Abstract:
A method for detecting aggregation or polymerization of molecules in a sample, which method comprises the steps of: a) exposing a sample containing the molecules to at least two labelling moieties which will bind to only a single mutually exclusive site on each of the said molecules; and b) determining whether multiply-bound labelling moieties are present. The method is particularly of use in differentiating pathogenic aggregated protein molecules from non-pathogenic, non-aggregated protein molecules.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to a method for distinguishing aggregated or polymerized forms of a given molecule from the unaggregated or unpolymerized form of the same molecule or a closely similar molecule.  
         BACKGROUND TO THE INVENTION  
         [0002]    Methods exist to detect the interaction between two different molecules. A dual bacteriophage method has been described in WO 99/63348 in which each molecule for which the interaction must be detected is labelled by a bacteriophage encoding a different selectable marker. As the molecules interact the phage are brought into proximity and can infect the same host bacilli thereby conferring the double selectable marker phenotype on the host. A yeast hybrid method has been described in which the proteins of interest are expressed in the yeast genome. If the proteins interact they activate a promoter which generates a measurable signal. A method called Fluorescence Excitation Transfer (FET) exists in which a transfer of energy can be measured between two ligands that are used to label each interacting molecule and are brought into close proximity by the interaction of these molecules (for a review of possible labels and approaches see Wood, P and Barnard, G. 1997. Fluoroimmunoassay in Principles and Practice of Immunoassay. Price and Newman Eds. Macmillan Reference Ltd., UK).  
           [0003]    In some cases, however, it is desirable to measure or detect a change in a given molecule that has causes the molecule to bind together or aggregate or polymerize. For example, in some disease such as the transmissible spongiform encephalopathies (TSEs) there has been a change in the normal cellular protein PrPc which is not aggregated to a form such as PrPsc which is aggregated or bound together. This aggregation and subsequent deposition in vivo may cause the pathology of these diseases. A considerable problem in diagnosis or detection of such diseases is the similarity of the normal and pathogenic proteins. Methods must be devised to differentiate the normal from the pathogenic form. It is difficult to raise antibodies, for example, which are specific for the pathogenic form and most antibodies cross-react with the normal cellular protein. Consequently, existing diagnostic methodologies must employ a complicated enrichment sample preparation procedure involving detergent extraction, protease treatment and/or solvent extraction in order to preferentially select the pathogenic protein prior to performing the assay.  
           [0004]    The present invention seeks to provide an improved method of differentiating aggregated molecules from unaggregated normal molecules.  
         DISCLOSURE OF THE INVENTION  
         [0005]    According to an aspect of the present invention there is provided a method for detecting aggregated or polymerized molecules in a sample, which method comprises the steps of:  
           [0006]    a) exposing a sample containing the molecules to at least two labelling moieties which will bind to only a single mutually exclusive site on each of the said molecules; and  
           [0007]    b) determining whether multiply-bound labelling moieties are present.  
           [0008]    Preferably the labelling moieties comprise a first virus and a second virus which are genetically different from each other. Both viruses are capable of binding directly or indirectly to only a single site on each of the said molecules. In addition, the viruses bind directly or indirectly to the same site on the said molecule so that binding of one virus excludes the binding of the other virus. The viruses either bind to the same identical site or a similar overlapping site such that when one virus is bound the other virus is prevented from binding. The latter can be achieved by steric hindrance by the first-binding virus thus preventing the second virus from binding or conformational change in the target material thus preventing the second virus binding.  
           [0009]    In the preferred embodiment of the method the viruses bind indirectly to an identical or overlapping site through monoclonal antibodies that are specific for an identical or overlapping sites. Hereafter, this property of the binding of one labelling moiety to the target molecule excluding the binding of the second or other labelling moieties is termed ‘mutually exclusive binding’, and the term ‘mutually exclusive site’ refers to a site on a molecule on which mutually exclusive binding occurs. The binding of two or more labelling moieties to the target material forms a material that has a distinctive property when it includes both the first and the second virus linked together via aggregated or polymerized molecules.  
           [0010]    The term “virus” is used herein to denote true viruses and organisms which infect bacteria in manner similar to a true virus. Thus, the term virus includes:  
           [0011]    a. Components of a virus which have the characteristics of the virus from which they are derived;  
           [0012]    b. Packaged phagemids, which are crosses between plasmids and viruses and can grow as plasmids in bacterial hosts, but which can be packaged and secreted as if they were viral particles in the presence of a helper virus although they cannot independently produce viral progeny;  
           [0013]    c. Viruses which are lysogenic for bacteria and can grow, replicate and produce progeny in the bacteria without lysis of the bacteria which can continue to grow and replicate.  
