Patent Grant
7659079
Transmissible spongiform encephalopathies (TSEs) cause spongy degeneration of the brain with severe and fatal neurological symptoms in humans and animal. TSEs include scrapie, which affects sheep and goats; bovine spongiform encephalopathy (BSE), which affects cattle; transmissible mink encephalopathy; feline spongiform encephalopathy; chronic wasting disease (CWD) of cervids including mule deer, white-tailed deer, black-tailed deer, and elk; and kuru, Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, fatal familial insomnia, and variant Creutzfeldt-Jakob disease (vCJD), which affect humans.
The only identified component of the agent causing TSEs is PrPSc, an abnormal aggregating isoform of PrPc. Current methods of detecting PrPSc subject a sample to proteolysis with proteinase K to destroy PrPc. The presence of surviving PrPSc is then determined by an immunoassay using an antibody that is not selective for PrPSc in the presence of PrPc. See Serban et al., Neurology, 40:110 1990. This methodology excludes the use of an antibody for capture or detection during the proteolysis step. Proteinase K must be removed or deactivated before any antibodies can be introduced to the assay.
Methods are needed that can rapidly identify samples containing TSEs with minimal sample handling, discriminate between the normal and disease-associated conformer of Prp independent of proteinase K digestion, and that can be automated for high throughput applications.
One embodiment of the invention provides a method for selective binding of an aggregating abnormal form of a protein in the presence of a non-aggregating normal form of the protein. The method comprises contacting, under selective binding conditions, brain tissue suspected of comprising the aggregating abnormal form and the non-aggregating normal form of the protein with a polyionic material having a binding avidity for the abnormal aggregating form of the protein, a zwitterionic agent, and a discriminating agent, wherein the aggregating abnormal form of the protein selectively binds to the polyionic material. The polyionic material can be protease resistant.
The polyionic material can be a polyanionic material having a multiplicity of anionic groups or a polycationic material having a multiplicity of cationic groups. The polyionic material can have a multiplicity of anionic groups that are sulphate, carboxyl or phosphate groups or a multiplicity of cationic groups that are amino groups, imine groups or quaternary ammonium groups.
The discriminating agent can have a lesser density of anionic groups than the polyionic material. The discriminating agent can be an anionic detergent or an amino acid amide of a fatty acid such as laurylsarcosine.
The aggregated abnormal form of the protein that is selectively bound to the polyionic material can be captured with an immobilized capture agent. The capture agent can be an antibody specific for the aggregated abnormal form of the protein.
The polyionic material which is selectively bound to the aggregated abnormal form of the protein can be captured with an immobilized capture agent. The capture agent can be a lectin or an antibody.
The selective binding conditions can comprise a pH from about 8 to about 9 or from about 8.2 to about 8.6.
The polyionic material can comprise a selectively bindable tag moiety and the capture agent can selectively bind to the tag moiety. The aggregated abnormal form of the protein comprises a selectively bindable tag moiety and the capture agent selectively binds to the tag moiety. The bindable tag moiety can be biotin, fluorescein, dinitrophenol, digoxyrenin, a nucleic acid or nucleic acid analogue sequence or (His) 6.
The polyionic material can be immobilized to a solid support material prior to contacting the brain tissue. The solid support material can have the polyionic material coated thereon. The polyionic material can be immobilized on the solid support through direct adsorption to the support. The polyionic material can comprise a selectively bindable tag moiety and can be immobilized to the solid support material via the tag moiety. The bindable tag moiety can be biotin, fluorescein, dinitrophenol, digoxyrenin, a nucleic acid or nucleic acid analogue sequence or (His) 6. The polyionic material can be a solid that provides a surface having said binding avidity.
