Abstract:
The present invention relates to agents comprising non-natural protein sequences with at least two protein or polypeptide epitopes that are relationally linked by a common or shared functional relationship such as being components in a series of components of the same metabolic or signal transduction pathway or a pathway associated with interaction between two systems such as host-parasite or host-pathogen. The invention further includes a method of simultaneously calibrating and investigating the quantitative relationships between the at least two protein or polypeptide epitopes.

Description:
[0001]    The present invention relates to agents comprising non-natural protein sequences, the sequence comprising a scaffold portion and at least two protein or polypeptide epitopes, the proteins or polypeptides being relationally linked by a functional relationship such as and without limitation, being components in a series of components of the same metabolic or signal transduction pathway or a pathway associated with interaction between two systems such as host-parasite or host-pathogen. The protein or polypeptide may also be, for example and without limitation, related by their interaction with a given protein or they may occupy a common site in a cell or they may be isoforms of the same protein or allelic variations thereof. The invention further includes a method of simultaneously calibrating and investigating the quantitative relationships between the at least two proteins or polypeptides. 
       BACKGROUND 
       [0002]    Biological science commonly involves the study of components which are in some way linked, i.e. they possess an important biological relationship with one another. The link or relationship could be that the proteins are: 
         [0000]    1. Isoforms of one another, that is to say a protein that has the same function as another protein but is encoded by a different gene and may have small differences in its sequence. For example, transforming factor beta (TGF-B) naturally exists in three versions, or isoforms (TGF-B1, TGF-B2, and TGF-B3), each of which can set off a signalling cascade that starts in the cytoplasm and terminates in the nucleus of the cell. Alternatively isoforms can be created by alternative splicing of RNA transcribed from a gene.
 
2. Allelic variants of one another. Polymorphisms of a gene exist in the human population, and other species, which in some cases lead to amino acid sequence differences in the protein between individuals within the species. These polymorphisms might be without functional consequence or they might deliver benefit to the organism in particular circumstances, or they might be associated with medical disease.
 
3. Components of a particular process, in biology most phenomena are achieved by the coordinated action of multiple components. These are frequently called pathways, where a series of linked reactions occur within a living cell to produce a specific product or products. There are numerous metabolic pathways for example glycolysis, tricarboxylic acid or Krebs cycle, urea cycle; see Nicholson for detailed metabolic pathway maps http://www.tcd.ie/Biochemistry/IUBMB-Nicholson/), pathways in signal transduction (e.g. hormones, cytokines, growth factors, stress; several examples can be found at http://www.grt.kyushu-u.ac.ip/spad/) or pathways associated with the interaction between two systems (e.g. host-parasite interactions, host-pathogen interactions).
 
4. Proteins sharing a common location in the cell. The relationship could be proteins which interact with a given protein, or proteins which occupy a common site in the cell (e.g. a lipid raft).
 
