Patent Grant
10288627
The invention relates to an assay for detecting prions. In particular the invention relates to an assay for detecting prion infection in blood or urine, most suitably blood.
Prion diseases are a group of fatal neurodegenerative disorders including variant Creutzfeldt-Jakob disease (vCJD) which originates from exposure to bovine spongiform encephalopathy (BSE). Iatrogenic transmission from asymptomatic patients with vCJD prion infection via blood transfusion has had a major impact on public health policy.
Prion Disease encompasses a range of closely related and uniformly fatal neurodegenerative disorders affecting the central nervous system of humans and animals. They include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI) and kuru in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) of deer and Elk and scrapie in sheep (1;2). The emergence of variant CJD (vCJD), and the confirmation that it originates from exposure to bovine spongiform encephalopathy (BSE) (3;4) has raised a plethora of public health concerns adversely affecting surgery, dentistry, organ transplantation and blood transfusion.
Exposure of the UK population to BSE has been widespread with over 181,000 cases confirmed to date in cattle (5) and yet the prevalence of human infection in the UK has yet to be determined with accuracy. A retrospective study of archived surgical lymphoreticular specimens estimated a prevalence of infection in the UK of 237 per million (95% confidence interval 49-692 per million)(6), far higher than the number of vCJD clinical cases thus far with further studies failing to refine these estimates(7). The demonstration of sub-clinical carrier states of prion infection in animal models (8-10) offers a potential explanation for the discrepancy between clinical cases and supports a prevalence which is likely to be in excess of 1 in 10,000 in the UK (11).
Concern regarding secondary transmission of vCJD has had extensive effects on public health policy in the UK and elsewhere. As the clinically silent incubation period of prion infection in humans can be prolonged, potentially exceeding 50 years (12), and the prevalence is poorly defined, the extent of future transfusion-transmission of vCJD cannot be estimated or accurately risk-assessed. Secondary infection from the clinically silent population has already been confirmed in four recipients of blood transfusion (13-15). Whilst the number of transfusion recipients positively identified as having received vCJD contaminated packed red cells is small, a much larger cohort of around 7000 recipients of contaminated plasma products have already been identified and notified of their at-risk status (16). Concern for this cohort has been heightened by post-mortem evidence for infection with vCJD prions in the spleen of a person with haemophilia (11).
The infectious agents, or prions, responsible for transmission of disease are composed principally if not entirely of a misfolded form of the host prion protein, PrPC. PrPC is ubiquitous, although expressed at highest levels in the central nervous system (CNS) and cells of the immune system. When recruited during prion propagation PrPC is remodelled to an aggregated, detergent insoluble isoform designated PrPSc chemically identical but conformationally distinct from PrPC (17). Detection of PrPSc in CNS and lymphoreticular tissues correlates widely with infection and the presence of prion infectivity (18-20) and is accepted as 100% specific for prion infection.
Although the quantities of PrPSc deposited in neural tissues are sufficient during the symptomatic phase of illness for detection by conventional immunoassays such as western blotting and ELISA, levels in peripheral tissues are significantly lower (21). Quantification of infectious titre using rodent models has indicated that the levels of infectivity, and by inference PrPSc, in blood may be extremely low, with buffy coat fractions containing between 2-10 LD50 Units ml−1 during the asymptomatic phases of disease, rising to 100 LD50 Units ml 1 during the clinical stage (22;23).
In order to successfully identify infection in blood an assay must be able to detect PrP in a range which is several orders of magnitude below the sensitivity of conventionally employed immunoassays. Furthermore, the ratio of background PrPC, which is chemically identical to PrPSc, is higher in blood than any other tissue and the high lipid and protein content of blood also contribute to non-specific background signals. Conventionally, immunoassays for PrPSc have been dependent upon protease pre-treatment of tissues to degrade PrPc and other proteins, thereby reducing cross-reactivity (24). It has now been shown in a number of different studies that the majority of disease associated PrP may well be sensitive to proteolytic digestion with proteinase K (PK) (25-31).
The observation that prions can bind avidly to metal surfaces (32;33) has been used to develop quantitative assays for infectivity (34) that approach the high levels of sensitivity required to detect the low levels of prions and abnormal PrP associated with blood. However, prior art assays typically assess infectivity in an animal model or cell culture system which is labour intensive and time consuming as well as raising ethical issues about use of animals.
Current evidence clearly indicates there is risk of iatrogenic vCJD from transfusion of blood and purified blood components (13-15;36) and by inference from many forms of surgical and dental interventions. Taken together with the knowledge that sub-clinical carrier states of prion disease can exist (8;9) alongside protracted incubation periods for clinical disease which may span several decades (12) the implications for current and future public health are substantial. Current risk reduction strategies in the UK are extremely costly involving leucodepletion of transfused packed red cells and the sourcing of plasma from the USA based on estimates of approximately 40% of infectivity being associated with leucocytes with the remainder in plasma (37). Studies modelling the use of leucocyte depletion have demonstrated reductions in blood infectivity of between 58% (37) and 72% (38). These and related issues also apply outside the UK, for example blood donation is not permitted in the USA if the prospective donor has spent a cumulative period of 6 moths or more in Europe. This causes significant issues for US public and military blood transfusion services.
It remains a major problem that a non-invasive and specific test for prion infection is not available.
The invention seeks to overcome problem(s) associated with the prior art.
The invention provides a method for the capture and detection of abnormal PrP from whole blood or urine using a solid-state matrix. However, rather than assay this material for infectivity by transfer to cell-culture or amplify the material for visualisation by western blotting we have developed an assay for the direct immunodetection of surface-bound material, avoiding the use of any proteolytic processing to ensure the complete ensemble of abnormal PrP isoforms are available for detection.
Central to these methods are the provision of specific buffer systems for the dilution of whole blood or urine leading to the successful capture of abnormal PrP from the sample on substrate such as steel particles.
Here we present the results of applying this assay to a blind panel of 190 whole blood samples which contained 21 samples obtained from vCJD patients, 100 normal control samples obtained from National Blood Service and 69 neurological disease controls. We also present results from urine.