The zwitterionic agent can comprise a zwitterionic detergent. The zwitterionic agent can comprise 3-(N,N-Dimethylocyl-ammonio) propanesulfonate, 3-(Decyldimethylammonio) propanesulfonate, 3-(Dodecyldimethylammonio) propanesulfonate, 3-(N,N-Dimethylmyristylammonio) propanesulfonate, 3-(N,N-Dimethylpalmitylammonio) propanesulfonate, n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, a sulfobetaine, 3-(1-pyridino)-1-propane sulfonate, dimethyl-2-hydroxyethyl-1-propane sulfonate, 3-(1-methylpiperidinium)-1-propane sulfonate, dimethylbenzylammonium-1-propane sulfonate, dimethylethylammonium-1-propane sulfonate n-dodecyl-N,N-dimethylglycine, lauryldimethylamine oxide, or combinations thereof. The zwitterionic agent can comprise a sulfobetaine. The zwitterionic agent can comprise a sulfonate group or a carboxyl group. The abnormal aggregated form of the protein can be PrPSc and the non-aggregated normal form of the protein can be PrPc.
Another embodiment of the invention provides a method of determining presence or absence of an abnormal aggregating form of a protein in a brain tissue sample in the presence of the non-aggregating normal form of the protein. The method comprises contacting, under selective binding conditions, brain tissue suspected of comprising the aggregating abnormal form of the protein and the non-aggregating normal form of the protein with a polyionic material having a binding avidity for the abnormal aggregating form of the protein, a discriminating agent and a zwitterionic agent; and determining the presence or absence of the abnormal aggregating form of the protein bound to the polyionic material.
The amount of the abnormal aggregating form of a protein can be determined. Determining the presence or absence of the abnormal aggregating form of the protein bound to the polyionic material can be qualitatively or quantitatively determined by conducting an immunoassay for the aggregating form of the protein.
The abnormal aggregated form of the protein can be PrPSc and the non-aggregated normal form of the protein can be PrPc.
The zwitterionic agent can comprise a zwitterionic detergent. The zwitterionic agent can comprise 3-(N,N-Dimethylocyl-ammonio) propanesulfonate, 3-(Decyldimethylammonio) propanesulfonate, 3-(Dodecyldimethylammonio) propanesulfonate, 3-(N,N-Dimethylmyristylammonio) propanesulfonate, 3-(N,N-Dimethylpalmitylammonio) propanesulfonate, n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, a sulfobetaine, 3-(1-pyridino)-1-propane sulfonate, dimethyl-2-hydroxyethyl-1-propane sulfonate, 3-(1-methylpiperidinium)-1-propane sulfonate, dimethylbenzylammonium-1-propane sulfonate, dimethylethylammonium-1-propane sulfonate n-dodecyl-N,N-dimethylglycine, lauryldimethylamine oxide, or combinations thereof. The zwitterionic agent can comprise a sulfobetaine. The zwitterionic agent can comprise a sulfonate group or a carboxyl group.
It has recently been discovered that selective binding of an aggregating abnormal form of a protein can be achieved by contacting a sample containing both aggregating abnormal forms of a protein and non-aggregating normal forms of the protein, under selective binding conditions, with a polyionic material having a binding avidity for the aggregating abnormal form of the protein. See WO 03/073106 A2 (Lane et al., “Binding of Pathological Forms of Prion Proteins”), which is incorporated by reference herein in its entirety. The binding conditions can include a discriminating agent such as laurylsarcosine. Among the advantages to this method is the fact that the proteolysis step is eliminated. Therefore, antibodies or other proteins can be added to the selective binding conditions at any time. The polyionic materials, selective binding conditions, other conditions, other materials, assay methods, and other methods described in WO 03/073106 can be used in the instantly disclosed methods and are specifically incorporated by reference in their entirety herein.
The instant invention provides an improvement to the compositions and methods described in WO 03/073106. The improvement comprises addition of a charged agent, such as charged detergent agent, for example, a zwitterionic agent to the selective binding conditions.