         [0003]    It is known from the prior art to use biochemical assays or antibodies specific for each individual protein as herein before described to elucidate the underlying mechanisms of pathways or to detect their presence. 
         [0004]    It is also known in the prior art to use single or multiple or his tagged proteins to detect or aid in the isolation and purifications of any of the proteins as herein before described so as to permit their quantification. However, these tagged proteins do not posses the appropriate properties which would allow them to be used for the study of components which are in some way linked and possess an important biological relationship with one another nor can they be used to study the “natural” molecule by virtue of the addition of extra protein sequence (which could influence structure or function) in the form of the epitope tag. Moreover, they do not provide any means with which to simultaneously calibrate quantitatively the amount of test product within a sample. 
         [0005]    PCT/GB2005/00015 describes calibration complexes comprising a scaffold protein and a method of using the complexes for quantifying the amount of a target protein in a sample. A target moiety, such as a protein, is covalently attached to one or more cysteine or lysine groups on the scaffold protein and the scaffold material has a controlled property such as relative molecular mass or weight or a pH value for the isoelectric point. In this way the target moiety and scaffold protein can be used to detect not only the presence of a target protein in a sample but also as a positive control, an internal standard or may it be used to generate a calibration curve. 
         [0006]    In the present invention we provide agents that have advantageously combined the property of a calibration standard with the ability to quantify multiple related or linked components in a biological system. In addition we describe a method using the agents of the present invention to detect not only the presence of individual relationally linked proteins or polypeptides but the quantity of each protein in a relationally linked series in a sample, in this way the method and agents of the invention may be used to provide stiochiometric information about the linked series of components in a sample. 
       BRIEF SUMMARY OF THE DISCLOSURE 
       [0007]    According to a first aspect of the invention there is provided a non-natural protein sequence, the sequence comprising a removable scaffold portion and at least two or more protein or polypeptide epitopes, the proteins or polypeptides epitopes being relationally linked. 
         [0008]    Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. 
         [0009]    Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. 
         [0010]    Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. 
         [0011]    Reference herein to “the proteins or polypeptides being relationally linked” is intended to include a shared or common functional relationship such as being components of the same metabolic or catabolic or signal transduction pathway, or isoforms of the same protein or polypeptide, or allelic variations thereof, or proteins or polypeptides that share a common location within a cell or that interact with the same target or protein or homologue thereof. 
         [0012]    Reference herein to an “epitope” relates to a specific chemical domain on an antigen that stimulates the production of, and is recognized by, an antibody. Each epitope on a molecule such as protein elicits the synthesis of a different antibody. aka antigenic determinant. 
         [0013]    Reference herein to “removable scaffold” is intended to include a non-natural protein sequence which may, as an end product contain a scaffold portion or which may prior to its use have the scaffold portion removed by, for example and without limitation enzymatic means. It will be appreciated that the non-natural protein sequence of the present invention when being constructed will comprise a backbone or scaffold on which the multiple epitopes can be arranged and that the epitopes may be manufactured so as to be adjacent one another without an intervening scaffold portion or that the scaffold portion may be cleaved out of the protein sequence so that the end product may exist as a string of multiple relationally linked epitopes. 
         [0014]    Preferably, the non-natural protein sequence of the invention comprises a plurality of epitopes, the number of epitopes may be more than 5 or 10 or 20 or 30 or 40 or 50 or more, there being no upper limit for their number rather the number being dictated by practical capabilities of their manufacture and a user&#39;s requirements. 
         [0015]    Preferably, the epitopes may also function as a calibration entity in so far as an epitope may have a selected and known property such as and without limitation a known molecular weight. 
         [0016]    It will be appreciated that the non-natural protein sequences of the present invention provide a product capable of serving simultaneously as a calibrant for two or more different biologically related components as well as providing a means of identification of the same. 
         [0017]    Preferably, the epitopes are linked in series in a continuous length of sequence. The epitopes could be linked directly to one another or they could be attached to a backbone sequence comprising non-reactive or inert molecules. 
         [0018]    In one embodiment of the invention, where the sequence allows, the epitopes could overlap. Again the epitopes could directly overlap one another or they may be interspersed with regions of an inert backbone sequence. 
         [0019]    In an alternative embodiment of the invention, the epitopes could be discontinuous in the primary sequence of the product, that is to say some may be present at the N-terminus, others at the C-terminus and some may be present within the sequence of the scaffold portion. 
         [0020]    It will be appreciated that in the instances that the epitopes are linked in series they are essentially non-branched non-natural protein sequences and as such there is no requirement for chemically reactive groups in the scaffold portion. 
         [0021]    However in other embodiments, where the epitopes are not linked in series the one or more epitopes could preferably be covalently attached to the calibration portion at a site other than utilising the α-carbon backbone sequence. Such “branched” non-natural protein sequences could preferably be fabricated using covalent bonding through unique (or controlled numbers of) reactive residues such as cysteine, lysine, aspartate, glutamate, tyrosine. 
         [0022]    Antibodies recognise relatively few amino acids in the particular protein target, which in one embodiment of the invention comprises a linear stretch of amino acids in the primary sequence (continuous epitope), and in another embodiment comprises amino acids in discontinuous in primary sequence, but contiguous in 3-dimensional space (discontinuous epitope). 
         [0023]    In the present invention we use antibodies specific for individual epitopes of a number of relationally linked proteins or polypeptides in the non-natural protein sequence to understand the workings of, for example and without limitation a biochemical pathway. A quantitative understanding of such pathways is most desirable, and this also requires calibration of the output of studies using the protein/polypeptide specific antibodies. Since the non-natural protein sequence of the present invention also includes a calibration portion which comprises known amounts of epitope specific for each antibody in the series, it conveniently provides a means of investigating the quantitative relationships between components within a biochemical pathway or other series of proteins such as isoforms, allelic variations or proteins located within a common intra-cellular compartment. 
         [0024]    The linking of a number of epitopes in series is an attractive advantage of the present invention as it reduces the cost of production of each product substantially (virtually in proportion with the number of epitopes in series) and thus makes the product profitable at reasonable price to the consumer. 
         [0025]    Reference herein to a “scaffold portion” refers to a protein or concatamer within the non-natural sequence of the agent of the present invention which is non-reactive or innocuous and contributes to the calibration capability only in terms of its dominant physical properties for example by its molecular weight, and/or its pI, and/or its good production (expression) characteristics. 
         [0026]    Preferably the scaffold portion of the non-natural protein sequence has a controlled property, preferably this property is relative molecular mass (Mr) or weight (Mwt) or the pH value for the isoelectric point of a given substance in solution (pI). 
         [0027]    Preferably, the scaffold portion is a protein. 
         [0028]    Preferably the scaffold portion comprises at least one natural or unnatural amino acid with at least one or more chemically reactive groups, preferably within the side chain of a residue. 
         [0029]    Preferably the scaffold portion comprises one or more chemically reactive groups, for example, the carbonyl on glutamic acid or aspartic acid or the hydroxyl on tyrosine and more preferably still comprises at least one cysteine and/or lysine amino acid groups. Thus it will be appreciated that the scaffold portion may be a polymer containing a thiol or primary amine functional group or any other protein in which there are suitable reactive side chain groups such as aspartic acid, glutamic acid, cysteine and/or lysine groups available for covalent conjugation with the epitopes. It is desirable that the covalent conjugation of the epitopes to the chemically reactive groups of the scaffold portion be controlled. 
         [0030]    Preferably, the number of reactive cysteine and/or lysine groups may be controlled by selecting the scaffold portion protein from a natural source which contains the desired number of reactive cysteine and/or lysine groups. 
         [0031]    Preferably, the scaffold portion protein is selected from the group comprising: 127, from titin which contains two cysteine residues; I39 domain which is a subunit (subunit 5) of splicing factor 3b and which contains one cysteine residue, organ of Corti protein ( Mus musculus ) Swiss-Prot/TrEMBL Primary Accession Number Q8R448 which contains one cysteine and one lysine residue; heat shock protein, mitochondrial ( Mus musculus ) Swiss-Prot/TrEMBL Primary Accession Number Q64433 which contains eleven lysine residues; splicing factor 3B subunit 5 ( Mus musculus ) Swiss-Prot/TrEMBL Primary Accession Number Q923D4 which contains one cysteine and five lysine residues; ubiquinol-cytochrome C reductase complex ubiquinone-binding protein QP-C ( Schizosaccharomyces pomme ) Swiss-Prot/TrEMBL Primary Accession Number P50523 which contains one cysteine and six lysine residues; E1B protein (Human adenovirus type 11) Swiss-Prot/TrEMBL Primary Accession Number Q8B8U6 which contains one cysteine residue; chaperonin ( Arabidopsis thaliana ) Swiss-Prot/TrEMBL Primary Accession Number P34893 which contains nine lysine residues; photosystem 11 reaction centre H protein ( Arabidopsis thaliana ) Swiss-Prot/TrEMBL Primary Accession Number P56780 which contains three lysine residues; a NADH-ubiquinone oxidoreductase subunit, mitochondrial [Precursor] ( Homo sapiens ) Swiss-Prot/TrEMBL Primary Accession Number P56181 which contains one cysteine and nine lysine residues; signal recognition particle protein ( Mus musculus ) Swiss-Prot/TrEMBL Primary Accession Number P49962 which contains two cysteine and eight lysine residues; DNA polymerase delta subunit 4( Mus musculus ) Swiss-Prot/TrEMBL Primary Accession Number Q9CWP8 which contains two cysteine and six lysine residues. 
         [0032]    In a particular embodiment, the scaffold portion may comprise one or more domains, such as 127, from titin. Titin contains a number of β-sandwich domains belonging to the Ig family. The I27 domains usually contain two cysteine residues and fold to form stable structures of 10 kDa. In Nature, the I27 domain contains two cysteine residues (the site for covalent attachment of peptide); however mutation of these cysteine residues, to serine for example, is compatible with domain folding. Thus a presentation system of I27 can be formed where the molecular weight step size is a convenient unit (10 kDa steps) and where one unit (or more if required) can be engineered to possess a single cysteine residue for peptide attachment while all other units of I27 will lack cysteine residues. In alternative embodiments, the units of I27 may lack other reactive residues. These residues may include, but not be limited to lysine, glutamate and aspartate. A copy of I27 could contain one or more of these reactive residues, offering a controlled number of sites for the covalent attachment of epitopes. 
         [0033]    Preferably, the number of reactive cysteine and/or lysine groups may be controlled by modifying any of the aforementioned scaffold portion proteins by selectively mutation by adding in or out or rendering ineffective any one or more of the reactive cysteine and/or lysine residues. 
         [0034]    Alternatively, one or more of the titin domains may be mutated to possess either one or no cysteine residues. In one embodiment, the non-natural protein sequence comprises one or more I27 domains and a plurality of epitopes, wherein one of the I27 domains comprises a single cysteine residue and the other I27 domains lack a cysteine residue. In an alternative embodiment, the scaffold portion may comprise an I39 domain which is a subunit (subunit 5) of splicing factor 3b. The I39 domain is a 10 kDa domain. Preferably, the scaffold portion is of a convenient molecular weight and is typically selected as 10 kDa for convenience. 
         [0035]    Preferably, the scaffold portion of the non-natural protein sequence of the present invention is blind to the antibody/antibodies specific to epitopes i.e. non-reactive thus, the scaffold portion may be considered capable of discrimination so that it is absolutely or relatively “immunologically blind” or “reactively inert” or substantially so. 
         [0036]    Many proteins are covalently modified in a transient manner in response to a number of stimuli. This form of covalent modification can alter the function of the protein in question. Covalent modification can take the form of phosphorylation (of serine, threonine, tyrosine, lysine, arginine, histidine, aspartic acid and glutamic acid residues), sulphation of tyrosine residues, nitrosylation of cysteine residues (http://download.cell.com/supplementarydata/cell/106/6/675/DC1/TableS1.pdf) glycosylation (of threonine, serine residues), ubiquinatinylation and related modifications (of lysine residues). A full understanding of the process of transient covalent modification includes description of the proportion of molecules covalently modified at any particular point in time, this is termed the stoichiometry or modification and it represents the number of moles of modified protein per mole of total target protein (i.e. mol/mol). 
         [0037]    In one embodiment of the invention the non-natural protein sequence of the present invention comprises at least two independent epitopes relating to a single protein. 
         [0038]    Preferably, the at least two independent epitopes relating to a single protein are both situated on the backbone sequence comprising non-reactive or inert molecules or one may be situated there and the other associated with the scaffold portion. 
         [0039]    The agents of the present invention can be used to determine the stoichiometry of modification of a particular site on a protein target. For example, total protein can be determined using a scaffold portion protein containing the epitope for the protein (where antibody binding to this epitope is not affected by any of the covalent modifications the protein can experience) as described above. In addition, the scaffold portion or concatamer portion can contain a single reactive residue to allow the covalent attachment of a second peptide epitope containing the covalent modification site, to which a modification-specific antibody exists. The non-natural protein sequence will thus contain two independent epitopes relating to this one protein, one (in series with the backbone of the scaffold protein) which records the total protein content of a sample, and a second (forming a branch in the polymer) peculiar to the covalent modification site. The use of this agent comprising a non-natural protein sequence with an experimental (test) sample will permit the collection of calibrated data relating to the binding of both antibodies to the agent and will facilitate calculation of the stoichiometry of modified proteins in the sample. 
         [0040]    Preferably, the non-natural protein sequence comprises a single (or controlled number of) attachment site(s) for the second epitope, which will result in the creation of a branched polymer. Cysteine, lysine, aspartic acid, glutamic acid, tyrosine are the principal reactive residues found in proteins. “In line epitope” sequences (i.e. those continuous with the α-carbon backbone of the scaffold portion) will dictate whether the protein contains controlled numbers of these residues to facilitate attachment of the second epitope (modification specific) without corruption of the first (in line series). For example, where an epitope is being displayed by covalent attachment to a thiol group within the scaffold portion, the presence of thiol groups within the in line epitope(s) would be highly undesirable. In some cases the “in line epitope” sequence might prove incompatible with this approach, and alternative in line epitope sequences will be required. 
         [0041]    In an alternative embodiment of the invention, the “branched” sequence may contain information for one or more epitope. 
         [0042]    Preferably, multiple epitopes may be a contiguous sequence or in an overlapping sequence or they may contain hapten epitopes (e.g. phosphoamino acid, dyes, other modified amino acids) or alternatively they could be linear or branched structures. 
         [0043]    For example, protein isoforms, generated from alternative genes, by alternative splicing, or by post-translational processing (e.g. proteolysis) can be used to create functional versatility from a limited genetic resource. Isoforms can differ in terms of their functional properties, their mode of regulation (partners, modification sites), their location in a cell, or alter their stability and life-time (1). 
         [0044]    Thus it will be appreciated that the non-natural protein sequence of the present invention has the ability to bind partners specific to the selected epitopes and simultaneously to provide calibration qualities. 
         [0045]    In a further aspect of the present invention, there is provided a non-natural protein sequence for use in detecting the presence of one or more relationally linked proteins or polypeptides in a sample and calibration of the said sample, the non-natural protein sequence product comprising a plurality of epitopes to proteins or polypeptides that are functionally related or linked and a scaffold portion comprising a non-reactive sequence that contributes to the calibration capability only in terms of its dominant physical properties as herein before described. 
         [0046]    In a further aspect of the present invention, there is provided a non-natural protein sequence for use in detecting the presence of one or more relationally linked proteins or polypeptides and the absolute concentration of said epitope in a sample, the non-natural protein sequence product comprising a plurality of epitopes to proteins or polypeptides that are functionally related or linked and a scaffold portion comprising a non-reactive sequence that contributes to the calibration capability only in terms of its dominant physical properties as herein before described. 
         [0047]    By determining the absolute concentration of an epitope in the sample we are able to equate this amount to a particular protein or protein modification. 
         [0048]    In a further aspect of the invention, the non-natural protein sequence of the present invention can be used to calibrate a sandwich ELISA style experiment, wherein one antibody bound to a physical surface captures an antigen and then a second antibody, specific for a second feature on the same antigen, binds to the captured antigen. A schematic representation is presented in  FIGS. 3 and 4 . The immobilisation of the second antibody on the physical surface may then be detected using any of a number of means (calorimetric end point, chemiluminescent end point, fluorescent end point, radioactive end point). This experiment can in principle be performed in solution, and the end point measured using the diffusional characteristics of the second antibody. This antibody, labelled in a detectable way (e.g. with a fluorophore) will move more slowly when complexed to the antigen and first antibody than when it is present in solution alone. The change in rate can be detected by for example fluorescence correlation spectroscopy or fluorescence polarisation. 
         [0049]    The present non-natural protein sequence or calibration material can incorporate multiple epitopes for antibodies specific for the same protein, spaced within the calibration product in such a way that more than one antibody can bind to its epitope simultaneously. In the example shown in  FIG. 4 , two epitopes from the same protein antigen (epitopes  1  and  2 ) are incorporated in the calibration product to allow capture of the product on a surface by interaction with antibody  1  (immobilised on the surface) and simultaneous interaction with antibody  2  (via epitope  2 ) leading to detection of bound antibody  2 . Additional epitopes relating to the same protein target molecule may be incorporated in the calibration material (e.g. phospho-epitopes), and simultaneous binding of more than two antibodies to their epitopes could be achieved. 
         [0050]    In a yet further aspect of the invention, there is provided a kit for identifying the presence of and quantifying the amount of at least two relationally linked proteins or polypeptides in a sample, the kit comprising a non-natural protein sequence product comprising a scaffold portion comprising a non-reactive sequence that contributes to the calibration capability only in terms of its dominant physical properties as herein before described and plurality of epitopes to proteins or polypeptides that are functionally related or linked. Optionally, the kit may comprise instructions for use thereof. 
         [0051]    In a yet further aspect of the invention, there is provided a method of simultaneously detecting the presence of at least two relationally linked proteins or polypeptides and quantifying the amount of said functionally related or linked proteins or polypeptides in a sample, the method comprising:
       a) providing a non-natural protein sequence, the sequence comprising a scaffold portion and at least two protein or polypeptide epitopes, the proteins or polypeptides being relationally linked and mixing this non-natural protein sequence with a test sample that may or may not contain binding partners to the epitopes;   b) adding at least two antibodies specific for the at least two epitopes;   c) carrying out a separation detection technique on said non-natural protein sequence; and optionally   d) generating at least one comparison point comprising intensity of a signal produced by the epitope portion of the non-natural protein sequence. Preferably, the method includes any one or more of the features hereinbefore described. The types of separation techniques that may be used in the method of the present invention are those as described in PCT/GB2005/00015.       
 