Provision of a blood or urine test which can identify asymptomatic patients according to the present invention provides the ability to manage the risk of transmission and allow early entry into therapeutic clinical trials.
Assay sensitivity was found to be several orders of magnitude higher than any previously reported and was able to distinguish a 10−10 dilution of exogenously spiked vCJD-infected brain from normal control brain with a significance of <0.0001. Analysis of the blind panel of 190 samples indicated the assay has a sensitivity for the detection of vCJD-infected patient blood of 71% and predicted overall specificity of approximately 99.97%.
Thus in one aspect the invention provides a method for detection of abnormal PrP in a sample of blood or urine, said method comprising:
Suitably step (a) comprises diluting the sample with buffer to comprise final concentrations of
Suitably step (a) comprises diluting the sample with buffer to comprise final concentrations of
In one embodiment, suitably the sample is blood and is diluted with buffer in the range 1:1 to 1:100. More suitably the blood sample is diluted with buffer in the range 1:10 to 1:100. Most suitably
In one embodiment, suitably the sample is urine and is diluted with buffer in the range 10:1 to 1:5. More suitably the urine sample is diluted with buffer at 1:1.
Suitably the buffer further comprises protease inhibitors.
Suitably the antibody of step (d) is selected from the group consisting of ICSM10, ICSM18, ICSM33 and ICSM35. More suitably, the sample is from a tga20 mouse and the antibody is ICSM10; or
Suitably step (c) comprises washing the steel particles to remove diluted sample and subjecting the steel particles to a heat treatment for 5 minutes. More suitably, the sample is from a tga20 mouse and heat treatment is at 50 to 110 degrees Celsius; or
Suitably the sample is blood and the steel particles comprise AISI 304 stainless steel.
Suitably the sample is urine and the steel particles comprise AISI 316 stainless steel.
Suitably the buffer agent is Tris.
Suitably the pH is 8.4.
In another aspect, the invention relates to a dry composition comprising Tris:BSA:CHAPS in the weight ratio 1:1.65:1.65.
In another aspect, the invention relates to a solution comprising Tris:BSA:CHAPS in the molar ratio 1:0.003:0.32.
In another aspect, the invention relates to a composition as described above or a solution as described above for use in detection of abnormal PrP.
In another aspect, the invention relates to use of a composition as described above or a solution as described above for detection of abnormal PrP in a sample.
In another aspect, the invention relates to a kit comprising
In another aspect, the invention relates to a method of aiding the diagnosis of prion infection in a subject, the method comprising
In another aspect, the invention relates to a method for detecting prion infection having sensitivity of at least 71% and specificity of at least 99.9%.
Preferred Aspects
In one preferred aspect the invention provides a method for detection of abnormal PrP in a blood sample, said method comprising:
Suitably step (a) comprises diluting the blood sample with buffer to comprise final concentrations of
Suitably step (a) comprises diluting the blood sample with buffer to comprise final concentrations of
Suitably the blood sample is diluted with buffer in the range 1:1 to 1:100.
Suitably the blood sample is diluted with buffer in the range 1:10 to 1:100.
Suitably the blood is from a tga20 mouse and the blood sample is diluted with buffer at 1:10; or
Suitably the buffer further comprises protease inhibitors.
Suitably the antibody of step (d) is selected from the group consisting of ICSM10, ICSM18, ICSM33 and ICSM35.
Suitably the blood is from a tga20 mouse and the antibody is ICSM10; or
the blood is from a CD1 mouse and the antibody is ICSM33; or
Suitably the blood is from a human and the antibody is ICSM18.
Suitably step (c) comprises washing the steel particles to remove diluted blood sample and subjecting the steel particles to a heat treatment for 5 minutes.
Suitably the blood is from a tga20 mouse and heat treatment is at 50 to 110 degrees Celsius; or
Suitably the steel particles comprise AISI 304 stainless steel.
Suitably the buffer agent is Tris.
Suitably the pH is 8.4.
In another aspect, the invention relates to a dry composition comprising Tris:BSA:CHAPS in the weight ratio 1:1.65:1.65.
In another aspect, the invention relates to a solution comprising Tris:BSA:CHAPS in the molar ratio 1:0.003:0.32.
In another aspect, the invention relates to a composition as described above or a solution as described above for use in detection of abnormal PrP.
In another aspect, the invention relates to use of a composition as described above or a solution as described above for detection of abnormal PrP in a sample.
In another aspect, the invention relates to a kit comprising
In another aspect, the invention relates to a method of aiding the diagnosis of prion infection in a subject, the method comprising
In another aspect, the invention relates to a method for detecting prion infection having sensitivity of at least 71% and specificity of at least 99.9%.
The methods of the invention involve manipulation of the high avidity of abnormal PrP for metal and plastic surfaces using a solid-state matrix for capture coupled to direct immunodetection of surface bound material. Quantitative assay sensitivity was first determined using a dilution series of exogenous spikes of vCJD-infected brain into whole blood before analysing a blind panel of authentic patient blood samples. The panel comprised 190 samples, including those from vCJD and sporadic CJD patients, non-prion neurological disease controls and normal control blood samples.
An assay using a sample of blood or urine, most suitably blood, with high sensitivity and a specificity sufficient for clinical use is provided. The introduction of such a test would provide a breakthrough in the management of public health risks associated with vCJD and allow the identification of asymptomatic individuals, mitigating the risk of iatrogenic transmission and facilitating early referral for specialist care and entry into future clinical trials.
Definitions
The term ‘comprises’ (comprise, comprising) should be understood to have its normal meaning in the art, i.e. that the stated feature or group of features is included, but that the term does not exclude any other stated feature or group of features from also being present.
Prion Protein/PrP/PrPSc/PrPC
The invention is concerned with the detection of prion infection or prion infectivity. More precisely, the invention in concerned with the detection of abnormal PrP or disease associated PrP. Detection of the presence of abnormal PrP or disease associated PrP is indicative of prion infection. Thus the invention is primarily concerned with the detection of abnormal PrP and the presence of abnormal PrP aids the diagnosis of prion infection in the subject being analysed. Presence of abnormal PrP may be taken as an indication of prion infection. Abnormal PrP includes PrPSc within the complete ensemble of abnormal PrP isoforms; thus abnormal PrP comprises PrPSc. Most suitably the abnormal PrP may be PrPSc.