For example, brain tissue comprising an aggregating abnormal form of a protein and a non-aggregating normal form of the protein can be contacted, under selective binding conditions, with a polyionic material having a binding avidity for the abnormal aggregating form of the protein, a zwitterionic agent, and a discriminating agent such as laurylsarcosine.
Selective binding means that an aggregating abnormal form of a protein binds to a polyionic material having a binding avidity for the aggregating abnormal form of the protein, while the non-aggregating abnormal form of the protein does not substantially bind the polyionic material.
Selective binding conditions provide conditions under which polyionic materials bind aggregated abnormal proteins forms, for example, PrPSc, but do not substantially bind the non-aggregated normal form of the protein, for example PrPc. Selective binding conditions provide binding that is sufficiently strong and selective to be useful in assays for the presence of the aggregated abnormal form of the protein. Selective binding conditions can be determined by one of skill in the art and can be obtained by, for example, suitable adjustment of the reaction conditions, particularly the presence and concentration of a discriminating agent, a charged agent such as a zwitterionic agent, the pH, and the detergency. Suitable selective binding conditions are described in, for example, WO 03/073106 and in the examples below. In one embodiment of the invention selective binding conditions comprise a pH from about 8 to about 9, and more particularly a pH from about 8.2 to about 8.6.
Binding avidity means the overall binding strength of a molecule with many binding sites with a multivalent binding agent (e.g., the polyionic material), which is in contrast to “affinity”, which is the binding strength between each individual binding site of the molecule and the binding agent (e.g., the polyionic material).
Suitable polyionic materials having a binding avidity for the aggregating abnormal form of the protein are described in WO 03/073106, which is incorporated by reference herein in its entirety. A polyionic material can be protease resistant. The polyionic material can be a polyanionic material having a multiplicity of anionic groups or a polycationic material having a multiplicity of cationic groups. Anionic groups can be, for example, sulphate, carboxyl or phosphate groups. Cationic groups can be, for example, amino groups, imine groups or quaternary ammonium groups.
In one embodiment of the invention a detergent is part of the selective binding conditions and promotes selective binding either by virtue of detergency or by acting as a discriminating agent.
Aggregating Abnormal and Non-Aggregating Normal Forms of Proteins
Methods of the invention can detect or selectively bind an aggregating abnormal form of a protein in the presence of the non-aggregating normal form of the protein. In particular, methods of the invention can detect or selectively bind an aggregating abnormal form of a protein in the presence of a non-aggregating normal form of the protein wherein the proteins are present in or derived from brain tissue. The methods of the invention comprise contacting, under selective binding conditions, a sample, such as brain tissue, suspected of comprising the aggregating abnormal form and the non-aggregating normal form of the protein, with a polyionic material having a binding avidity for the abnormal aggregating form of the protein, a zwitterionic agent, and a discriminating agent such as laurylsarcosine.
One example of a protein that has aggregating abnormal forms and non-aggregating normal forms is PrP. The only identified component of the agent that causes transmissible spongiform encephalopathies (TSEs) is PrPSc, which is an abnormal aggregating isoform of the non-aggregating normal form of PrPc. Therefore, in one embodiment of the invention, the disclosed methods can be used to detect or selectively bind PrPSc in the presence of PrPc.
One example of aggregating abnormal forms of proteins are abnormal protein aggregates dominated by beta-sheet structures such as beta-peptides that form amyloid deposits in Alzheimer's disease, alpha-synuclein protein that produces amyloid-like deposits in Lewy bodies of Alzheimer's and Parkinson's patients, and the ABri peptide that form amyloid deposits in familial British dementia (FBD).
The test samples can be, for example, mammalian brain tissue. In one embodiment of the invention the obex is used. Methods of the invention can detect or selectively bind TSEs in samples suspected of comprising TSEs that cause scrapie, BSE, transmissible mink encephalopathy, feline spongiform encephalopathy, CWD, kuru, Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, fatal familial insomnia, and variant Creutzfeldt-Jakob disease (vCJD).