         [0056]    The present invention is therefore of great utility to a researcher who may wish to study multiple related components in their system and at the same time provide a positive control, an internal standard or generate a calibration curve. 
         [0057]    The present invention is of also great utility in industry pathway systems which can be altered to accomplish the manufacture of new products, those which occur in nature and others not normally produced in nature. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0058]      FIG. 1  shows a schematic representation of multiple epitopes in series. 
           [0059]      FIG. 2  shows a schematic representation of a series of epitopes comprising isoforms of a single protein. 
           [0060]      FIG. 3  shows a schematic representation of a sandwich ELISA assay. 
           [0061]      FIG. 4  shows a schematic representation of the non-natural protein sequence of the present invention in use as a calibrant in a sandwich ELISA assay. 
           [0062]      FIG. 5  shows cardiac ryanodine receptor and the sites of interaction of some of its associated proteins (Bers, D. M. (2004). Journal of Molecular and Cellular Cardiology, 37: 417-429). 
           [0063]      FIG. 6  shows a cartoon block diagram of the protein expressed by  E. coli ., which contains the antibody epitopes for selected proteins involved in the Ryanodine receptor 2 (RyR2) macromolecular complex (Table 14: Notations 2-9). 
           [0064]      FIG. 7  shows a cartoon diagram of the pGS-21a expression vector and the genetic sequence of the cloning region (GenScript). 
           [0065]      FIG. 8  shows a Western blot of 12% SDS-PAGE gel of RyR2Macromolecular Complex Calibrant, loaded in doubling dilutions and with a uninduced control. Western blot probed with a rabbit anti-Calstabin2 (FKBP12.6) polyclonal primary antibody at 1 in 1000 dilution. Secondary antibody=GAR-HRP. 
           [0066]      FIG. 9  shows a Western blot of the RyR2Macromolecular Complex Calibrant used to produce calibration plot—probed with a rabbit anti-Calstabin2 (FKBP12.6) polyclonal primary antibody and a secondary antibody=GAR-HRP. 
           [0067]      FIG. 10  shows a calibration plot using the densitometry measured from the western blot in  FIG. 9 . 
           [0068]      FIG. 11  shows a Western blot of the RyR2Macromolecular Complex Calibrant used to produce calibration plot—probed with a rabbit anti-PKA polyclonal primary antibody. Secondary antibody=GAR-HRP. 
           [0069]      FIG. 12  shows a calibration plot using the densitometry measured from the western blot in  FIG. 11 . 
           [0070]      FIG. 13  shows a cartoon block diagram of the protein expressed by  E. coli ., which contains the antibody epitopes for proteins involved in a MAPKinase pathway (Table 15: Notations 2-10). 
           [0071]      FIG. 14  shows a cartoon diagram of the pGS-21a expression vector and the genetic sequence of the cloning region (GenScript). 
           [0072]      FIG. 15  shows a Western blot of 12% SDS-PAGE gel of MAPKinase Pathway Calibrant, loaded in doubling dilutions and with a uninduced control. Western blot probed with a rabbit anti-EGFR polyclonal primary antibody at 1 in 500 dilution. Secondary antibody=GAR-HRP. 
           [0073]      FIG. 16  shows a Western blot of the MAPKinase Pathway Calibrant used to produce calibration plot—probed with a rabbit anti-EGFR polyclonal primary antibody. Secondary antibody=GAR-HRP. 
           [0074]      FIG. 17  shows a calibration plot using the densitometry measured from the western blot in  FIG. 16 . 
           [0075]      FIG. 18  shows a Western blot of the MAPKinase Pathway Calibrant used to produce calibration plot—probed with a rabbit anti-Ras polyclonal primary antibody. Secondary antibody=GAR-HRP. 
           [0076]      FIG. 19  shows a calibration plot using the densitometry measured from the western blot in  FIG. 18 . 
           [0077]      FIG. 20  shows a Western blot of the MAPKinase Pathway Calibrant used to produce calibration plot—probed with a rabbit anti-ERK1+ERK2 polyclonal primary antibody. Secondary antibody=GAR-HRP. 
           [0078]      FIG. 21  shows a calibration plot using the densitometry measured from the western blot in  FIG. 20 . 
       
    
    
     MATERIAL AND METHODS 
       [0079]    Antibodies specific for epitope sequences can be sourced from commercial or collaborative sources or produced using published procedures (Drago G. A., &amp; Colyer, J. (1994) J. Biol. Chem. 269, 25073-25077; Hudson L., &amp; Hay, F. C. (1980) Practical Immunology, 3 rd  Ed., Blackwell Scientific Publications, Oxford). Conversely, the epitope of an antibody can be defined using published methods (Morris, G. L., Cheng, H-C., Colyer, J., &amp; Wang, J. H. (1991) J. Biol. Chem. 266, 11270-11275). 
       Design &amp; Production of a Calibration Product for Relationally Linked Components: 
       [0080]    The epitope of each antibody for a component in a relationally linked series must be established, either by virtue of the immunisation material (i.e. a peptide) or by empirical characterisation of the antibody. The peptide sequence of each chosen epitope (e.g. five sequences from different SERCA enzyme isoform in example 1) will be selected on the basis of their recognition by an appropriate antibody, and their chemical dissimilarity with other sequences in the calibration product. A gene encoding these peptide epitope sequences in series will be designed using codon usage information for  Escherichia coli , or other relevant protein expression host. The gene (epitope gene) would be synthesised using published methods (Maniatis, Fritsch and Sambrook. 1st Ed 1982, 2nd Ed. 1989, Molecular Cloning: A laboratory manual. Cold Spring Harbor Press.). 
         [0081]    The epitope gene was cloned into a further gene encoding the scaffold portion and an affinity purification tag, at an unique restriction site (Brockwell, D. J., Beddard, G. S., Clarkson, J., Zinober, R., Blake, A. W., Trinick, J., Olmsted, P. D., Smith, D. A., &amp; Raadford, S. E. (2002) Biophys. J. 83, 458-472). Inserts with the correct orientation were identified by polymerase chain reaction using appropriate primer sets, and the gene product was expressed in  E. coli  as described in Brockwell et al. (2002). 
         [0082]    The calibration product was purified by affinity chromatography (Brockwell et al., 2002) followed by preparative SDS-PAGE, and the amount of product was determined using a standard protein assay (Smith P K, Krohn R1, Hermanson G T, Mallia A K, Gartner F H, Provenzano M D, Fujimoto E K, Goeke N M, Olson B J, Klenk D C. Measurement of Protein Using Bicinchoninic Acid. Anal. Biochem. 1985; 150: 76-85) 
       Calibration of Western Blot Experiments: 
       [0083]    Known amounts of purified calibration product, in a serial dilution series from 10 pmol to 0.01 pmol were resolved on a 10% SDS-PAGE gel, transferred to PVDF membrane and stained with an antibody specific for the SERCA1a epitope. A series of biological samples containing SERCA proteins were analysed in parallel. Identical experiments were performed in series, or parallel and stained with antibodies specific for each of the other SERCA isoforms in turn, Immunosignals were quantified by densitometry (Rodriguez, P., Bhogal, M. S., &amp; Colyer, J. (2003) J. Biol. Chem. 278, 38593-38600) and a calibration curve relating the immunosignal to calibrant loading (pmol) was used to determine the amount of epitope present in each biological sample. 
       DETAILED DESCRIPTION 
       [0084]    Taking, by way of example only a process A→B→C→D→E, catalysed by enzymes f, g, h, and i. Antibodies specific to each enzyme exist (called f′, g′, h′, and i′) and the epitope for each of these antibodies has been defined (f″, g″, h″, and i″). A calibration standard material could be constructed in which the chemical constituent of each antibody epitope (f″, g″, h″, and i″) is linked in series to form an unnatural protein sequence. This sequence is then linked in series to an additional protein sequence or scaffold portion, which is not recognised by any of the antibodies in this particular experimental series. This additional protein mass or scaffold portion functions in controlling the molecular weight of the non-natural protein sequence of the present invention. The non-natural protein sequence product will contain known amounts of each epitope, and can thus be used in experiments in known amounts to calibrate the signals generated by the experiment. The non-natural protein sequence of the present invention is shown schematically in  FIG. 1 , in which a series of 11 epitopes are linked in series to a scaffold portion also termed as an irrelevant protein or concatamer as in the Figure. 
       EXAMPLE 1 
     Isoforms 
       [0085]    Many proteins can be expressed in a variety of isoforms, either from the expression of closely related genes or from the production of alternatively spliced forms of an individual gene, or by combination of both of these mechanisms. 
         [0086]    The multifunctional SERCA Sarcoplasmic/endoplasmic reticulum (Ca 2+ —Mg 2+ )-Adenosine triphosphoatase exists in a number of isoforms generated from different genes (1,2,3), with alternative splicing products of genes SERCA1 and SERCA2 resulting in further diversity. See Table 1 below for details. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Isoform 
                 Molecular detail 
                 Functional detail 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SERCA1a 
                 FIARNYLE 994 G 994   
                 Adult skeletal 
                   
               
               
                   
                 (SEQ ID NO:1) 
                 isoform, fast- 
               
               
                   
                   
                 twitch muscle (2) 
               
               
                   
               
               
                 SERCA1b 
                 Excision of exon 42, 
                 Foetal/neonatal 
               
               
                   
                 C-terminal sequence 
                 developmental 
               
               
                   
                 FIARNYLE 994 DPEDERRK 1001   
                 expression 
               
               
                   
                 (SEQ ID NO:2) 
                 pattern, skeletal 
               
               
                   
                   
                 muscle (3) 
               
               
                   
               
               
                 SERCA2a 
                 LEP 994 AILE 997   
                 Cardiac muscle 
               
               
                   
                 (SEQ ID NO:3) 
                 isoform (4) 
               
               
                   
               
               
                 SERCA2b 
                 LEP 994 GKECVQPATKSCSFSACT 
                 Smooth muscle 
               
               
                   
                 DGISWPFVLLIMPLVIWVYSTDTN 
                 isoform (5) 
               
               
                   
                 FSDMFWS 1024   
               
               
                   
                 (SEQ ID NO:4) which en- 
               
               
                   
                 codes a domain that 
               
               
                   
                 transverses the mem- 
               
               
                   
                 brane. 
               