The abnormal PrP detected may be quantified. Quantities of abnormal PrP detected may be expressed as masses, or as equivalents of LD50 Units of infectivity.
An exemplary or reference sequence for human PrP is given in SEQ ID NO: 7.
Sample
The sample may be blood or urine. For convenience, the assay is mostly described in relation to blood. However, unless otherwise indicated, the disclosures herein may be applied equally to the assay whether the sample is blood or is urine. Regarding two specific factors (dilution and steel type) there may be advantages to using a specific condition when the sample is blood and a different specific condition when the sample is urine. For example, although all of the steels used herein may be applied to blood or to urine, there is an advantage to using steel AISI 304 when the sample is blood and there is an advantage to using steel AISI 316 when the sample is urine. Thus it can be appreciated that the disclosure provided is applied equally to blood or to urine, except where specifically mentioned otherwise.
It is a key advantage of the invention that detection of abnormal PrP in a sample of blood or urine, most suitably blood, is disclosed. In particular this is accomplished whilst avoiding protease treatment. In particular this is accomplished whilst avoiding immunoprecipitation steps. Thus suitably the sample analysed comprises blood or urine, most suitably blood.
The sample may be from a subject. The subject is suitably a mammal, most suitably a livestock animal or a human, most suitably a human.
The sample of blood or urine, most suitably blood may be collected as a preliminary step of the method of the invention. More suitably the method of the invention begins with a sample of blood or urine, most suitably blood previously collected so that collection of the sample is suitably not a part of the invention. This has the advantage that the subject need not be present for the methods of the invention.
Suitably the sample of blood or urine, most suitably blood is diluted for detection. Dilution of the sample is very important to the invention. The capture buffer has been carefully developed for diluting the sample to provide excellent results. Dilution of the sample over particular ranges is important to signal generation.
Blood Dilution
Typically blood is diluted 1:1 to 1:100. Most suitably the optimal dilution is chosen according to the species as in the following table:
For species not listed in the table, the skilled worker can determine the optimal dilution by trial and error using the given values as a start point. In example 5 there is provided a protocol for determination of optimised dilutions.
The volume of blood required may be chosen by the operator according to the volume(s) available and the processing requirements. A typical volume used is 8 μl of whole blood per aliquot/sample.
Without wishing to be bound by theory, the actual volume used is not thought to be critical. For example, doubling the volume used does not necessarily double the signal detected—the dilution of the blood is more important as explained above.
Blood derivatives such as blood fractions may be used in place of blood. For example, the red cell fraction may be used, the mononucleocyte fraction may be used, or the plasma fraction may be used. Typically such blood fractions perform in the method of the invention, but whole blood is preferred since it typically provides a stronger signal than blood fractions.
Urine Dilution
In contrast to blood, higher dilutions of urine such as 1:100 do not typically give robust signal for urine.
Typically urine is diluted 10:1 to 1:5. More suitably urine is diluted 2:1 to 1:2. Most suitably the optimal dilution of urine is 1:1.
If necessary, the skilled worker can determine the optimal dilution by trial and error using the given values as a start point. In example 5 there is provided a protocol for determination of optimised dilutions.
The volume of urine required may be chosen by the operator according to the volume(s) available and the processing requirements. A typical volume used is 400 μl of whole urine per aliquot/sample.
Without wishing to be bound by theory, the actual volume used is not thought to be critical. For example, doubling the volume used does not necessarily double the signal detected—the dilution of the sample is more important as explained above.
When the sample comprises urine, most suitably the urine is human urine.
Diagnosis
The invention provides methods for aiding the diagnosis of prion infection. Such methods are suitably performed on an isolated sample from the subject being investigated. Thus, suitably the methods are methods which may be conducted in a laboratory setting without the need for the subject to be present. Suitably the methods are carried out in vitro i.e. suitably the methods are in vitro methods. Suitably the samples are in vitro i.e. suitably the samples are in vitro samples.
Capture
According to the invention, abnormal PrP is captured on steel particles. The inventors have found that specific form(s) and/or amount(s) of steel particles can provide advantageously good results.
Suitably the steel particles used comprise steel powder.
Suitably the steel used is an austenitic stainless steel.
Suitably the steel used is a ‘3-series’ austenitic steel.
Suitably the steel used comprises AISI 316 or AISI 304; most suitably the steel used comprises AISI 304.
Suitably when the sample is blood the steel particles comprise AISI 304 stainless steel.
Suitably when the sample is urine the steel particles comprise AISI 316 stainless steel.
AISI is an international standard for the composition of steel. AISI 304 and AISI 316 are austenitic stainless steels with the chemical compositions:
Steels of these compositions from any manufacturers/suppliers may be used in the invention.
Most suitably steel is obtained from the commercial supplier Goodfellow.
Steel can be used within a concentration range of 5 mg to 90 mg ml−1. It is most effective in the range 10 to 50 mg ml−1 and is optimal at 23 mg ml−1.
The assay performance can be improved by preparing the steel before use. Suitably the steel is prepared by washing in aqueous detergent solution followed by washing in water, and then washing in an aqueous alcohol solution followed by washing in water. Finally the steel particles may be resuspended in phosphate buffered saline (PBS) ready for use.
An exemplary preparation method is as follows:
Prepare stock solution of 100 mg/ml of new batch of steel powder (AISI 304). Wash approximately 3 g in 30 ml 2% triton-x-100 (sigma)/sterile ddH2O in a falcon tube for 2 hours at room temperature. Remove liquid and wash powder 5×10 mins in 30 ml sterile ddH2O on rocking platform. Wash steel in 30 ml 70% ethanol and incubate for 10 mins on rocking platform, then wash again in 3×30 ml sterile ddH2O. Remove small aliquots of 1 ml into eppendorf tubes. Remove all liquid and weigh powder. Resuspend in PBS to give a final concentration of 100 mg/ml powder, aliquot into replica eppendorf tubes giving final mass of steel powder of 23 mg (230 μl of 100 mg/ml stock), then remove all liquid.