Charged Detergent Agents
A charged detergent or detergent-like agent can be added to the selective binding conditions of the methods of the invention to improve sensitivity and detection of an aggregating abnormal form of a protein. A charged detergent or detergent-like agent can be an anionic, cationic, or zwitterionic detergent or detergent-like agent. A zwitterionic agent is a molecule carrying both a positive and a negative charge. Any zwitterionic agent can be used in the methods of the invention, for example, a zwitterionic agent can be, for example, ZWITTERGENT® 3-08 (n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), ZWITTERGENT® 3-10 (n-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), ZWITTERGENT® 3-12 (n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), ZWITTERGENT® 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), ZWITTERGENT® 3-16 (n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). In one embodiment of the invention the zwiterionic compound is a Zwitterionic detergent.
Other zwitterionic agents are sulfobetaines, including, for example, 3-(1-pyridino)-1-propane sulfonate, dimethyl-2-hydroxyethyl-1-propane sulfonate, 3-(1-methylpiperidinium)-1-propane sulfonate, dimethylbenzylammonium-1-propane sulfonate, dimethylethylammonium-1-propane sulfonate. Other zwitterionic agents include n-dodecyl-N,N-dimethylglycine, and lauryldimethylamine oxide. See also, the zwitterionic agents listed in Examples 2 and 3.
About 0.1% to about 10% of a charged agent, such as a zwitterionic agent is added to the selective binding stock solution or working plate diluent. Therefore, about 0.02, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, or 20% of a zwitterionic agent is present in a selective binding reaction.
Discriminating Agents
A discriminating agent is an agent that allows selective binding of PrPSc to a polyionic material, as described above, and/or prevents PrPc from binding to the polyionic material. The discriminating agent can have a lesser density of anionic groups than the polyionic material. The discriminating agent can be an anionic detergent, an amino acid amide of a fatty acid, or laurylsarcosine. A discriminating agent can comprise about 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, or 10% of the selective binding conditions.
Detection of Aggregating Abnormal Forms of a Protein
Once the aggregating abnormal form of the protein has been selectively bound to the polyionic material and optionally after the normal form of the protein has been removed, the presence or absence and/or quantity of the aggregating abnormal form the protein can be determined. See e.g., WO 03/073106. Any assay type to detect the selectively bound abnormal form of the protein can be used. For example, enzyme linked immunosorbent assay (ELISA), western blot, indirect fluorescent antibody assay (IFA), radioimmunoassay (RIA), hemagglutination (HA), and fluorescence polarization immunoassay (FPIA) can be used. Any antibody specific for PrP can be used in these assays. Several of such antibodies are known in the art. In some cases, a denaturant, such as guanidine thiocyanate (GuSCN), is used to expose PrP epitopes before or during the addition of an antibody specific for PrP to the assay.
The polyionic material can be immobilized to a solid support material either before or after being contacted with a sample. Separation of the sample from the solid support material can then be used to remove the non-aggregating normal form of the protein from the assay leaving only the aggregated abnormal of the protein. Solid support materials are well known in the art and include, for example, include microtiter plates, dipsticks, laminar flow devices, microbeads and superparamagnetic microbeads.
Biotin or other tags can be conjugated to the polyionic material by methods well known in the art. Biotin is a bindable tag moiety that can be used to bind the polyionic material to a solid support material derivatized with avidin or a material with avidin binding properties such as steptavidin, Neutravidin or Captavidin.
Other molecules can be used as bindable tag moieties and include those that are readily conjugated to a polyionic material and that can be captured or bound by a suitable capture agent such as fluorescein dinitrophenol DNP, digoxygenin, nucleic acid or nucleic acid analog sequences, and (His) 6. A capture agent can be used that selectively binds the polyionic material itself rather than through a tag moiety. For instance, polyglycosides can be bound by a suitable lectin or by a suitable antibody.