           [0014]    If viral infection of bacterial cells is carried out using an excess of bacteria over that required to achieve parity between the infecting viruses and the infectable bacterial cells, a given bacterium cell is unlikely to be infected by more than one virus particle. The amount at which such dual infection becomes sufficiently unlikely that it will not distort the results of the assay method of the invention can be calculated statistically and is denoted herein as the statistical amount. Such a statistical calculation can be confirmed by simple trial and error tests.  
           [0015]    In a preferred embodiment of the invention, two viral particles are physically linked together through the target material and can thus each infect the same bacterial cell so as to endow that cell with both of the characteristic properties of the infecting viruses. A bacterial cell infected by both viruses and possessing the sum of the two characteristic properties can readily be distinguished from cells which possess only one of those properties. For example, the infected cells can be cultivated under conditions under which the cells possessing only one of the properties cannot survive, for example in the presence of specific antibiotics or specific temperature or pH conditions. The infected cells having both characteristic properties survive and will replicate. Thus, in the presence of tagged target material a cascade of bacterial growth indicates that the target material was present in the initial sample. If no tagged material is present in this cultivation stage, little or no bacterial growth will take place. In addition, if lysogenic viruses are used as the tags to be attached to the target material, the viruses will replicate within the infected bacterial cells and produce progeny virus particles which will be released to begin further cycles of infection and replication.  
           [0016]    In a particularly preferred method of the invention a ligand such as a monoclonal antibody (mAbs) is used which recognizes and binds to one site on the molecule of interest. In the unaggregated or non-pathogenic state only one ligand will bind to one molecule in isolation from all other ligands. However, when the molecule is in the pathogenic form and aggregated, a plurality of ligands bind to the aggregate through recognition of the binding sites on each individual molecule comprising the aggregate. In this case the aggregate contains many copies of the ligand. A single ligand can be differentiated from multiply bound ligands using various detection techniques. A proportion of the ligand can be labelled with a first moiety and a proportion of the ligand labelled with a second moiety such that in the aggregate with multiple ligand binding the energy transfer between the two moieties that have been brought into close proximity can be measured (for a review of possible labels and approaches see Wood, P and Barnard, G. 1997. Fluoroimmunoassay in Principles and Practice of Immunoassay. Price and Newman Eds. Macmillan Reference Ltd., UK). In another example, the ligands could be labelled with the inactive components or subunits of a functional moiety such that when the components or subunits are brought together the moiety is endowed with a measurable function. In this example the subunits of an enzyme would be used as labels such that when brought together in proximity they partially restore or fully restore or alter the activity of the enzyme. It is preferred to use the double bacteriophage method described in WO 99/63348, the contents of which are hereby incorporated by reference, with the two bacteriophage being labelled with the same ligand. In this example, when the two bacteriophage labelled with ligand are added to the assay the normal molecule will only bind one ligand and one phage so there will be no signal. However, an aggregate of the pathogenic molecules will bind many molecules of the ligand that will be labelled with either phage type. It is likely that a mixture of the two phage types will be bound to the aggregate through the ligand. The linking of the two phage types through the ligand and the aggregated pathogenic molecules generates a signal as described in WO 99/63348.  
           [0017]    In a further embodiment of the invention a nucleic acid label may be used. In this case a ligand such as a monoclonal antibody is coupled to a single or double stranded nucleic acid molecule that acts as the label. Coupling can be undertaken by standard techniques, such as linking a 5′ or a 3′ amino DNA oligonucleotide to an antibody using bis succinimide or bis maleimide cross-linkers or glutaraldehyde. Short oligonucleotides of 15 to 25 bases are preferred; longer DNA molecules of several hundred bases may also be employed. Under appropriate conditions the oligonucleotide coupled to one antibody via the 5′ end is able to hybridize to the oligonucleotide coupled via the 3′ end to another antibody molecule; this is preferably achieved using a prior cleavage with a restriction endonuclease that generates short complementary sequences (‘sticky ends’) at each end of the oligonucleotides prior to coupling to the antibodies.  