               
                   
               
               
                 SERCA3 
                 75-77% identity with 
                 Non-muscle isoform 
               
               
                   
                 SERCA1 and SERCA2. 
                 (primarily) (6) 
               
               
                   
                 Sequence 
               
               
                   
                   990 HHVDEKKDLk 999   
               
               
                   
                 (SEQ ID NO:5) is iso- 
               
               
                   
                 form specific 
               
               
                   
               
             
          
         
       
     
         [0087]    With reference to  FIG. 2 , there is shown a schematic representation of a non-natural protein sequence of the present invention which can be used to investigate isoforms of SERCA Sarcoplasmic/endoplasmic reticulum (Ca 2+ —Mg 2+ )-Adenosine triphosphoatase 
       EXAMPLE 2 
     Isoforms 
       [0088]    The multifunctional protein phosphatase, calcineurin (CaN) is an example of an enzyme expressed in a variety of isoforms. CaN is involved in a large variety of biological events including programmes of gene expression in response to extracellular signals (CaN/NFAT). The calcineurin isoforms are as follows: Calcineurin alpha (CaN alpha); Calcineurin beta (CaN beta); and Calcineurin gamma (CaN gamma) sequence not commercially available. A product of the invention comprises the three calcineurin isoforms and Table 2 below for details. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Antibody 
                   
                 Cross 
                   
               
               
                   
                 for: 
                 Epitope 
                 Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 CaN alpha 
                 Synthetic peptide ( CDPKLSTTDRVV ) 
                 All 
                   
               
               
                   
                   
                 SEQ ID NO:6 coupled to KLH 
                 mammals 
               
               
                   
               
               
                 2 
                 CaN beta 
                 25 residue synthetic peptide 
                 Bovine, 
               
               
                   
                   
                 corresponding to amino acids 
                 Human, 
               
               
                   
                   
                 500-524 SEQ ID NO:7 
                 Mouse, 
               
               
                   
                   
                 ( VQQDGFNSLNTAHTTENHGTGNHSA ) of 
                 Rat. 
               
               
                   
                   
                 rat Calcineurin/PP2B Aβ 
               
               
                   
               
             
          
           
               
                 3 
                 CaN gamma 
                 Antibodies to this protein with published epitope 
                   
               
               
                   
                   
                 characteristics are not currently available from 
               
               
                   
                   
                 commercial sources. An antibody might be available 
               
               
                   
                   
                 by collaboration with an academic group, wherein 
               
               
                   
                   
                 the epitope of the antibody would be known, or 
               
               
                   
                   
                 could be discovered. Alternatively a new antibody 
               
               
                   
                   
                 could be created to an immunogenic sequence 
               
               
                   
                   
                 peculiar to this protein, and this sequence could 
               
               
                   
                   
                 be included in the calibration product 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 3 
     Isoforms 
       [0089]    The multifunctional protein kinase, calmodulin-dependent kinase II is an example of an enzyme expressed in a variety of isoforms. CaM kinase II is involved in a large variety of biological events including memory, regulation of vesicle movement, and maladaptive responses in heart failure. The isoforms of CAMKII are listed below: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 1. CaMKII alpha, 
                   
               
               
                   
                 (SEQ ID NO:8 KWQIVHFHRSGAPSVLPH) 
               
               
                   
                   
               
               
                   
                 2. CamKII beta 
               
               
                   
                 (SEQ ID NO:9 RRGSGTPEAEAPRQW) 
               
               
                   
                   
               
               
                   
                 3. CamKII delta 
               
               
                   
                 (SEQ ID NO:10 QNVHFHRSGSPTVPIK) 
               
               
                   
                   
               
               
                   
                 4. CamKII gamma 
               
               
                   
                 (SEQ ID NO:11 RSPEGRSSRDRTAPS) 
               
               
                   
                   
               
               
                   
                 5. CamKII antibody 
               
               
                   
                 (SEQ ID NO 12 CTRFTDEYQLFEEL) 
               
               
                   
                   
               
               
                   
                 6. CamKII alpha phospho (T286) 
               
               
                   
                 (SEQ ID NO 13: MHRQET(PO 4 )VDCLKKFN) 
               
             
          
         
       
     
         [0090]    A calibration product could be constructed from a series of epitope sequences, where each sequence represents the epitope for an antibody specific for an isoform of Cam kinase II. Some epitopes are shared between all or several isoforms, these epitopes could be incorporated in the calibration standard to calibrate multiple isoforms with a single antibody (e.g. module 5). A number of phosphorylation sites exist in the protein. Epitopes for phosphorylation site specific antibodies could be incorporated in the product (e.g. module 6 above) to calibrate the status of phosphorylation too. Details of the antibodies and epitopes are set out below in Table 3. 
       EXAMPLE 4 
     Polymorphisms 
       [0091]    Polymorphisms occur within biological species in probably every gene. In some cases the polymorphisms occur with altered probability in disease situations, and in those case are of particular interest and use. 
         [0092]    Genetic variation exists within the population of a species, which at the individual gene level is manifest as polymorphisms of a gene. Polymorphisms represent typically single base changes in the sequence of a gene which can occur in the coding or non-coding regions. These deviations in sequence can be without consequence to the gene, or can alter the level of expression of the gene, or can alter the polymer encoded by the gene. In many instances the probability of disease is linked to particular polymorphisms, which serves as a useful screening tool, and as a basis for hypothesis driven research into the cause and management of disease. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Antibody 
                 Epitope 
                 Specificity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 CAMK2 alpha Antibody (C- 
                 KWQIVHFHRSGA 
                 Human 
                   
               
               
                   
                 term) (Purified Rabbit 
                 PSVLPH (from 
               
               
                   
                 Polyclonal Antibody) 
                 Human) 
               
               
                   
                   
                 SEQ ID NO:8 
               
               
                   
               
               
                 2 
                 CAMK2 beta Antibody (C- 
                 RRGSGTPEAEAPRQW 
                 Human, 
               
               
                   
                 term) (Purified Rabbit 
                 (from human) 
                 Primate 
               
               
                   
                 Polyclonal Antibody) 
                 SEQ ID NO:9 
               
               
                   
               
               
                 3 
                 CAMK2 delta Antibody (C- 
                 QNVHFHRSGSPTVPIK 
                 Human, 
               
               
                   
                 term) (Purified Rabbit 
                 (from human) 
                 Primate 
               
               
                   
                 Polyclonal Antibody) 
                 SEQ ID NO:10 
               
               
                   
               
               
                 4 
                 CAMK2 gamma Antibody (C- 
                 RSPEGRSSRDRTAPS 
                 Human 
               
               
                   
                 term) (Purified Rabbit 
                 (from human) 
               
               
                   
                 Polyclonal Antibody) 
                 SEQ ID NO:11 
               
               
                   
               
               
                 5 
                 CAMK2 Antibody (N-term) 
                 CTRFTDEYQLFEEL 
                 This antibody recognizes 
               
               
                   
                 (Purified Rabbit Poly- 
                 (from Rat delta 
                 alpha, delta, and gamma 
               
               
                   
                 clonal Antibody) 
                 isoform) 
                 (NOT beta) isoforms of 
               
               
                   
                   
                 SEQ ID NO:12 
                 human, mouse, rat, 
               
               
                   
                   
                   
                 bovine, hamster, guinea 
               
               
                   
                   
                   
                 pig, chicken, and rabbit 
               
               
                   
                   
                   
                 CaMKII 
               
               
                   
               
               
                 6 
                 CaMKII alpha (phospho 
                 MHRQET(PO 4 )VDCLKK 
                 Rat 
               
               
                   
                 T286) antibody 
                 FN (from Rat) 
               
               
                   
                 (monoclonal) 
                 SEQ ID NO:13 
               
               
                   
               
             
          
         
       
     
         [0093]    Certain polymorphisms in the RYR2 gene, which encodes an ion channel expressed in the heart, are associated with a disorder known as catecholaminergic polymorphic ventricular tachycardia (CPVT) which can provoke electrical irregularity and sudden death when an individual exercises. To date, over 20 separate mis-sense polymorphisms (those which alter the primary sequence of the protein) have been discovered in the human RYR2 gene, which are linked to CPVT. These include: 
         [0000]    
       
         
               
             
               
               
             
           
               
                   
               
               
                 Mutation (ref 6) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 S2246L 
               
               
                   
                 E2311D 
               
               
                   
                 R2474S 
               
               
                   
                 L3778F 
               
               
                   
                 G3946S 
               
               
                   
                 N4104K 
               
               
                   
                 R4497C 
               
               
                   
                 V4771I 
               
               
                   
                 A4860G 
               
               
                   
                 I4867M 
               
               
                   
                 N4895D 
               
               
                   
                 E4950K 
               
               
                   
                   
               
             
          
         
       
     
         [0094]    Which are residues conserved between man (and mouse),  Drosophila  and  Caenorhabditis elegans . They exist in regions which are also highly conserved, both across these species, and between isoforms of RYR (1,2, and 3). 
       EXAMPLE 5 
     Polymorphisms 
       [0095]    A large number of mutations in RyR2 are found in patients with arrhythmogenic right ventricular dysplasia type 2 (ARVD2) and catecholaminergic polymorphic ventricular tachycardia (CPVT). These are believed to play a causal role in disease. A sub-set of known disease associated mutations of RYR2 include: (1) R176 Q, (2) V2306 I, (3) G3946 S and (4) V4653 F. To our knowledge antibodies specific for these mutations do not exist, however it is likely that they can be generated using short synthetic peptide immunogen incorporating the mutation site, using techniques known in the art. Calibration of such antibodies could be achieved using a product comprising SEQ ID NOs 19-22 (see Table 4 below). 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Antibody 
                   
                   
                 Epitope after 
                   
               
               
                   
                 for: 
                 Domain 
                 Pre-mutation 
                 mutation 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 R 176 Q 
                 N- 
                 EGEKVRVGDDL 
                 EGEKVQVGDDL 
                   
               
               
                   
                   
                 terminal 
                 (SEQ ID NO: 14) 
                 (SEQ ID NO: 18) 
               
               
                   
               
               
                 2 
                 V2306 I 
                 Central 
                 FLRFAVECNGE 
                 FLRFAIFCNGE 
               
               
                   
                   
                   
                 (SEQ ID NO: 15) 
                 (SEQ ID NO: 19) 
               
               
                   
               
               
                 3 
                 G3946 S 
                 C- 
                 WDAVVGFLHVF 
                 WDAVVSFLHVF 
               
               
                   
                   
                 terminal 
                 (SEQ ID NO: 16) 
                 (SEQ ID NO: 20) 
               
               
                   
               
               
                 4 
                 V4653 F 
                 C- 
                 FVKRKVMDKYG 
                 FVKRKFMDKYG 
               
               
                   
                   
                 terminal 
                 (SEQ ID NO: 17) 
                 (SEQ ID NO: 21) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 6 
     Polymorphisms 
       [0096]    Protein p53 (also called TP53) is associated with a high proportion of cancers in man. For example in human liver cancer, 26% of cases (559 of 2153 tumours) show mutation in TP53 according to 64 studies (Jackson et al; 2006 Toxicology Science 90, 400-418). Similarly TP53 mutations occur in 42% of spontaneous lung tumours in man. Missense polymorphisms result in mutant proteins, some of which are associated with cancer, such as: (1) R 249 S—most frequent TP53 mutation in hepatocellular carcinoria (HCC), (2) R172P, (3) R172H and (4) R270H. To our knowledge antibodies specific for these mutations do not exist, however it is likely that they can be generated using short synthetic peptide immunogen incorporating the mutation site, using techniques known in the art. Calibration of such antibodies could be achieved using the following product comprising SEQ ID NOs 26-29 of the epitopes after mutation, see Table 5 below: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 Antibody 
                 Type of 
                   