Suitably prepared steel particles are not allowed to dry out before use. Suitably steel particles are used within about 4 days of preparation.
Capture Buffer
The capture buffer comprises three core elements—detergent, buffer agent and background protein. The capture buffer may also contain one or more optional elements as discussed below.
Without wishing to be bound by theory, the buffer described herein have been arrived at to provide an advantageous balance between the need to disrupt membranes and release PrP, but without being so stringent as to disrupt PrP conformation and/or binding to steel.
Suitably chaotropic agents such as guanidine are not used. Without wishing to be bound by theory, it is believed that guanidinium is ineffective, which may be due to the chaotropic action releasing bound material from the steel surface. Suitably chaotropic agents such as guanidine are omitted from the methods and compositions of the invention.
Concentrations referred to are FINAL concentrations in use. This means final concentrations when diluted with the sample of blood or urine, most suitably blood to form the sample being analysed. Most typically a stock solution of capture buffer is prepared having correspondingly higher concentrations of the constituents so that when it is combined with sample of blood or urine, most suitably blood the concentrations are the final concentrations. For the avoidance of doubt, the concentrations being discussed are FINAL concentrations (i.e. concentrations when diluted with sample of blood or urine, most suitably blood) unless otherwise specified.
Detergent
The detergent is suitably a mild detergent such as a detergent suitable for use in NMR.
Suitably the detergent is a weak zwitterionic detergent. Suitably the detergent may be selected from any similar class of detergent such as sulphobetaine in any of its forms (3-06, 3-08, 3-10, 3-12, 3-14, 3-16), alkyldimethylamine oxides, alkyl glucosides, alkyl maltosides, alkyl sulphates, alkyl thioglucosides, Big CHAPS, CHAPSO, Bile acids, digitonin, glucamides, lecithins, lysolecithins, polyoxyethylenes or quaternary ammonium compounds such as CTAB.
The detergent is suitably CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate).
CHAPS is suitably used at between 1% and 8% w/v, being effective at these concentrations.
More suitably CHAPS is used at between 2% and 4% which has the advantage of working most effectively.
Most suitably CHAPS is used at 2%, which has the advantage of being optimal.
Buffering
The capture buffer may comprise buffer agent such as tris(hydroxymethyl)aminomethane (Tris) e.g. Tris.Cl or alternative buffer. In principle it is expected that any buffer would be functional. Phosphate buffered saline (PBS) may be used but Tris buffer offers improved performance relative to PBS. Suitably the buffer agent comprises Tris e.g Tris.Cl.
Suitably the buffer agent is used at a concentration of 10 to 500 mM.
More suitably the buffer agent is used at a concentration of between 50 and 200 mM, which has the advantage of producing best results.
When calculating the concentration of, or how to prepare, the buffer agent such as Tris, account must be taken of the molecular weight and/or the salt used. For example Tris has a molecular weight of 121.14, but is often supplied as Tris.Cl/Tris.HCl, and so molecular weights must be taken into account in preparation of the final concentration desired. For example 1 g of Tris may need to be adjusted to 1.3 g if using Tris.HCl (since Tris.HCl has a molecular weight of 157.56). Such adjustments are well within the abilities of the person skilled in the art. Unless otherwise indicated, weights given are for Tris.
The buffer is suitably pH 8.4.
Protein
Background protein is included in the capture buffer.
Suitably the background protein is Bovine Serum Albumin (BSA). Suitably this is used between 1% and 10% w/v.
More suitably this is used between 1 and 4%, which has the advantage of operating best.
Most suitably this is used at 2%, which has the advantage of being optimal.
Protease Inhibitors
Optionally protease inhibitors such as commercially available ‘Complete Inhibitors’ are included in the capture buffer. Most suitably the protease inhibitors are ‘Roche Complete protease inhibitor cocktails tablets’ (Roche Diagnostics GmbH) and are used according to the manufacturer's instructions. This has the advantage of eliminating protease treatments which have formed part of prior art methods. This also has the technical advantage of preserving more abnormal PrP signal. It is believed that some abnormal PrP types are in fact susceptible to protease degradation. Therefore the invention advantageously specifically excludes protease treatment. Suitably the invention relates to methods from which protease step(s) are omitted. Suitably the methods of the invention comprise inhibition of protease activity.
Nuclease
Optionally nuclease such as commercially available ‘Benzonase’ is included in the capture buffer.
Chelator
Optionally a chelator such as EDTA may be included in the capture buffer.
DMSO
Abnormal PrP may be aggregated into fibrillar structures. A solvent for amyloid is dimethyl sulphoxide (DMSO) and this may optionally be used as a pre-treatment for samples such as blood samples.
Thus, optionally a DMSO pretreatment may be applied to the sample before dilution into capture buffer. When optional DMSO pretreatment is used, it does not affect the final dilution ratio into capture buffer. When used, optional DMSO pretreatment can be considered as the first step in a two stage dilution. For example, if a final dilution of 1:100 is desired and an optional DMSO pretreatment is used, then a first step of 1:10 with DMSO may be used followed by a second step 1:10 into a final concentration of capture buffer may be used.
When used, it is desired to dilute the sample with DMSO first, and then secondly dilute into capture buffer. Simply adding DMSO to capture buffer with the sample in a one-step procedure is not desired.
Thus, when used, the dilution step can be carried out as two sub-steps
For example 8 ul of blood can be diluted to 80 ul in 50% v/v DMSO in PBS. This is optionally incubated at room temperature (21 C) for 4 hours with agitation. The samples are then diluted to final volume of 800 ul in DDA capture buffer containing 15% v/v DMSO. The assay can then be performed as described.
Stock Solutions of Capture Buffer
As noted above, higher concentrations of buffer may be prepared for use as stock solutions in order to ease preparation of blood/urine dilutions.
For example a final concentration of 100 mM Tris, 2% BSA, 2% CHAPS may be desired and a 5× stock may be prepared which would comprise 500 mM Tris, 10% BSA, 10% CHAPS. Thus the invention relates to buffers having these components in fixed ratios, independent of their precise concentrations. These have the advantage of facilitating dilutions and sample preparation. Thus for example compositions comprising this molar ratio, whether they be a 1×, 2× or any other concentration are provided by the invention.