           [0018]    When the monoclonal antibody labelled with either form of nucleic acid label binds to the aggregated form of a molecule the oligonucleotide labels will be juxtaposed and hybridization will be promoted. This will be a more favourable reaction than that taking place in either free solution (the background signal) or when binding to non-aggregated molecules. After hybridization of the oligonucleotides a ligation reaction, catalyzed either chemically or with DNA ligase can proceed; for single stranded DNA molecules a prior hybridization step is preferred with a complementary ‘bridging’ oligonucleotide that generates a double stranded DNA region as the substrate for the ligase. Blunt end ligation can also take place between two non-overlapping DNA oligonucleotide labels.  
           [0019]    After ligation molecules of the type described by Cantor (U.S. Pat. No. 5,849,878) are generated, that is two antibody molecules linked by a ligated DNA molecule. The product of ligation may then be detected by various means, such as hybridization of labelled probes across the ligated region, or by DNA amplification techniques such as PCR using primers situated either side of the ligation region. Other suitable DNA amplification techniques include RCA (rolling circle amplification) and LCR (ligase chain reaction).  
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0020]    The invention will now be described by way of illustration in terms of preferred embodiments thereof. In these examples the Dual Phage detection method is used to demonstrate the presence of polymerized bovine serum albumin (BSA) in the first example and polymerized tubulin in the second example. In the third example it is shown that the method can differentiate the aggregated PrP molecules associated with the Transmissible Spongiform Encephalopathy (TSE) disease scrapie from the normal unaggregated PrP molecules present in the normal non-diseased brain. The Dual Phage method uses two phagemid which encode two different antibiotic resistance genes: resistance to ampicillin in phage A and resistance to chloramphenicol in phage C. The phagemid are packaged into infectious particles using a M13-derived helper phage.  
         [0021]    Method 1: In this method a model system was used to demonstrate that artificially cross-linked molecules could be differentiated from identical molecules that were not cross-linked.  
         [0022]    Preparation of the Assay Reagents  
         [0023]    Anti-BSA monoclonal antibody (clone no. BSA-33, Sigma-Aldrich Company Ltd, UK) and the phage A and C were biotinylated using Biotinamidocaproic acid 3-sulfo-N-hydroxysuccinimide ester (Biotin ester) (Sigma-Aldrich Company Ltd, UK). The antibody was dialysed against PBS and 2.5 μl of biotin ester stock at 0.1 mg/ml in PBS added (this gives an average of approximately 1 biotin label per antibody molecule) and incubated at room temperature for 2 hours. Excess ester was removed by dialysis. Phage A and phage C were prepared following standard procedures (Phage Display of Peptides and Proteins: A Laboratory Manual. 1996. Kay, Winter and McCafferty, Eds. Academic Press, UK) and purified by caesium chloride density gradient centrifugation. After dialysis against PBS to remove the caesium chloride 10 11  plaque forming units of each phage were labelled in a volume of 100 μl with 10 μl of biotin ester (10 μg/ml in PBS) for 2 hours at room temperature. Excess ester was removed by polyethylene glycol precipitation of the phage following standard procedures (Phage Display of Peptides and Proteins: A Laboratory Manual. 1996. Kay, Winter and McCafferty, Eds. Academic Press, UK) and the phage pellet was resuspended in 100 μl PBS.  
         [0024]    Detection of Cross-Linked BSA  
         [0025]    1. BSA, Fraction V (Sigma-Aldrich Company Ltd, UK) was cross-linked using serial dilutions of glutaraldehyde (Grade 1, 25% stock) (Sigma-Aldrich Company Ltd, UK). The BSA was prepared at 10 mg/ml in PBS and 100 μl aliquots cross-linked at room temperature for 2 hours with serial 5-fold dilutions of glutaraldehyde of final concentrations 2-0.0032% (v/v). A no-glutaraldehyde and a no-glutaraldehyde, no-BSA control were also included in the experiment.  
         [0026]    2. After incubation the volume of each reaction was made up to 1 ml with TBS, 5 mM ethanolamine (Sigma-Aldrich Company Ltd, UK) and incubated for 2 hours at room temperature in order to quench the glutaraldehyde.  
         [0027]    3. 1 μl of each reaction was added to 10 μl of 4% (w/v) sodium dodecylsulfate (Sigma-Aldrich Company Ltd, UK), 2.5% (v/v) B-mercaptoethanol (Sigma-Aldrich Company Ltd, UK) and heated at 95° C. for 5 mins in order to denature the protein and expose the epitopes for antibody binding.  