                 Epitope after 
                   
               
               
                   
                 for: 
                 TP53 
                 Pre-mutation 
                 mutation 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 R 249 S 
                 Human 
                 GGMNRRP ILTI 
                 GGMNRSP 
                   
               
               
                   
                   
                   
                 (SEQ ID NO:22) 
                 ILTI 
               
               
                   
                   
                   
                   
                 (SEQ ID NO:26) 
               
               
                   
               
               
                 2 
                 R 172 P 
                 Mouse 
                 TEVV RRCPHHE 
                 TEVV 
               
               
                   
                   
                   
                 (SEQ ID NO:23) 
                 RPCPHHE 
               
               
                   
                   
                   
                   
                 (SEQ ID NO:27) 
               
               
                   
               
               
                 3 
                 R 172 H 
                 Mouse 
                 TEVV RRCPHHE 
                 TEVV 
               
               
                   
                   
                   
                 (SEQ ID NO:24) 
                 RHCPHHE 
               
               
                   
                   
                   
                   
                 (SEQ ID NO:28) 
               
               
                   
               
               
                 4 
                 R 270 H 
                 Mouse 
                 DSFEVR VCACP 
                 DSFEVH 
               
               
                   
                   
                   
                 (SEQ ID NO:25) 
                 VCACP 
               
               
                   
                   
                   
                   
                 (SEQ ID NO:29) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 7 
     Components with a Functional Relationship; Pathways 
       [0097]    Proteins are involved in natural processes such as metabolism, blood clotting, and hormone action. For example, glycolysis: achieves the following chemical reaction 
         [0000]      C 6 H 12 O 6 +2ADP+2P i +2NAD + →2CH 3 COCOO+2ATP+2H 2 O+2NADH+2H +   
         [0000]    It utilises 10 enzymes, and an 11 th  under anaerobic conditions, specifically the enzymes are as follows: 
         [0098]    Hexokinase, Phosphoglucose isomerase, 6-phosphofructose-1-kinase, fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehydes 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, (lactate dehydrogenase). 
         [0099]    A calibration standard comprising epitopes for antibodies to each of these proteins would be useful in the study of glycolytic processes in biology, biotechnology and medicine. 
       EXAMPLE 8 
     Components with a Functional Relationship; Pathways 
       [0100]    The eukaryotic cell cycle is an essential pathway necessary for all proliferative responses. This cycle involves a number of protein kinases and their partner regulatory proteins (cyclins), the concentration of the cyclins change throughout the cell cycle to allow passage of the cell through specific controlling check-points. Cell cycle engine parts include: (1) Cyclin A; (2) cdK1; (3) Cyclin D; (4) cdK4; (5) cdK6; (6) Cyclin E; (7) cdK2; and (8) Cyclin B. A product comprising the non-natural protein sequence comprising two or more epitopes selected from 1-8 as defined in Table 6 below would be useful in the study of cell cycles in biology, biotechnology and medicine. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Antibody for: 
                 Epitope 
                 Cross Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 Cyclin A 
                 Synthetic peptide: C- 
                 Human, Mouse, Rat 
                   
               
               
                   
                   
                 KYHGVSLLNPPETLNL, (SEQ ID 
                   
               
               
                   
                   
                 NO:30) corresponding to amino acids 
               
               
                   
                   
                 417-432 of Human Cyclin A 
               
               
                   
               
               
                 2 
                 cdK1 
                 Recombinant fragment ( Xenopus   
                 Human, Mouse, Rat, 
               
               
                   
                   
                   laevis ). C-Terminal 2/3rds of  Xenopus   
                   Xenopus laevis , 
               
               
                   
                   
                 cdc2 expressed in  E .  coli  (220-227) 
                 Chicken, Guinea pig 
               
               
                   
                   
                   
                 and Woodchuck 
               
               
                   
               
               
                 3 
                 Cyclin D 
                 peptide corresponding the 11 C- 
                 Human, Mouse, Rat 
               
               
                   
                   
                 terminal amino acids, residues 285- 
               
               
                   
                   
                 295, of human Cyclin D1 
               
               
                   
                   
                 (CTPTDVRDVDI), (SEQ ID NO:31) 
               
               
                   
               
               
                 4 
                 cdK4 
                 MATSRYEPVA EIGVGAYGTV (From 
                 Human, Rat, Mouse, 
               
               
                   
                   
                 Human), (SEQ ID NO:32) 
                 Pig 
               
               
                   
               
               
                 5 
                 cdK6 
                 Synthetic peptide: C-SQNTSELNTA, 
                 Human, Mouse, Rat 
               
               
                   
                   
                 (SEQ ID NO:33) corresponding to 
               
               
                   
                   
                 amino acids 317-326 of Human Cdk6 
               
               
                   
               
               
                 6 
                 cyclin E 
                 Synthetic peptide (amino acid 
                 Human, Mouse 
               
               
                   
                   
                 sequence considered to be 
               
               
                   
                   
                 commercially sensitive) (Human) (C 
               
               
                   
                   
                 terminal 
               
               
                   
               
               
                 7 
                 cdK2 (cdc2) 
                 The epitope is thought to be residues 
                 Human, Rat, Mouse, 
               
               
                   
                   
                 220-227 of mouse cdc2, LGTPNNEV, 
                 Chicken,  Xenopus   
               
               
                   
                   
                 (SEQ ID NO:34) 
                   laevis  and 
               
               
                   
                   
                   
                 Amphibians 
               
               
                   
               
               
                 8 
                 Cyclin B 
                 generated from 14-168 of cyclin B 
                 Human, Rat 
               
               
                   
                   
                 (human) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 9 
     Components with a Functional Relationship; Pathways 
       [0101]    A series of components acting in consort can form a pathway. Large numbers of pathways exist in biochemistry. One example is a pathway of interactions which control the expression of cell cycle regulators, cyclin A and cyclin E. G1 cyclins that overcome inhibitors of cell cycle progression are: (1) P16; (2) Cyclin D; (3) Retinoblastoma Protein; (4) E2F; (5) Cyclin E; (6) Cyclin A; and (7) P27. A product comprising the non-natural protein sequence comprising two or more epitopes selected from 1-7 as defined in Table 7 below would be useful in the study of cell cycles in biology, biotechnology and medicine. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 Antibody 
                   
                   
                   
               
               
                   
                 for: 
                 Epitope 
                 Cross Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 P16 
                 human p16 protein (missing the first 7 amino 
                 Human. Cross 
                   
               
               
                   
                   
                 acids) SME PSADWLATAA ARGRVEEVRA 
                 reactivity may occur 
               
               
                   
                   
                 LLEAGALPNA PNSYGRRPIQ VMMMGSARVA 
                 with p15 when 
               
               
                   
                   
                 ELLLLHGAEP NCADPATLTR PVHDAAREGF 
                 immunoprecipitating 
               
               
                   
                   
                 LDTLVVLHRA GARLDVRDAW GRLPVDLAEE 
                 in vitro translated 
               
               
                   
                   
                 LGHRDVARYL RAAAGGTRGS NHARIDAAEG 
                 product but not in 
               
               
                   
                   
                 PSDIPD (SEQ ID NO:35) 
                 immunoblots. 
               
               
                   
                   
                   
                 Potentially cross 
               
               
                   
                   
                   
                 reacts with p15.5. 
               
               
                   
               
               
                 2 
                 Cyclin D 
                 peptide corresponding the 11 C-terminal amino 
                 Human, 
               
               
                   
                   
                 acids. residues 285-295, of human Cyclin D1 
                 Mouse, Rat 
               
               
                   
                   
                 (CTPTDVRDVDI) (SEQ ID NO:36) 
               
               
                   
               
               
                 3 
                 Retino- 
                 Antibodies to this protein with published epitope 
                 Human 
               
               
                   
                 blastoma 
                 characteristics are not currently available from 
               
               
                   
                 Protein 
                 commercial sources. An antibody might be 
               
               
                   
                   
                 available by collaboration with an academic group, 
               
               
                   
                   
                 wherein the epitope of the antibody would be 
               
               
                   
                   
                 known, or could be discovered. Alternatively a new 
               
               
                   
                   
                 antibody could be created to an immunogenic 
               
               
                   
                   
                 sequence peculiar to this protein, and this 
               
               
                   
                   
                 sequence could be included in the calibration 
               
               
                   
                   
                 product 
               
               
                   
               
               
                 4 
                 E2F 
                 SPPPSSPPS SLTTDPSQSL LSLEQEPLLS 
                 Human 
               
               
                   
                   
                 RMGSLRAPVD EDRLSPLVAA DSLLEHVRED (from 
               
               
                   
                   
                 human E2F-1)(SEQ ID NO:37) 
               
               
                   
               
               
                 5 
                 Cyclin E 
                 Antibodies to this protein with published epitope 
                 Human, 
               
               
                   
                   
                 characteristics are not currently available from 
                 Mouse 
               
               
                   
                   
                 commercial sources. A new antibody could be 
               
               
                   
                   
                 created to an immunogenic sequence peculiar to 
               
               
                   
                   
                 this protein, and this sequence could be included 
               
               
                   
                   
                 in the calibration product 
               
               
                   
               
               
                 6 
                 Cyclin A 
                 Synthetic peptide: C-KYHGVSLLNPPETLNL, (SEQ ID 
                 Human, 
               
               
                   
                   
                 NO:38)corresponding to amino acids 417-432 of 
                 Mouse, Rat 
               
               
                   
                   
                 Human Cyclin A 
               
               
                   
               
               
                 7 
                 p27 
                 MSNVRVSNGS PSLERMDARQ AEHPKPSACR 
                 Human, 
               
               
                   
                   
                 NLFGPVDHEE LTRDLEKHCR DMEEASQRKW 
                 Mouse, Rat 
               
               
                   
                   
                 NFDFQNHKPL EGKYEWQEVE KGSLPEFYYR 
               
               
                   
                   
                 PPRPPKGACK VPAQESQDVS GSRPAAPLIG 
               
               
                   
                   
                 APANSEDTHL VDPKTDPSDS QTGLAEQCAG 
               
               
                   
                   
                 IRKRPATDDS STQNKRANRT EENVSDGSPN 
               
               
                   
                   
                 AGSVEQTPKK PGLRRRQT (SEQ ID NO:39) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 10 
     Components with a Functional Relationship; Pathways 
       [0102]    An example of a pathway of interactions which communicate extracellular stimuli to changes in gene expression, involving NFAT and calcineurin is: (1) CHP; (2) FK506; (3) MCIP/calcipressin; (4) AKAP79; (5) CsA/CyA; (6) Cabin 1/CAIN; (7) NFAT(P); (8) PKA; (9) CKI; (10) GSK-3beta; (11) JNK; (12) P38; (13) MEF2; and (14)NFAT. Table 8 below shows the details for the manufacture a non-natural protein sequence of the present invention using two or more epitopes selected from the group comprising epitopes  1 - 14 . 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 8 
               