The captured aggregating abnormal form of the protein can be, if necessary, eluted from the polyionic material prior to the assay. Sodium dodecyl sulphate (SDS) is suitable for this purpose and is preferably used at a concentration of about 0.5 to about 1% by weight, preferably above about 0.75%.
The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
The following are provided for exemplification purposes only and are not intended to limit the scope of the invention described in broad terms above. All references cited in this disclosure are incorporated herein by reference.
Brain samples (ovine or bovine) were collected from known negative or TSE-positive animals and homogenized to prepare 10-20% lysates in water. Before applying the homogenate to an ELISA assay plate, the sample was diluted into a working plate diluent, which is composed of plate diluent component 1 and plate diluent component 2. Plate diluent component 1 contains 250 mM TrisHCl, pH 8.3, 5% bovine serum albumin, 5% laurylsarcosine and 5% TRITON® X-100. Plate diluent component 2 (1 mg/ml DNAse I and 2.5 mg/ml trypsin) was mixed thoroughly with component 1, and 25 μl of the final working plate diluent was mixed with 100 μl of brain homogenate to prepare the sample for assay.
ELISA assays were performed using antigen capture plates that were coated with a charged polyionic polymer. See WO 03/073106. One hundred μls of brain homogenate-working plate diluent mixture were applied to each well and incubated for two hours at room temperature without agitation. After two hours, the lysates were aspirated from the plates, and the plates were washed 6 times with 1× wash solution. The last wash was aspirated, tapped on absorbent pad, and 100 μl of conditioning buffer containing 4M guanidine thiocyanate added to the wells. After a 10 minute incubation at room temperature, the plates were aspirated and washed 3 times with 1× wash solution.
To detect bound PrPSc, the plates were incubated with 100 μl of HRPO-conjugated anti-PrP antibody solution for one hour at room temperature. After aspiration of the detection antibody solution, the plates were washed 5 times with 1× wash solution. The plates were then tapped dry onto an absorbent pad, 100 μl of TMB substrate added, and the plates incubated for 15 minutes to allow color development. The assay was then stopped with an HCl stop solution, and absorbance of the microwells read at 450 nm and 650 nm (for background compensation).
To determine if the addition of ZWITTERGENT® 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) to the working plate diluent would improve the performance of the TSE assay, either 5% or 0.5% ZWITTERGENT® 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) were added to the formulation for working plate diluent component 1. Assays were performed using normal sheep brain, or scrapie sheep brain homogenate that was diluted into normal sheep brain homogenate at either 1:25 or 1:100 ratios; the ELISA assay protocol used was the one described above. The results are shown in
The experiments summarized in
The data in
To determine how the addition of ZWITTERGENT® 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) to the working plate diluent influences the sensitivity of a BSE ELISA assay, a series of dilutions of BSE brain homogenate into normal bovine brain homogenate were prepared and tested with the working plate diluent. In
Evaluation of Different Zwitterionic Detergents in Plate Diluent
The addition of zwitterionic detergents to TSE EIA Plate Diluent 1 was investigated for the ability to increase the ability to detect PrPSc in brain homogenate as compared to the non-ionic detergent TRITON® X-100.