         [0028]    4. After denaturation, each reaction was made up to 1 ml with 50 mM sodium carbonate pH 9.6.  
         [0029]    5. Each reaction was diluted a thousand-fold in the carbonate buffer and 100 μl of each was used to coat a maxisorb (Nunc, Denmark) microtiter plate well at 4° C. overnight.  
         [0030]    6. After coating, the wells were washed x3 with TBS 0.2% (v/v) Tween 20. The last wash was left in the well for 60 mins at room temperature.  
         [0031]    7. The biotinylated anti-BSA monoclonal antibody was diluted 10 −4  in TBS Tween20 and 100 μl added to each well.  
         [0032]    8. After 1 hour at room temperature the wells were washed x3 with TBS Tween20 and 100 μl of streptavidin (Sigma-Aldrich Company Ltd, UK) (a stock solution of 1 mg/ml diluted 10 −4  in TBS Tween20) added.  
         [0033]    9. After 1 hour at room temperature the wells were washed x3 with TBS Tween20 and 100 μl of TBS Tween20 containing 108 plaque forming units of each of the biotinylated phage C and phage A was added.  
         [0034]    10. After incubation at room temperature for 1 hour the wells were washed x3 with TBS Tween20 and then x2 with TBS.  
         [0035]    11. 1 ml of log phase growth XL-1 Blue (Stratagene)  E. coli  cells are then added to each well and the microtiter plate incubated at 37° C. for 60 minutes.  
         [0036]    12. The cell suspension from each well was then plated out on a 2xYT (Sigma Aldrich Chemical Company Ltd) 1.5% (w/v) agar containing 100 μg/ml ampicillin and 50 μg/ml chloramphenicol.  
         [0037]    13. After incubation overnight at 37° C. the number of colonies on each plate were counted.  
         [0038]    Results  
                                                                 Percentage of glutaraldehyde   Number of colonies           used for cross-linking   on the plate                                        2   3           0.4   66           0.08   50           0.016   Greater than 1000           0.003   142           0 (no glutaraldehyde)   70           Control (no BSA)   80                      
 
         [0039]    Conclusion  
         [0040]    This experiment demonstrates that the cross-linked BSA could be differentiated from the monomeric (uncross-linked) BSA. The monoclonal antibody can only bind to one site on each BSA molecule. Consequently, this results in the capture of only one phage A or C to each molecule of BSA. This does not bring phage A and C into close proximity to allow the subsequent dual infection of the  E. coli . Consequently, the  E. coli  remains uninfected or singly infected and does not grow on agar plates containing both antibiotics. However, when the BSA is cross-linked there are multiple molecules bound together in close-proximity each of which can bind one phage A or C. Thus phage A and phage C are brought into close proximity which allows dual infection of the  E. coli  that can then grow on the agar plates containing both antibiotics.  
         [0041]    From the results, it can be seen that under these conditions and in this model system 0.016% (v/v) glutaraldehyde gave the highest signal. Lower concentrations of glutaraldehyde probably yielded few cross-linked molecules while at a higher concentration of glutaraldehyde the epitope detected by the antibody could be modified by the agent and destroyed preventing recognition by the antibody.  
         [0042]    Method 2.  
         [0043]    In this method the aggregated form of a self-aggregating molecule was distinguished from the unaggregated form.  
         [0044]    1. Anti-tubulin monoclonal antibody TUB 2.1 (Sigma Aldrich Company Ltd) was cross linked to each of the two bacteriophage, phage A and phage C, using standard conjunction chemistry protocols (Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences. 1998. Aslam and Dent, Eds. Macmillan Reference Ltd., UK).  
         [0045]    2. Two tubes containing 100 μl 2 mg/ml tubulin (T238, Cytoskeleton, Inc, Denver USA) were prepared. To one 10 μl of distilled water was added; this is the unpolymerized control tube. To the other tube 10 μl of glycerol was added to initiate the polymerization process.  
         [0046]    4. The tubes were incubated at 37° C. for 30 mins.  
         [0047]    5. Each reaction was diluted 10 −6  and to 10 μl of each dilution 10 μl of phage buffer (50 mM Na 3 PO 4 , 0.15 M NaCl, 1 MM MgCl 2  pH 7.2) containing 10 5  plaque-forming units of anti-tubulin antibody-conjugated phage A and phage C was added and incubated 30 min at room temperature.  