               
                   
                   
               
               
                   
                 Antibody 
                   
                   
                   
               
               
                   
                 for: 
                 Epitope 
                 Cross Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                  1 
                 CHP 
                 MESHSVTQAGVQWRDLGSLQPLPPGFKQFSHLSL 
                 Human 
                   
               
               
                   
                   
                 PSSWDYRRVPPYLGNFCIFSGEGVSPCWPGWS (SEQ 
                   
               
               
                   
                   
                 ID NO:40) 
               
               
                   
               
               
                  2 
                 FK506 
                 Antibodies to this protein with published epitope 
                 Recognizes 
               
               
                   
                   
                 characteristics are not currently available from 
                 conjugated and 
               
               
                   
                   
                 commercial sources. A new antibody could be 
                 non-conjugated 
               
               
                   
                   
                 created to an immunogenic sequence peculiar to 
                 FK-506 with 
               
               
                   
                   
                 this protein, and this sequence could be included 
                 apparent 
               
               
                   
                   
                 in the calibration product 
                 dissociation 
               
               
                   
                   
                   
                 constant 
               
               
                   
                   
                   
                 Kd = 10-9 M 
               
               
                   
               
               
                  3 
                 MCIP/ 
                 Synthetic peptide: EEVDLQDLPSAT(SEQ ID 
                 Human and 
               
               
                   
                 calci- 
                 NO:41), corresponding to N terminal amino acids 
                 Mouse 
               
               
                   
                 pressin 
                 2-13 of Human Calcipressin 1 
               
               
                   
               
               
                  4 
                 AKAP79 
                 Sythetic peptide produced from human AKAP79 
                 Human 
               
               
                   
                   
                 aa180-427 
               
               
                   
                   
                 QDLSEGISQKDGDEVCESNVSNSITSGEKVISVELGLDN 
               
               
                   
                   
                 GHSAIQTGTLILEEIETIKEKQDVQPQQASPLETSETDH 
               
               
                   
                   
                 QQPVLSDVPPLPAIPDQQIVEEASNSTLESAPNGKDYES 
               
               
                   
                   
                 TEIVAEETKPKDTELSQESDFKENGITEEKSKSEESKRM 
               
               
                   
                   
                 EPIAIIITDTEISEFDVTKSKNVPKQFLISAENEQVGVF 
               
               
                   
                   
                 ANDNGFEDRTSEQYETLLIETASSLVKNAIQLSIEQLVN 
               
               
                   
                   
                 EMASDDNKINNLLQ (SEQ ID NO:42), 
               
               
                   
               
               
                  5 
                 CsA/CyA 
                 Full length human recombinant cyclophilin A 
                 Human, Mouse, 
               
               
                   
                   
                 MVNPTVFFDITADDEPLGRVSFELFADKVPKTAENFRAL 
                 Rat 
               
               
                   
                   
                 STGEKGFGYKGSSFHRIIPGFMCQGGDFTRHNGTGGRSI 
               
               
                   
                   
                 YGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTA 
               
               
                   
                   
                 KTEWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKI 
               
               
                   
                   
                 TISDCGQL (SEQ ID NO:43) 
               
               
                   
               
               
                  6 
                 Cabin 
                 MIRIAALNASSTIEDDHEGSFKSHKTQTKEAQEA 
                 Human 
               
               
                   
                 1/CAIN 
                 EAFALYHKALDLQKHDRFEESAKAYHELLEASLL 
               
               
                   
                   
                 REAVSSGDEKEGLKHPGLILKYSTYKNLAQLAAQ 
               
               
                   
                   
                 REDLETAM (SEQ ID NO:44) 
               
               
                   
               
             
          
           
               
                  7 
                 NFAT(P) 
                 Antibodies to this protein with published epitope characteristics 
                   
               
               
                   
                   
                 are not currently available from commercial sources. A new 
               
               
                   
                   
                 antibody could be created to an immunogenic sequence peculiar to 
               
               
                   
                   
                 this protein, and this sequence could be included in the 
               
               
                   
                   
                 calibration product 
               
               
                   
               
             
          
           
               
                  8 
                 PKA 
                 KKGSEQESVKEFLAK (SEQ ID NO:45), (aa 8-22 
                 Human, Bovine, 
                   
               
               
                   
                   
                 human alpha isoform of the catalytic subunit) 
                 Mouse, Porcine, 
               
               
                   
                   
                   
                 Rat 
               
               
                   
               
               
                  9 
                 CKI 
                 synthetic peptide selected from aa 324~337 of 
                 Human 
               
               
                   
                   
                 human CK1a 
               
               
                   
               
               
                 10 
                 GSK- 
                 Rat GSK3 beta synthetic peptide (aa. 335-349)1, 
                 Bovine, Human, 
               
               
                   
                 3beta 
                 conjugated to KLH; identical to human GSK3 beta 
                 Mouse, Rat 
               
               
                   
                   
                 sequence over these residues (CAHSFF 
               
               
                   
                   
                 DELRDPNVK) (SEQ ID NO:46) 
               
               
                   
               
               
                 11 
                 JNK 
                 Fusion protein: HIDQWNKVIEQ LGTPSAEFMKK 
                 Human, Mouse 
               
               
                   
                   
                 LQPTVRNYVEN RPKYPGIKFEE LFPDWIFPSES 
                 and Rat 
               
               
                   
                   
                 ERDKIKTSQAR DLLSKMLVIDPD KRISVDEALRHP 
               
               
                   
                   
                 YITVWYDPAEAE APPPQIYDAQ LEEREHAIEEW 
               
               
                   
                   
                 KELIYKEVMDWE ERSKNGVVKDQ PSDAAVSSNATP 
               
               
                   
                   
                 SQSSSINDISSMST EQTLASDTDSSLD ASTGPLEGCR 
               
               
                   
                   
                 (SEQ ID NO:47), corresponding to amino acids 231-424 
               
               
                   
                   
                 of Human JNK2 
               
               
                   
               
               
                 12 
                 p38 
                 Synthetic peptide: TYDEVISFVPPPLDQEEMES, (SEQ 
                 Human, Mouse, 
               
               
                   
                   
                 ID NO:48) corresponding to amino acids 341/360 
                 Rat, Rabbit, 
               
               
                   
                   
                 of Human p38 
                 Cow, Chicken, 
               
               
                   
                   
                   
                 Dog, Guinea 
               
               
                   
                   
                   
                 pig, Hamster, 
               
               
                   
                   
                   
                 Monkey, Pig 
               
               
                   
                   
                   
                 and Sheep 
               
               
                   
               
               
                 13 
                 MEF2 
                 Synthetic peptide derived from a region between 
                 Human 
               
               
                   
                   
                 residues 227-276 of human Myelin expression 
               
               
                   
                   
                 factor 2 (QAGR LGSTIFVANL DFKVGWKKLK 
               
               
                   
                   
                 EVFSIAGTVK RADIKED) (SEQ ID NO:49) 
               
               
                   
               
               
                 14 
                 NFAT 
                 Synthetic peptide: 
                 Human, Rat 
               
               
                   
                   
                 (51)AISSPSGLAYPDDVLDYGL(69).(SEQ ID NO:50) 
                 and Mouse 
               
               
                   
                   
                 The immunizing peptide corresponds to amino acid 
               
               
                   
                   
                 residues 51-69 of mouse NFAT1-A, B and C 
               
               
                   
                   
                 isoforms 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 11 
     Components with a Functional Relationship; Pathways 
       [0103]    A further example pathway of interactions is control of the production of cytokines downstream of the toll-like receptor. Lipopolysaccharide is a ligand for the Toll-like receptor and the proteins involved in the signalling network include: (1) TLR; (2) TRIF; (3) TRAF6; (4) JNK; (5) IL10; (6) IL6; (7) RANTES; (8) GCSF; (9) TNF alpha; and (10) MIPI alpha. A product comprising the non-natural protein sequence comprising two or more epitopes selected from 1-10 as defined in Table 9 below would be useful in the study of cytokine production in biology, biotechnology and medicine. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Antibody 
                   
                 Cross 
                   
               
               
                   
                 for: 
                 Epitope 
                 Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                  1 
                 TLR 
                 Amino acids 215-410 of TLR2 (VIRDLL HSAIWLDVRK 
                 Human 
                   
               
               
                   
                   
                 LAFSVPEKIQLLRIMSSSFA KKISLKQCLF TDATVPEIVS 
               
               
                   
                   
                 ILEGMPKLME VEMKDCTLLG 
               
               
                   
                   
                 TGKWYKQIHANQSQSLRILT IENLSIEEFY 
               
               
                   
                   
                 LFTDLQSVLD LLSLFRKVTV ENTKVFLVPC 
               
               
                   
                   
                 KLSQHLLSLEYLDLSANLLG DQSLEHSACQ 
               
               
                   
                   
                 GAWPSLQTLN LSQNSLSDLK MTGKSLFHLR) (SEQ ID 
               
               
                   
                   
                 NO: 51) 
               
               
                   
               
               
                 2 
                 TRIF 
                 Synthetic peptide, corresponding to amino acids 143-158 
                 Human and 
               
               
                   
                   
                 of Mouse TRIF (ERGLSGLFRN LTLERVVE) (SEQ ID 
                 Mouse 
               
               
                   
                   
                 NO: 52) 
               
               
                   
               
               
                 3 
                 TRAF6 
                 Synthetic peptide: DQSEAPVRQNHEEI, (SEQ ID NO: 53) 
                 Human and 
               
               
                   
                   
                 corresponding to amino acids 436-449 of Human TRAF6 
                 Mouse 
               
               
                   
               
               
                 4 
                 JNK 
                 Fusion protein: HIDQWNKVIEQ LGTPSAEFMKK 
                 Human, Mouse 
               
               
                   
                   
                 LQPTVRNYVEN RPKYPGIKFEE LFPDWIFPSES 
                 and Rat 
               
               
                   
                   
                 ERDKIKTSQAR DLLSKMLVIDPD KRISVDEALRHP 
               
               
                   
                   
                 YITVWYDPAEAE APPPQIYDAQ LEEREHAIEEW 
               
               
                   
                   
                 KELIYKEVMDWE ERSKNGVVKDQ PSDAAVSSNATP 
               
               
                   
                   
                 SQSSSINDISSMST EQTLASDTDSSLD ASTGPLEGCR, 
               
               
                   
                   
                 (SEQ ID NO: 54) corresponding to amino acids 231-424 
               
               
                   
                   
                 of Human JNK2 
               
               
                   
               
               
                 5 
                 IL10 
                 Fragment, corresponding to amino acids 19-178 of 
                 Human 
               
               
                   
                   
                 Human IL10 (ALLSLRLGSD AHGTELPSPP 
               
               
                   
                   
                 SVWFEAEFFH HILHWTPIPN QSESTCYEVA (SEQ ID 
               
               
                   
                   
                 NO: 55) 
               
               
                   
               
               
                 6 
                 IL6 
                 COS-7-expressed, recombinant mouse IL-6 
                 Mouse 
               
               
                   
               
               
                 7 
                 RANTES 
                 Synthetic peptide: 
                 Human, Mouse 
               
               
                   
                   
                 SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAV 
                 and Rat 
               
               
                   
                   
                 VFVTRKNRQVCANPEKKWVREYINSLEM, (SEQ ID NO: 
               
               
                   
                   
                 56) corresponding to amino acids 24-90 of Human 
               
               
                   
                   
                 RANTES. 
               