Heat Treatment
A heat treatment is optionally applied to the steel particles after washing and before detection. This has advantage of exposing (‘retrieving’) epitopes on the captured PrP and thereby making it more amenable to detection by antibody.
It is possible that a chemical treatment could be used to expose epitopes on the captured PrP. However, guanidinium is ineffective, which may be due to the chaotropic action releasing bound material from the steel surface. Therefore it is preferred that the optional exposing/retrieving epitopes step is performed as a heat treatment.
The duration of the heat treatment is typically 5 minutes.
The heat treatment may be applied by any suitable means known in the art such as by water bath (for temperatures up to 100 degrees Celsius), oil bath, heating block, or any other device.
The heat treatment may be optimised depending on the species of PrP being detected (i.e. the species from which the sample of blood or urine, most suitably blood is derived). The table below provides exemplary values. Values for species not listed may be determined by trial and error using these values as a start point.
Detection
Detection of the actual abnormal PrP captured on the steel particles can in principle be carried out by any suitable method known in the art.
The term ‘antibody’ should be understood to be an especially suitable reagent or embodiment—it must be noted that antibody fragments, sc-Fv, fused or humanised antibodies or other antibody-derived reagents or affybodies or aptamer-type binding reagents may be used if desired. The important element is to have a reagent which can selectively, specifically bind abnormal PrP so that the presence or absence or abnormal PrP can be read out from the steel particles.
Thus the precise mode of detection of the abnormal PrP bound to the steel particles is a matter for the skilled worker. An exemplary approach is to contact the steel particles with a primary antibody capable of binding abnormal PrP, and incubating to allow binding. Excess antibody is then washed away. Binding of this primary antibody may then be detected e.g. by use of secondary antibody with chemiluminescence or similar.
Suitably the primary antibody is biotinylated which allows any avidin-based secondary reagent to be used for detection. However, if desired the primary antibody may equally be conjugated to an enzyme for detection such as horseradish peroxidise (HRP) or other well known moiety.
In a preferred embodiment detection is by application of one or more of the primary antibodies ICSM10, ICSM18, ICSM33 or ICSM35 to the steel particles. The ICSM antibodies are publicly availably e.g. from D-Gen Ltd (UK).
Suitably the antibody or antibody derived detection reagent comprises CDRs of ICSM10, ICSM18, ICSM33, or ICSM35 as appropriate. I.E. suitably the antibody or antibody derived detection reagent comprises CDR amino acid sequence as shown herein.
A designation of “B” following an antibody indicates that it is biotinylated. For example, ICSM10B means ICSM10 biotinylated antibody.
ICSM10 (D-Gen Product No. 0130-01001) is a monoclonal anti-prion protein (PrP); purified mouse immunoglobulin. The antibody isotype is IgG1 K. ICSM10 specifically reacts with human native and denatured PrPC and denatured PrPSc. The antibody also reacts with sheep, mouse, hamster and bovine PrP. By Western blotting and immunoprecipitation ICSM 10 does not bind to diglycosylated PrP; binding only un-glycosylated PrP and a subset of mono-glycosylated PrP. The mono-glycosylated PrP detected by ICSM 10 has a higher relative molecular mass than detected by ICSM 3 (Beringue et al., 2003 for mouse PrP).
The detection reagent of the present invention may comprise one or more antibodies or antibody fragments capable of binding abnormal PrP, mimetics thereof or small molecule(s) capable of binding abnormal PrP or combinations thereof. Preferably the detection reagent of the invention is an antibody or fragment thereof, preferably a monoclonal antibody or fragment thereof. Preferably the agent of the invention comprises an antibody or antibody fragment capable of binding abnormal PrP, such as ICSM10, ICSM18, ICSM33 or ICSM35 antibody or a fragment thereof.
Suitably the antibody comprises at least the CDRs of one or more antibodies shown in the sequence listing.
Advantageously when the agent is an antibody, said antibody is a humanised antibody. Humanisation of antibodies is well known in the art and can be easily accomplished by the skilled worker. For example, an antibody may be humanised with reference to the sequences encoding the CDRs presented herein. In this regard,
Moreover, reference is made to
Guidance regarding humanisation may be found for example in the literature as published by Greg Winter et al., and techniques for the manipulation and production of recombinant antibodies may be found in Harlow and Lane ‘Antibodies-A Laboratory Manual’, Cold Spring Harbour press.
In one embodiment, the antibodies (or fragments) may advantageously be humanised by manufacture of chimaeric antibodies.
In another embodiment, the antibodies (or fragments) may advantageously be CDR-grafted.
In another embodiment, the antibodies (or fragments) may advantageously be fully humanised to the extent that the technology permits.
In a preferred embodiment this primary antibody is biotinylated. More preferably, this is deliberately over-biotinylated e.g. to approximately 14× (i.e. approx 14 biotins per antibody molecule). This may be easily accomplished by conventional biotinylation but using approx. 10× the usual amount of biotin precursor/biotin reagent.
The invention may be applied to any species susceptible to prion infection. For example the invention may also be applied to bovine species such as dairy or beef cattle; capreoline species such as Capreolus or Cervidae (e.g. elk or venison deer); or any of the species in the tables herein.
Especially advantageous primary antibodies for detection of abnormal PrP for particular species are shown in the following table. Others may be selected by trial and error with reference to the examples section.
In more detail, method for detection of abnormal PrP in a sample of blood or urine, most suitably blood may be carried out as follows:
Ideally the 23 mg ‘dry’ steel particle aliquot is combined with a final volume of sample (blood-buffer or urine-buffer mix) of 800 μl.
In practice the 23 mg steel has a ‘dry’ volume of approx. 25 μl. Thus in practice the total volume of the sample (blood-buffer or urine-buffer mix) with the steel added is approx. 825 μl. It should be noted that ‘dry’ means merely that the excess liquid has been removed; it does not infer that the prepared steel is dried; in fact it is better to avoid the drying of the prepared steel, for example by drawing off the liquid just before adding the sample or by covering the steel (e.g. by placing a lid on the container) after the liquid has been drawn off so that it does not dry out.