         [0048]    6. 1 ml of log phase growth XL-1 Blue (Stratagene) E. coli cells were then added to each tube and incubated at 37° C. for 60 minutes.  
         [0049]    7. The tubes were centrifuged for 5 minutes at 4000×g and the pellets resuspended in 50 μl of phage buffer.  
         [0050]    8. The resuspended pellets were then plated out on two plates containing 2xYT media (Sigma Aldrich Chemical Company Ltd), 1.5% (w/v) agar and 100 μg/ml ampicillin and 50 μg/ml chloramphenicol.  
         [0051]    9. The plates were incubated overnight at 37° C. and the resulting numbers of colonies of  E. coli  counted.  
         [0052]    Result:  
         [0053]    The plate derived from the tube containing unpolymerized tubulin contained no colonies of  E. coli  growth. The plate derived from the tube of polymerized tubulin showed many colonies of  E. coli  growth (greater than 1000 colonies). The polymerized tubulin was able to link the two phage types such that they could infect the same bacterial host and confer resistance to both antibiotics which were subsequently used for selection in the agar plate.  
         [0054]    This experiment shows that unaggregated or unpolymerized molecules, in this example of tubulin, can clearly be differentiated from the aggregated or polymerized form. The latter gives a clear positive signal when using a monoclonal antibody in the Dual Phage detection assay.  
         [0055]    Method 3  
         [0056]    In this example material for the assay was prepared from scrapie-infected and uninfected ovine brain homogenates by detergent solubilization and low speed centrifugation clarification of debris as described in Saborio, G. P., Permanne, B., and Soto, C. 2001. Nature, 411: 810-813. No proteinase K step was performed.  
         [0057]    To prepare the phage conjugates, the same anti-PrP monoclonal antibody was cross linked to each of the two bacteriophage, phage A and phage C, using standard conjunction chemistry protocols (Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences. 1998. Aslam and Dent, Eds. Macmillan Reference Ltd., UK).  
         [0058]    1. The wells of a Reacti-Bind Maleic Anhydride microtiter plate (Pierce, UK) were coated with 100 ng of an anti-PrP monoclonal antibody in carbonate buffer overnight at 4° C.  
         [0059]    2. The wells were filled with 0.2 M ethanolamine pH 7.5 and incubated at room temperature for 2 hours.  
         [0060]    3. 100 μl of the material extracted from the uninfected and the infected brain was added to separate wells and incubated at room temperature for 1 hour.  
         [0061]    4. The wells were then washed x3 with PBS and then filled with 0.016% (v/v) glutaraldehyde in PBS.  
         [0062]    5. After incubation for 2 hours at room temperature the wells were washed with PBS and then filled with 6 M urea.  
         [0063]    6. After 30 mins the wells were emptied and filled with 0.2 M ethanolamine pH 7.5.  
         [0064]    7. After 30 mins the wells were washed x3 with TBS Tween20.  
         [0065]    8. 100 μl of TBS Tween20 containing 108 plaque forming units of each of the anti-PrP conjugated phage C and phage A was added.  
         [0066]    9. After incubation at room temperature for 1 hour the wells were washed x3 with TBS Tween20 and then x2 with TBS.  
         [0067]    10. 1 ml of log phase growth XL-1 Blue (Stratagene) E. coli cells were then added to each well and the microtiter plate incubated at 37° C. for ˜60 minutes.  
         [0068]    11. The cell suspension from each well was then plated out on a 2xYT (Sigma Aldrich Chemical Company Ltd) 1.5% (w/v) agar containing 100 μg/ml ampicillin and 50 μg/ml chloramphenicol.  
         [0069]    12. After incubation overnight at 37° C. the number of colonies on each plate were counted.  
         [0070]    Results  
         [0071]    The plate derived from the well containing uninfected brain extract contained no colonies of  E. coli  growth. The plate derived from the well containing infected brain extract showed many colonies of  E. coli  growth (greater than 1000 colonies). The aggregated PrP was able to link the two phage types such that they could infect the same bacterial host and confer resistance to both antibiotics which were subsequently used for selection in the agar plate. This shows that the method can be used to differentiate the aggregated disease form of a molecule from the unaggregated normal form.  
         [0072]    Although the invention has been described with reference to preferred embodiments thereof, the invention is not limited to these embodiments and many modifications may be made within the scope of the claims.