gly-R8) -Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly- Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly- (Gly-R9-R? O-Trp) (Va) wherein, Rs and Rg are each independently selected from Gly or absent; Rio is either Gly orThr; and wherein one or more residues in parentheses may be present or absent with the proviso that if present they are attached to the remainder of the peptide in the sequence. This can be prepared as described in EP-B-616613. Particularly preferably, the antibody is prepared using a conjugate of a polypeptide of the sequence. This antibody can be prepared as described in EP-B-616613. Particularly preferably, the antibody is prepared using a conjugate of a polypeptide with the sequence GGWNTGGSRYPGQGSPGGNRYPPQGGGC (Va ') with an immunogenic vehicle, for example, tetanus toxoid, ovalbumin, etc. The conjugation can be carried out using a standard binding agent, for example SMBS. The antibody is preferably polyclonal. Standard techniques for the production of antibodies can be used. When the test result for ante mortem TSE is positive, it is preferable to treat the subject with a therapeutic agent comprising a Vc binding antibody. This can be "the antibody alone or it can be an antibody conjugate with an agent that serves to prevent the transformation of niPrP into iPrP or to degrade iPrP." In the case of human TSE, the therapeutic antibody is preferably a selected antibody ( that is, an anti-Vc or anti-Va antibody as defined above.) Administration is preferably by injection or infusion, for example in CSF.The doses can be determined conventionally using models of animals infected with TSE. The therapeutic aspect forms a further aspect of the present invention Seen from a further aspect, the invention provides a kit for use in the assay method of the invention, the kit comprising: (i) a Vc binding antibody (ii) optionally and preferably a binding antibody to -Va; (iii) optionally and preferably proteinase K; (iv) a material capable of chemical, biological or biochemical amplification, preferably amplif biological or biochemical, and the detection or causing the chemical, biological or biochemical amplification, preferably biological or biochemical amplification, of a detectable species, the material being optionally conjugated to the antibody of (i); and (v) optionally and preferably instructions for carrying out the test method. Although the material of (iv) may be an enzyme or another catalyst, it is preferably a substance which in turn is replicated in the amplification process. The invention will now be described further with reference to the following non-limiting examples.

EXAMPLE 1 Preparation of Samples Initial tissue disruption is carried out using suitable methods such as homogenization in a detergent pH regulator using a pistil and centrifuge tube to produce a crude homogenate which can be rinsed by centrifugation. The tissue homogenate sample is diluted to a working concentration in a suitable pH buffer in preparation for digestion with proteinase K.

Example 2 Digestion of Prion Protein To distinguish iPrP from niPrP the detection systems are based on the relative resistance of iPrP to digestion by proteinase K (PK) enzyme (see Kretzschmar, Clin Lab Med. P109-128 (2003) ). Typical conditions of PK treatment would be: diluted homogenate sample divided into two equal portions and PK added to a portion and incubated accordingly (eg, for 30 minutes at 37 ° C).

Example 3 Addition of Vc binding antibody Purified serum or IgG is prepared from the blood of animals immunized with Vc peptide conjugate vaccine. These sera would then be diluted to a working concentration, brought into contact with the homogenized sample digested with PK and incubated accordingly (eg, for one hour at 20-25 ° C).

Example 4 Signal Amplification and Detection Signal amplification would typically require the application of biochemical techniques, secondary molecular antibody techniques or conjugates. An example would be the incubation of an antibody conjugated with de-biotin molecules (for example for one hour at 20-25 ° C) amplified by the addition of Avidin (biotin-binding agent) conjugated to horseradish peroxidase (for example during a hour at 20-25 ° C) and detection using a chromogenic substrate, read at a specific absorbance in a spectrophotometer.

EXAMPLE 5 Efficacy of the antibodies The polyclonal Vc-binding and Va-binding antibodies prepared by immunizing animals with antigenic conjugates of Vc 'and Va' respectively, were contacted with brain homogenate prepared as described above from human brain. infected and not infected with CJD, with and without digestion with proteinase K. The samples were then subjected to Western blot analysis and the results are shown in figures 1 and 2 appended. Figures 1 and 2 are for Va binding and Vc binding antibodies respectively. In each figure, line 1 is homogenate not infected, line 2 is homogenate uninfected treated with PK, line 3 is homogenate infected and line 4 is homogenate infected treated with PK. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (5)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A test method for detecting an infectious prion protein in a sample from a mammalian subject, the method comprising: obtaining a sample containing prio.n-protein from the subject; contacting the sample with an agent that serves to digest non-infectious prion protein and to partially digest infected prion protein, to produce a prion protein polypeptide residue; contacting the digested sample with an antibody capable of binding to a polypeptide having the amino acid sequence Vc (Gly-Gly-Gly-Trp) -Gly-Gln-Gly-Gly-R1-R2-His-R3-Gln-Trp-Asn-Lys-Pro-R4-Lys-Pro-Lys-Thr-R5-R6- ys (-His-R7-Ala-Gly) (Vc) (where Ri is either Gly or absent, R2 is either Thr or Ser, R3 is an amino acid residue selected from Gly, Ser and Asn; R4 and R5 are each independently either Asn or Ser; R6 is an amino acid residue selected from Met, Leu and Phe; R7 is either Val or Met; and where one or more of the - residues in parentheses can be present or absent, with the proviso that if they are present they are attached to the remainder of the peptide in the sequence); and detecting conjugates of the antibody and the prion protein polypeptide residue; further characterized in that the detection of the conjugates comprises the chemical, biological or biochemical amplification of a detectable species, and the detection of the amplified species.

2. The method according to claim 1, characterized in that the subject is human, preferably animated.

3. The method according to any of claims 1 and 2, characterized in that it is for detecting infectious prion protein associated with CJD, nvCJD or kuru.

4. Equipment for use in the assay method according to any of claims 1 to 3, the kit is characterized in that it comprises: (i) a Vc-binding antibody (ii) optionally a Va-binding antibody; (iii) optionally proteinase K; (iv) a material capable of chemical, biological or biochemical amplification, and of the detection or of causing the amplification, chemical, biological or biochemical of a detectable species, the material being optionally conjugated to the antibody of (i); and (v) optionally instructions for carrying out the test method.

5. The use of an iPrP binding antibody in the preparation of a medicament for use in the. treatment of human TSE.