The plate diluent 1 base solution comprises, TRIZMA® Base (pH 8.3), N-Lauroyl Sarcosine detergent, Bovine Serum Albumin Solution, and Deionized water. The zwitterionic detergents used in the experiment include:
TSE—EIA Assay:
Approved BSE EIA Test Kit components
Normal sheep brain homogenate (20% w/v)
Scrapie brain homogenate diluted into normal sheep brain homogenate@1:1500
Scrapie brain homogenate diluted into normal sheep brain homogenate@1:300
Scrapie brain homogenate diluted into normal sheep brain homogenate@1:40
Eleven zwitterionic detergents were added to a plate diluent base solution 1 at concentrations of 0.1%, 1.0%, & 10%. A control non-ionic detergent (TRITON® X-100) was also added to the plate diluent base at 5%, the amount previously determined to be the optimal concentration for this detergent. Each formulation of plate diluent 1 was mixed with the standard plate diluent 2 (2.5 mg/mL Trypsin; 1 mg/mL Dnase 1; 250 mM Tris HCl (pH 8.3) (final concentration in working plate diluent)) and then added to the 20% brain homogenate samples (100 ul brain homogenate to 25 ul working plate diluent). The sample/diluent mixture was added to a polyionic capture microtiter plate. Assays were performed according to standard BSE assay protocol using four samples as indicators: normal ovine brain homogenate and ovine scrapie brain homogenate that was diluted into normal sheep brain homogenate at 1:1500, 1:300, and 1:40 fold dilutions. A standard BSE EIA assay protocol was used. The sample was mixed with the working diluent while avoiding any bubbles. The diluted sample is added to the polyionic capture microplate along with the controls. The plate is covered and incubated for 2-3 hours. The plates are washed 6 times with 1× wash solution 1 (2.2 g/L Sodium Phosphate Monobasic, anhydrous; 11.9 g/L Sodium Phosphate Dibasic, anhydrous; 85 g/L Sodium Chloride; 10 g/L N-Lauroyl Sarcosine; deionized water). A conditioning buffer is added and the plates are covered and incubated for 10 minutes. The plates are washed 3 times with 1× wash solution 2 (2.2 g/L Sodium Phosphate Monobasic, anhydrous; 11.9 g/L Sodium Phosphate Dibasic, anhydrous; 85 g/L Sodium Chloride; 10 mL/L TWEEN® 20; deionized water). Conjugate (Conjugate Diluent:17.55 g/L Sodium Chloride; 0.22 g/L Sodium Phosphate Monobasic, anhydrous; 1.19 g/L Sodium Phosphate Dibasic, anhydrous; 0.5 mL/L Igepal CA-720; 2 mL/L 500 mM EDTA; 0.1% Bovine Serum Albumin; 3 mL/L Blue Dye; Deionized water. Conjugate Concentrate Stock:12F10: HRPO Conjugate (final working concentration: 0.1-1 ug/ml); Stabilzyme conjugate stabilizer) is added to the plate. The plate is covered and incubated for 15 minutes. The reaction is stopped with HCl solution.
The plate is read at 450 nm (reference wavelength AREF=620-650 nm). The cutoff is mean NC+0.120. For interpretation: if samples A450-AREF is less than the cutoff the result is negative. If samples A450-AREF is greater than or equal to the cutoff the result is initially reactive (retest in duplicate). If the duplicate retest mean A450-AREF is greater or equal to the cutoff the sample is positive.
Table 1 depicts the actual optical density values (OD) for testing the scrapie and normal ovine samples with the different plate diluent formulations. The test detergent OD values relative to the control detergent OD value for testing of the scrapie 1:300 sample are listed as an example of the increased signal obtained for the different zwitterionic detergents evaluated in this study. With only a few exceptions, the zwitterionic detergents result in a 2 to 2.8-fold increase in optical density for a positive sample with out increasing the signal on negative samples.
All eleven zwitterionic detergents evaluated in plate diluent 1 of the TSE-EIA at a concentration of between 0.1-10% enhanced the PrPSc specific signal by at least 2-fold as compared to the non-ionic TRITON® X-100 plate diluent formulation. This data demonstrates that a variety of zwitterionic detergents can be substituted into the TSE plate diluent 1 formulation and result in increased selective binding of PrPSc by the polyionic capture material.
The methodology of Example 2 was repeated using BSE samples. The results are shown in Table 2 and
| Number | Date | Country |
|---|---|---|
| 9830909 | Jul 1998 | WO |
| 9941280 | Aug 1999 | WO |
| 03073106 | Apr 2003 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20050239139 A1 | Oct 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60559815 | Apr 2004 | US |