               
                   
               
               
                 8 
                 GCSF 
                 Recombinant full length protein, express in mouse NSO 
                 Human 
               
               
                   
                   
                 cells 
               
               
                   
               
               
                 9 
                 TNF 
                 Highly pure (&gt;98%) recombinant full length protein 
                 Mouse 
               
               
                   
                 alpha 
                 (Mouse) 
               
               
                   
               
               
                 10 
                 MIP1 
                 Synthetic peptide: 
                 Human 
               
               
                   
                   
                 CVETFGTTSYYDDVGLLCEKADTRALMA (SEQ ID NO: 
               
               
                   
                   
                 57)(Human) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 12 
     Proteins Sharing a Common Location in the Cell 
       [0104]    Some biological components share a common location in a cell for all or some of their time. A number of signals contained within the primary sequence of proteins control their location in the cell. Residence of this location is typically dynamic (rather than static) and thus evaluation of the entire protein complement of that location would be valuable in biological research. 
         [0105]    Lipid raft domains of biological membranes are an interesting example of a discrete cellular location. Our present understanding places lipid rafts as subdomains of the plasma membrane, characterised by a gel phase lipid composition (lipid and cholesterol) which allows residence of some particular proteins and exclusion of others. Three distinct lipid raft types can be resolved, as summarised in table below (taken from http://www.bms.ed.ac.uk/research/others/smaciver/Cyto-Topics/lipid rafts and the cytoskeleton.htm): 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
               
               
                 Raft Type 
                 Constituent 
                 Properties 
                 Function 
               
               
                   
               
             
             
               
                 Caveolae 
                 Cholesterol, glycol- 
                 Invagina- 
                 Presumed to 
               
               
                   
                 sphingolipid, Arachidonic 
                 tions of 
                 be signalling 
               
               
                   
                 acid, Plasmenylethanol- 
                 membranes 
                 centres and 
               
               
                   
                 amine, Caveolin1 and 2, 
                 rich in 
                 perhaps 
               
               
                   
                 hetero- trimeric G- 
                 signalling 
                 regions of 
               
               
                   
                 proteins and monomeric 
                 molecules 
                 cholesterol 
               
               
                   
                 G-proteins, EGF &amp; PDGF 
                   
                 import 
               
               
                   
                 receptors, Fyn, GPI- 
               
               
                   
                 linked enzymes, integrins. 
               
               
                   
                 Flotillin 
               
               
                 Glycosphingo- 
                 Cholesterol, glycoshingo- 
                 Detergent 
                 Signalling 
               
               
                 lipid enriched 
                 lipid, low in PI and other 
                 resistant 
               
               
                   
                 anionic phospholipids 
                 membranes 
               
               
                 PIP 2  enriched 
                 PIP 2 , MARKS, CAP, 
                   
                 Signalling, 
               
               
                   
                 GAP-43 
                   
                 Structural. 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 13 
     Proteins Sharing a Common Location in the Cell 
       [0106]    The centrosome is a common physical location for some biological components. It is located adjacent to the eukaryotic nucleus and serves a variety of functions including the organisation of microtubules. The centrosome organises the assembly of the mitotic spindle which permits the correct segregation of chromosomes. Abnormalities in centrosome components can lead to centrosome dysfunction, which is often associated with proliferative diseases such as cancer. Centrosomes also play important roles in cell migration, the movement of cilia and the movement of vesicular membrane structures within cells. Centrosome contains a number of proteins, including: (1) Microtubule; (2) Pericentrin; (3) Centrin; (4) PCMI; (5) Ninein; (6) BBS4; (7) P150 Glued; (8) Dynein; (9) Centriolin; (10) Gamma Tubulin; (11) Polo Kinases; (12) Aurora Kinases; (13) Catanin; and (14) Katanin. 
         [0107]    A product comprising the non-natural protein sequence comprising two or more epitopes selected from 1-14 as defined in Table 10 below would be useful in the study of cell cycles in biology, biotechnology and medicine. Furthermore a calibration product comprising amino acid sequences to antibodies specific to multiple components below is envisaged. In some instances suitable antibodies with known epitope sequences have been described, in other instances such antibodies need to be identified. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 10 
               
               
                   
                   
               
               
                   
                 Antibody 
                   
                   
               
               
                   
                 for: 
                 Epitope 
                 Cross Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 Mircotubule 
                 K562 erythroleukemia cell line 
                 Human 
               
               
                 2 
                 pericentrin 
                 aa 142-592 of mouse pericentrin. It is expressed 
                 Human 
               
               
                   
                   
                 as a GST fusion protein. The corresponding amino 
               
               
                   
                   
                 acids are present in both pericentrin and kendrin 
               
               
                 3 
                 Centrin 
                 synthetic peptide corresponding to the C-terminus 
                 Human 
               
               
                   
                   
                 of human centrin 1 (amino acids 157-172) 
               
             
          
           
               
                 4 
                 PCM1 
                 Antibodies to this protein with published epitope characteristics are 
               
               
                   
                   
                 not currently available from commercial sources. A new antibody 
               
               
                   
                   
                 could be created to an immunogenic sequence peculiar to this 
               
               
                   
                   
                 protein, and this sequence could be included in the calibration 
               
               
                   
                   
                 product 
               
             
          
           
               
                 5 
                 Ninein 
                 Fusion protein containing amino acids 32-506 of 
                 Human. 
               
               
                   
                   
                 murine Ninein 
                 Predicted to 
               
               
                   
                   
                   
                 react with 
               
               
                   
                   
                   
                 Mouse due to 
               
               
                   
                   
                   
                 sequence 
               
               
                   
                   
                   
                 homology 
               
             
          
           
               
                 6 
                 BBS4 
                 Antibodies to this protein with published epitope characteristics are 
               
               
                   
                   
                 not currently available from commercial sources. An antibody might 
               
               
                   
                   
                 be available by collaboration with an academic group, wherein the 
               
               
                   
                   
                 epitope of the antibody would be known, or could be discovered. 
               
               
                   
                   
                 Alternatively a new antibody could be created to an immunogenic 
               
               
                   
                   
                 sequence peculiar to this protein, and this sequence could be 
               
               
                   
                   
                 included in the calibration product 
               
             
          
           
               
                 7 
                 p150 
                 Synthetic peptide produced from aa3-202 of rat 
                 Chicken, Dog, 
               
               
                   
                 Glued 
                 p150Glued 
                 Human, Mouse, 
               
               
                   
                   
                   
                 Rat 
               
               
                 8 
                 Dynein 
                 Cytoplasmic full length native protein (purified from 
                 Human and 
               
               
                   
                   
                 chick brain) (Chicken) 
                 Chicken 
               
             
          
           
               
                 9 
                 Centriolin 
                 Antibodies to this protein with published epitope characteristics are 
               
               
                   
                   
                 not currently available from commercial sources. An antibody might 
               
               
                   
                   
                 be available by collaboration with an academic group, wherein the 
               
               
                   
                   
                 epitope of the antibody would be known, or could be discovered. 
               
               
                   
                   
                 Alternatively a new antibody could be created to an immunogenic 
               
               
                   
                   
                 sequence peculiar to this protein, and this sequence could be 
               
               
                   
                   
                 included in the calibration product 
               
             
          
           
               
                 10 
                 gamma 
                 Synthetic peptide: EEFATEGTDRKDVFFY-C 
                 Reacts with 
               
               
                   
                 Tubulin 
                 (SEQ ID NO: 58) conjugated to KLH, 
                 Human, 
               
               
                   
                   
                 corresponding to C terminal amino acids 38-53 of 
                 Chicken and 
               
               
                   
                   
                 Human gamma Tubulin 
                 Hamster 
               
               
                 11 
                 Polo 
                 aa330-370 OF His-Plk1 
                 Human and 
               
               
                   
                 Kinases 
                   
                 Mouse 
               
               
                   
                 (Plk1) 
               
             
          
           
               
                 12 
                 Aurora 
                 Variety of different isoforms of kinases 1-3 and ABC 
               
               
                   
                 Kinases 
               
               
                 13 
                 Catanin 
                 Antibodies to this protein with published epitope characteristics are 
               
               
                   
                   
                 not currently available from commercial sources. An antibody might 
               
               
                   
                   
                 be available by collaboration with an academic group, wherein the 
               
               
                   
                   
                 epitope of the antibody would be known, or could be discovered. 
               
               
                   
                   
                 Alternatively a new antibody could be created to an immunogenic 
               
               
                   
                   
                 sequence peculiar to this protein, and this sequence could be 
               
               
                   
                   
                 included in the calibration product 
               
             
          
           
               
                 14 
                 Katanin 
                 Synthetic peptide produced from human Katanin 
                 Human 
               
               
                   
                   
                 p80 aa 295-415 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 14 
     Proteins Sharing a Common Location in the Cell 
       [0108]    Lipid rafts is another physical location, which is a domain of the plasma membrane phase separated from surrounding regions of membrane. The phase separation arises as a consequence of the concentration of cholesterol and sphingomelin lipids, which group together to form a gel phase. Transmembrane proteins typically cannot enter this microdomain, which is populated instead by proteins anchored through fatty acid, or lipid-like units, including: GPI (glycosylinositolphosphatidyl) anchored proteins and proteins which are both myristolated and palmitoylated. 
         [0109]    A series of proteins associated with lipid rafts include: (1) Lck (SRC kinase family members); (2) Fyn (SRC kinase family members); (3) H-Ras; (4) ZAP-70; (5) CD3ç; (6) LAT; (7) Flotillin-1; (8) CD2; (9) PAG; (10) F-actin; and (11) CD59. A calibration product comprising amino acid sequences to antibodies specific to multiple components above is conceivable. In some instances suitable antibodies with known epitope sequences have been described, in other instances such antibodies need to be identified. A product comprising the non-natural protein sequence comprising two or more epitopes selected from 1-11 as defined in Table 11 below would be useful in the study of lipid rafts in biology, biotechnology and medicine. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 11 
               
               
                   
                   
               
               
                   
                 Antibody for: 
                 Epitope 
                 Cross Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                  1 
                 LcK 
                 Synthetic peptide, corresponding to amino 
                 Human 
                   
               
               
                   
                   
                 acids 22-36 of Human Lck p56 (CHYPIVPLD 
               
               
                   