(b-ii) incubating to allow PrP binding
This may be done by incubating the steel powder with each sample for at 18° C. o/n at 650 rpm on a thermomixer.
This washing procedure may be done by capturing steel powder samples with magnetic block and removing supernatant; washing steel powder samples 2×1 ml PBS/0.05% tween capturing each time on magnetic block. Vortex samples in wash buffer and spin briefly. Wash a further 3×1 ml PBS/0.05% tween capturing each time on magnetic block. Spin samples briefly and remove all liquid.
At this stage, an optional heat treatment may be used to improve detection. If used, the heat treatment step may be carried out as follows: Heat treat all samples of steel powder at appropriate temperature (see table) for 5 minutes on a heat block. Allow samples to cool for 3 minutes.
The detection step may be accomplished by any suitable method known in the art. For example, it may be carried out as follows: incubate steel powder samples with 50 μl of detection reagent such as primary antibody capable of binding abnormal PrP (e.g. with an appropriate ICSM antibody-see table) prepared in PBS/1% tween at 1 μg/ml; for 1 hour at 37° C. and 750 rpm on a thermomixer; wash steel powder samples 1×1 ml PBS/0.05% tween capturing each time on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples in wash buffer and spin briefly. Remove buffer and wash a further 1×1 ml PBS/0.05% tween capturing each time on magnetic block. Spin briefly and remove all liquid. Incubate steel powder samples with 50 μl of Neutravidin-HRP (Pierce) prepared in PBS/1% tween at 1:100,000 for 45 minutes at 37° C. and 750 rpm on a thermomixer. Prepare a serial dilution series of secondary detection reagent such as secondary antibody (1:100,000, 1:1 million and 1:10 million). Wash steel powder samples 1×1 ml PBS/0.05% tween capturing each time on magnetic block. Add 1 ml PBS/0.05% tween then vortex samples in wash buffer and spin briefly. Remove buffer and wash a further 1 x 1 ml PBS/0.05% tween capturing each time on magnetic block. Mix equal volumes of Supersignal ELISA femto chemiluminescent substrate (Thermo Scientific). Add 60μl of chemiluminescent substrate to steel powder aliquots, mix thoroughly by pipetting and aliquot into 3×200 in 3 replica wells of black ELISA plates (Greiner). To 2 replica wells add 20 μl of each dilution of the secondary antibody dilution series. Add a further 80 μl per well of Supersignal ELISA femto chemiluminescent substrate. Place plate into Tecan M1000 plate reader immediately. Shake for 60 seconds on plate reader then read plate on luminescence, attenuation automatic settings.
Combinations
The methods of the invention may advantageously be combined e.g. with tonsil biopsy and/or with MRI scan. For example if a subject is indicated as having an increased likelihood of infection by a method of the invention, then it may be desired to perform a tonsil biopsy and/or MRI scan in order to confirm the diagnosis.
Application to Urine
The assay steps when urine is the sample are the same as the assay steps when blood is the sample. The only difference is the dilution. Details are given for preferred blood dilutions and preferred urine dilutions separately herein. There are also advantages to different steels for blood and for urine, although each of the steels disclosed herein works with both sample types.
To the knowledge of the inventors, abnormal PrP has never been detected in urine before the present invention.
Numerous commercial hormone products are prepared (purified) from urine. Therefore testing of urine is commercially desirable. For example, in Canada approx. 300,000 patients per year receive hormone products purified from urine. This is a further clear industrial application for the invention when the sample is urine.
Industrial Application
The invention provides a blood or urine, most suitably blood, based assay for the detection of prion infection such as vCJD prion infection.
The invention finds application in aiding clinical diagnostic testing in cases of neurological disease where prion disease forms part of the differential diagnosis.
The methods of the invention may be used in a high throughput format for example for use by the UK National Blood Service and/or other similar international agencies.
The invention finds application in pre-surgical screening of patients to reduce or eliminate the risk of contaminating instruments and medical devices with vCJD prions.
The invention may be applied to screening of tissue and/or organ donations.
The invention may be used for detection of various strains/species of prion disease for elimination from the human food chain, eg Chronic Wasting Disease, BSE, sheep scrapie.
The methods disclosed may be used in prevalence screening for vCJD in humans and BSE, CWD and scrapie in animals.
The invention can be used to assist in eradicating animal prion diseases in commercial herds. The invention may be applied to the testing of commercial herds such as of cattle and sheep.
The invention provides a laboratory based test to replace rodent bioassay in many areas of prion research where they are used for sensitivity.
The invention is now described by way of example. These examples are intended to be illustrative, and are not intended to limit the appended claims.
Methods
Collection and Storage of Human Blood Samples
These studies were approved by the local research ethics committee of University College London Institute of Neurology and National Hospital for Neurology and Neurosurgery. Blood samples were obtained with consent, collected in EDTA blood tubes and stored frozen at −70° C. Diagnosis of vCJD was made according to established criteria e.g. http://www.advisorybodies.doh.gov.uk/acdp/tseguidance/tseguidance_annexb.pdf.Definite sporadic CJD was diagnosed according to WHO criteria and diagnosis of probable sporadic CJD according to published WHO criteria with a high specificity (35). Normal control blood was obtained as EDTA samples from the National Blood Service of England and Wales (NBS).
Assaying Blood Spiked with vCJD and Normal Brain Homogenate:
Steel particles (AISI 304; Goodfellow) were prepared by washing in 2% v/v Triton-X-100 in ddH2O for 2 hours at room temperature. Steel was captured with a magnet and washed 5× with an excess of ddH2O and sterilised by washing in 70% v/v ethanol before washing again with an excess of ddH2O. Aliquots of 23 mg were removed to microfuge tubes, captured with a magnet and the liquid aspirated with a pipette. 5 μl of 10% w/v vCJD-infected brain homogenate or normal human brain homogenate was spiked into 9995 μl of whole human blood. From this a serial dilution series of 10−7 to 10−10 of vCJD-infected brain homogenate into whole human blood was prepared. Each sample was further diluted 2 fold into capture buffer [200 mM Tris pH 8.4, 4% w/v BSA, 4% w/v CHAPS, 2× Complete protease inhibitors (Roche), 80 Units Benzonase (GradeII, Merck)] for assay. To each aliquot of steel powder, 800 μl of spiked blood/buffer mix was added and incubated at 18° C. with agitation overnight.
Steel was captured on a magnetic rack and the supernatant discarded. Steel was washed 5× with 1 ml PBS+0.05% v/v Tween-20 (PBST). After the final wash all liquid was removed and the steel heat treated at 115° C. for 5 minutes. To each tube an aliquot of 50 μl of biotinylated primary antibody ICSM18 (D-Gen, Ltd) prepared at 1 μg/ml in PBS+1% v/v Tween-20 was added and incubated at 37° C. with agitation for 1 hour. Samples were washed 3× with 1 ml PBST capturing between washes on a magnetic rack. Each sample was then incubated with High Sensitivity NeutrAvidin-HRP (Pierce) prepared at a 1:100,000 dilution in PBS+1% v/v Tween-20 at 37° C. with agitation for 45 minutes.
Finally samples were washed 3× with 1 ml PBST capturing between washes on a magnetic rack. To each sample 60 μl of SuperSignal ELISA Femto chemiluminescent substrate (Pierce) was added and 20 μl of each steel powder sample was transferred into 3 replica wells of a black flat bottom ELISA plate (Greiner). Immediately prior to reading the plate a further 80 μl of SuperSignal ELISA Femto chemiluminescent substrate was added per well. A dilution series of 1:100,000 to 1:10 million of the High sensitivity NeutrAvidin-HRP (Pierce) was prepared to allow for correction in absolute chemiluminescent readings across multiple plates. Plates were scanned using a M1000 plate reader (Tecan).
Assaying of Patient Blood Samples:
Steel particles (AISI 304; Goodfellow) were prepared as detailed above. To each aliquot of steel, 800 μl of a 1:100 dilution of whole human blood into capture buffer (as above) was added and incubated at 18° C. with agitation overnight. Steel was captured on a magnetic rack and the supernatant discarded before processing as described above. Plates were scanned using a M1000 plate reader (Tecan) in chemiluminescence mode.
The Blind Panel and Scoring of Samples:
A panel of 190 whole blood samples comprising 21 from vCJD patients, 100 normal controls (provided by the NBS), 16 patients with probable sCJD, 11 confirmed sCJD cases as well as 69 samples from other neurodegenerative diseases (25 Alzheimer's Disease, 4 Frontal Temporal Dementia (FTD), 6 Familial Alzheimer's Disease (FAD) and 7 neurological referrals to the National Prion Clinic confirmed as not Prion Disease) were prepared as blind samples numbered 1 to 190 by parties independent of the assay and analysis. Each of the blind panel samples were tested twice in independent assays as described above. Samples were processed and analysed in groups of 19 blind panel samples per 96-well plate with a set of 8 quality control samples containing 6 normal control blood samples and 2 vCJD-infected patient blood samples.
Samples were scored as reactive if the ratio of the mean chemiluminescence signal from three replicate wells exceeded a cut-off threshold determined for each plate. The threshold was set at the mean plus 3×Standard Deviations from the mean of the 6 normal blood samples on each plate. Thus samples with a ratio of greater than 1 were considered reactive. Samples that were reactive in each of the two independent assays were scored as positive samples. On completion of testing all samples in duplicate the results were declared to an independent party and the samples decoded.
In order to determine the sensitivity of the assay relative to other methods we analysed serial dilutions of vCJD brain homogenate diluted into whole blood to provide a background diluent as close to endogenous patient samples as possible. A dilution range of 10−7 to 10−10 of 10% w/v vCJD brain homogenate was assayed and compared to a high background concentration (10−6) of normal brain homogenate (10% w/v) also diluted in whole blood. Although a non-linear response was seen with respect to dilution, vCJD-infected brain homogenate could clearly be distinguished from control even at a 10−10 fold dilution (
Initial studies performed using exogenous spikes of vCJD-infected brain homogenate in whole human blood demonstrated our assay was capable of discriminating between infected and non-infected samples with a sensitivity theoretically sufficient to detect infection in vCJD blood based upon estimates of titre obtained from rodent models (22;23). However, the biochemical nature of infectivity and abnormal PrP associated with blood is unknown and the results obtained from exogenous spiking experiments cannot be assumed to apply to authentic patient samples. To ensure this level of discrimination could be achieved with endogenous blood samples we tested a sub-set of confirmed vCJD patient bloods obtained from the National Prion Clinic and compared these to normal control bloods obtained from the NBS (
In order to confirm our results and remove any bias from the analysis a panel of 190 samples taken from 21 confirmed vCJD patients (National Prion Clinic), 69 patients with other neurological disease and 100 normal healthy controls (NBS) were blinded by parties independent to the testing and analysis. The samples were tested in batches of 19 samples with internal controls to determine a cut-off threshold for each plate, samples which gave chemiluminescence signals above the cut-off were deemed reactive. To eliminate potential false positive reactions originating not from the sample but from contamination or assay errors each sample was tested twice within independent assay runs (
From the panel of 190 samples tested a total of 19 and 22 were reactive in assays 1 and 2 respectively (
Samples from 6 vCJD patients were not identified during testing of the blind panel. Of those samples 3 had been singularly reactive in either assay run 1 or 2 (
None of the normal controls or other neurological disease controls were scored as positive suggesting a high level of specificity. Superficially this could be considered as 100% as none of the controls were scored positive. However, it is important to consider the probability of a negative sample reacting in duplicate assays purely by chance and independent of abnormal PrP content. This rate is provided by the frequency of single reactive samples in each independent assay run and provides a realistic estimation of specificity of 99.97% ([2/190]Assay 1×[6/190]Assay 2).
Our research efforts have focused upon sensitive methods for the detection of vCJD infection in whole blood as this is not only a sample easily obtained for clinical purposes but also the target for protecting the donated blood supply. Through the use of matrix capture and optimised immunodetection of captured material we have been able to achieve detection of vCJD brain homogenate diluted 1010-fold in exogenous spiking experiments (
As an orphan disease, blood samples from patients with vCJD are scarce and it is impossible to validate assay results with the high numbers of samples typically used in such circumstances. However, we have applied our assay to all the samples available to us, totalling 21, of which 15 were positively identified in our blinded panel, yielding a sensitivity of 71%. Whilst this represents a very significant step forward in prion diagnostics it must be interpreted within the context of associated specificity. Indeed for consideration as a screening assay of blood and tissue donations specificity must exceed a minimum of 99.9% to avoid large numbers of false positive tests resulting in serious ramifications for the individual donors and the related health care agencies.
Our panel contained 100 normal blood samples from the NBS in addition to 69 neurological disease controls. We included samples obtained from patients with Alzheimer's Disease as it has been suggested that abnormal PrP deposition may accompany Aβ accumulation in these diseases (41). Other disorders that may form part of the differential diagnosis for prion disease were also included in the panel. Encouragingly, none of the neurological disease controls provided any reactions in either of the two independent assay runs, single reactive samples being either vCJD or normal controls. Although no false positive results were recorded by our assay criteria a predictive specificity for large scale screening of controls would be approximately 99.97% based on single reactive sample frequencies observed. This level of specificity would be acceptable in clinical use. This could usefully be confirmed with large (1000+) numbers of negative controls.
The blood samples obtained from vCJD patients have to date been taken following the onset of symptoms as at present it is not possible to identify individuals who are definitely infected with vCJD but are currently asymptomatic. There remains therefore the question of when during the pre-clinical silent stage of disease blood is rendered infectious or differentiable from normal controls. The availability of a blood based assay for determining prion disease infection could make it possible to identify preclinical patients infected with vCJD and provide the samples necessary for further assay validation. However, this would be further confounded by our lack of knowledge regarding what a positive assay result would mean in such circumstances and whether a positive assay result would always result in an individual progressing to a clinical onset.
The invention thus represents a major advance in the ability to detect prion infection and provides an assay forming part of a viable clinical blood test.
Note: this protocol is sometimes referred to as the DDA protocol.
The designations “Hamster A” and “Hamster B” refer to two different preferred embodiments for assaying hamster samples. When the dilution is 1:10, advantageously only a RT heat shock is required; conversely when the dilution is 1:1 then advantageously a 115 Celsius heat shock is used.
In this example, the invention is demonstrated for sheep.
The standard DDA protocol (e.g. as in Example 8) using biotinylated ICSM33 as a primary antibody, a dilution of blood 1:100 into capture buffer and a heat treatment at 120 C was applied to a range of sheep blood samples taken from 8 known uninfected controls and 8 cases of confirmed scrapie.
All 8 positive samples were distinguishable from negative controls
Data are shown in
The standard DDA protocol (e.g. as in Example 8) using biotinylated ICSM18 as a primary antibody, a dilution of blood 1:100 into capture buffer and a heat treatment at 120 C was applied to a range of cattle blood samples taken from 8 known uninfected controls and 8 cases of confirmed BSE.
All 8 positive samples were distinguishable from negative controls
Data are shown in
In this example, we demonstrate discrimination of vCJD urine from control samples. A standard DDA protocol (e.g. as in Example 8) was carried out using biotinylated ICSM18 as a primary antibody, a dilution of urine 1:1 into capture buffer and a heat treatment at 110 C was applied to a panel of 12 control urine samples and 4 samples collected from vCJD patients. The standard DDA protocol was modified by the use AISI 316 steel in place of the standard AISI 304. AISI 316 gives better results when the sample is urine. A cut-off value was calculated as the mean chemiluminescence signal of the 12 control samples plus 3×standard deviations from the mean.
Three of the four vCJD patient samples were considered positive, i.e. had a ratio of greater than 1.
N.B. Although AISI 304 steel also functions with urine samples, AISI 316 gives superior results.
Data are shown in
In this example, optional DMSO pretreatment is demonstrated.
A potential issue for the methods of the invention is that abnormal PrP is likely to be aggregated into fibrillar structures. A solvent for amyloid is dimethyl sulphoxide (DMSO) and this was investigated as a pre-treatment for samples, in this example blood samples.
8 ul of blood was diluted to 80 ul in 50% v/v DMSO in PBS and incubated at room temperature (21 C) for 4 hours with agitation. The samples were then diluted to final volume of 800 ul in DDA capture buffer containing 15% v/v DMSO. The assay was then performed as described above.
Pre-treatment increases the signal levels obtained from vCJD samples but does not affect control samples, thereby advantageously increasing the levels of differentiation.
Data are shown in
A typical high throughput and/or automated assay uses a 96-well format. However, such wells are necessarily of a rather small size. This is an advantage for sample processing and space considerations, but the small size restricts the volume available for analysis.
According to the invention, the assay volume can be varied (such as increased) since it is a flexible part of the method. However, it is important to make any volume increases in conjunction with an increased amount of steel particles/powder (i.e. matrix). In other words the ratios of components of the assay are suitably kept within the ranges described above, but by increasing the amount of each of the individual components, a greater volume can be assayed whilst maintaining the ratios of the components within the described ranges.
To demonstrate this, the assay was performed as per the protocol described above (e.g. example 8) with the exception of:
Increasing the volume of blood used in the assay with a fixed steel concentration of 23 mg/ml gives no improvement in signal.
A marginal improvement can be seen with 12 ul of blood and increased steel concentrations.
A significant enhancement is obtained by increasing the input to 16 ul in conjunction with increasing the steel concentration to 46 mg/ml. In this embodiment the input is doubled (8 ul to 16 ul) and the steel is doubled (23 mg to 46 mg) so the proportions remain the same and the capture buffer concentrations remain the same; the net effect is to prepare more of the steel-captured-analyte (the PrP) by use of the larger volumes without altering the core of the method of the invention. Thus the method of the invention can easily be scaled up to larger volumes to suit alternate read-out or assay formats as desired by the skilled worker.
Data are shown in
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described aspects and embodiments of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1015569.5 | Sep 2010 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2011/001341 | 9/13/2011 | WO | 00 | 3/15/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/035296 | 3/22/2012 | WO | A |
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| Number | Date | Country | |
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| 20130196356 A1 | Aug 2013 | US |