                   
                 GKGTLL) (SEQ ID NO:59) 
               
               
                   
               
               
                  2 
                 Fyn 
                 Peptie sequence VQCKDKEATKLTE (SEQ ID 
                 Human 
               
               
                   
                   
                 NO:60) corresponding to the C-terminus of 
               
               
                   
                   
                 FYN (NP_002028 
               
               
                   
               
               
                  3 
                 H-Ras 
                 SAKSVFSSKALVKMQLLKDVVGNDTYRINNKY 
                 Human 
               
               
                   
                   
                 DETYPPLPVEEIIKRSEFVIGQEVAYNLLVNNCE 
               
               
                   
                   
                 HFVTLLRYGEGVSEQANR (SEQ ID NO:61) 
               
               
                   
               
               
                  4 
                 ZAP-70 
                 Synthetic peptide KDKKLFLKRDNLLIAD 
                 Human 
               
               
                   
                   
                 (SEQ ID NO:62) derived from ZAP-70 
               
               
                   
                   
                 sequence 
               
               
                   
               
               
                  5 
                 CD3epsilin 
                 Synthetic peptide: KAKAKPVTRGAGA, (SEQ 
                 Human, Baboon, 
               
               
                   
                   
                 ID NO:63) corresponding to amino acids 
                 Feline, Canine, 
               
               
                   
                   
                 156-168 of Human CD3 epsilon 
                 Horse and Monkey 
               
               
                   
               
               
                  6 
                 LAT 
                 Synthetic peptide: GAPDYENLQELN, (SEQ 
                 Human 
               
               
                   
                   
                 ID NO:64) corresponding to amino acids 
               
               
                   
                   
                 251-262 of Human LAT. 
               
               
                   
               
               
                  7 
                 Flotillin-1 
                 Synthetic peptide: SISQVNHKPLRTA, (SEQ 
                 Human. 
               
               
                   
                   
                 ID NO:65) corresponding to C terminal 
                 Predicted to react 
               
               
                   
                   
                 amino acids 415-427 of Human Flotillin 1. 
                 with Chimpanzee 
               
               
                   
                   
                   
                 and Pig due to 
               
               
                   
                   
                   
                 sequence 
               
               
                   
                   
                   
                 homology 
               
               
                   
               
             
          
           
               
                  8 
                 CD2 
                 Antibodies to this protein with published epitope characteristics 
                   
               
               
                   
                   
                 are not currently available from commercial sources. An antibody 
               
               
                   
                   
                 might be available by collaboration with an academic group, 
               
               
                   
                   
                 wherein the epitope of the antibody would be known, or could be 
               
               
                   
                   
                 discovered. Alternatively a new antibody could be created to an 
               
               
                   
                   
                 immunogenic sequence peculiar to this protein, and this 
               
               
                   
                   
                 sequence could be included in the calibration product 
               
               
                   
               
             
          
           
               
                  9 
                 PAG 
                 Recombinant fragment, corresponding to amino 
                 Human and 
                   
               
               
                   
                   
                 acids 97-432 of Human PAG (TLT CMQHYEEVQT 
                 Mouse 
               
               
                   
                   
                 SASDLLDSQD STGKPKCHQS RELPRIPPES 
               
               
                   
                   
                 AVDTMLTARS VDGDQGLGME GPYEVLKDSS 
               
               
                   
                   
                 SQENMVEDCL YETVKEIKEV AAAAHLEKGH 
               
               
                   
                   
                 SGKAKSTSAS KELPGPQTEG KAEFAEYASV 
               
               
                   
                   
                 DRNKKCRQSV NVESILGNSC DPEEEAPPPV 
               
               
                   
                   
                 PVKLLDENEN LQEKEGGEAE ESATDTTSET 
               
               
                   
                   
                 NKRFSSLSYK SREEDPTLTE EEISAMYSSV 
               
               
                   
                   
                 NKPGQLVNKS GQSLTVPEST YTSIQGDPQR 
               
               
                   
                   
                 SPSSCNDLYA TVKDFEKTPN STLPPAGRPS 
               
               
                   
                   
                 EEPEPDYEAI QTLNREEEKA TLGTNGHHGL 
               
               
                   
                   
                 VPKENDYESI SDLQQGRDIT RL) (SEQ ID NO:66) 
               
               
                   
               
               
                 10 
                 F-actin 
                 Human CAPZA1, amino acids 80- 
                 Human 
               
               
                   
                   
                 150(DLGNSRFLDPR NKISFKFDHL KEASDPQPE 
               
               
                   
                   
                 EADGGLKSWRESCDSALRAYVKDHYSNGFCTVYAKTI 
               
               
                   
                   
                 DGQ) (SEQ ID NO:67) 
               
               
                   
               
             
          
           
               
                 11 
                 CD59 
                 Antibodies to this protein with published epitope characteristics 
                   
               
               
                   
                   
                 are not currently available from commercial sources. An antibody 
               
               
                   
                   
                 might be available by collaboration with an academic group, 
               
               
                   
                   
                 wherein the epitope of the antibody would be known, or could be 
               
               
                   
                   
                 discovered. Alternatively a new antibody could be created to an 
               
               
                   
                   
                 immunogenic sequence peculiar to this protein, and this 
               
               
                   
                   
                 sequence could be included in the calibration product 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 15 
     Macromolecular Complex 
       [0110]    Macromolecular complexes which bring post-translational modification enzymes close to their target substrates are known. AKAPs (A-kinase anchoring proteins) are a good example of such complexes, which target enzymes involved in signalling with their substrate or effector proteins, creating local signalling circuits. The mAKAP macromoleular complex contains: (1) DE4D3; (2) Makap; (3) PKA; (4) Epac1; (5) Rap1; (6) MEKK; (7) MEK5; and (8) ERK5. A product comprising the non-natural protein sequence comprising two or more epitopes selected from 1-8 as defined in Table 12 below would be useful in the study of lipid rafts in biology, biotechnology and medicine. 
       EXAMPLE 16 
     Macromolecular Complex 
       [0111]    Many biological polymers assemble stably or transiently into macromolecular complexes, which typically exhibit function. One such complex is the dystrophin complex which forms a junction between the plasma membrane of a muscle cell and the underlying cytoskeletal structure. The Dystrophin complex contains a number of proteins, including: (1) Laminin; (2) Alpha dystroglycan; (3) Beta dystroglycan; (4) Caveolin; (5) Dystrobrevin; (6) Dystrophin; (7) Actin; (8) Alpha sacroglycan; (9) Beta sacroglycan; (10) Delta sacroglycan; (11) Gamma sacroglycan; and (12) Sacrospan. A product comprising the non-natural protein sequence comprising two or more epitopes selected from 1-12 as defined in Table 13 below would be useful in the study of lipid rafts in biology, biotechnology and medicine. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                 Antibody 
                   
                 Cross 
                   
               
               
                   
                 for: 
                 Epitope 
                 Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                  1 
                 PDE4D3 
                 Synthetic peptide from unique region near N- 
                 Human, 
                   
               
               
                   
                   
                 terminal of the PDE4D3 variant Antibodies to 
                 Mouse and 
               
               
                   
                   
                 this protein with published epitope 
                 Rat 
               
               
                   
                   
                 characteristics are not currently available 
               
               
                   
                   
                 from commercial sources. An antibody might 
               
               
                   
                   
                 be available by collaboration with an 
               
               
                   
                   
                 academic group, wherein the epitope of the 
               
               
                   
                   
                 antibody would be known, or could be 
               
               
                   
                   
                 discovered. Alternatively a new antibody 
               
               
                   
                   
                 could be created to an immunogenic 
               
               
                   
                   
                 sequence peculiar to this protein, and this 
               
               
                   
                   
                 sequence could be included in the calibration 
               
               
                   
                   
                 product 
               
               
                   
               
               
                  2 
                 mAKAP 
                 Synthetic peptide LTMSVTLSPLRSQ-C, (SEQ 
                 Human, 
               
               
                   
                   
                 ID NO:68) representing the N-Terminus of 
                 Mouse and 
               
               
                   
                   
                 the human protein according to NP_004265 
                 Rat 
               
               
                   
               
               
                  3 
                 PKA 
                 KKGSEQESVKEFLAK (SEQ ID NO:69) (aa 8-22 
                 Human, 
               
               
                   
                   
                 human alpha isoform of the catalytic subunit) 
                 Bovine, 
               
               
                   
                   
                   
                 Mouse, 
               
               
                   
                   
                   
                 Porcine, Rat 
               
               
                   
               
               
                  4 
                 Epac1 
                 Synthetic peptide: DHSVLTLQLPVTASVR, (SEQ 
                 Human, 
               
               
                   
                   
                 ID NO:70) corresponding to amino acids 526-541 
                 Mouse and 
               
               
                   
                   
                 of Epac1 
                 Rat 
               
               
                   
               
               
                  5 
                 Rap1 
                 Synthetic peptide: 
                 Human 
               
               
                   
                   
                 KAEEDPEAADSGEPQNKRTPDLPE, (SEQ ID 
               
               
                   
                   
                 NO:70) corresponding to amino acids 212-235 of 
               
               
                   
                   
                 Human RAP1 
               
               
                   
               
               
                  6 
                 MEKK 
                 Mouse Mekk-1 synthetic peptide (aa. 654-669; 
                 Human, 
               
               
                   
                   
                 Lange-Carter, 1993), conjugated to KLH 
                 Mouse and 
               
               
                   
                   
                 (LRAMLVYTPCHSLAER) (SEQ ID NO:71) 
                 Rat 
               
               
                   
               
               
                  7 
                 MEK5 
                 Synthetic peptide: AFEYEDEDGDRITVRSC (SEQ 
                 Human, 
               
               
                   
                   
                 ID NO:72), corresponding to amino acids 59/74 
                 Mouse, Rat, 
               
               
                   
                   
                 of Human MEK5 
                 Rabbit, 
               
               
                   
                   
                   
                 
                   Xenopus 
                 
               
               
                   
                   
                   
                   laevis , Dog, 
               
               
                   
                   
                   
                 Monkey and 
               
               
                   
                   
                   
                 Sheep 
               
               
                   
               
               
                  8 
                 ERK5 
                 Synthetic peptide: MAEPLKEEDG EDGS (SEQ ID 
                 Human, 
               
               
                   
                   
                 NO:73) conjugated to KLH, corresponding to N 
                 Mouse, Rat 
               
               
                   
                   
                 terminal amino acids 1-14 of Human ERK5 
                 and Cow 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 13 
               
               
                   
                   
               
               
                   
                 Antibody 
                   
                   
                   
               
               
                   
                 for: 
                 Epitope 
                 Cross Reactivity 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                  1 
                 Laminin 
                 Full length protein (Human) 
                 Cross-reacts with all 
                   
               
               
                   
                   
                   
                 Mammals 
               
               
                   
               
               
                  2 
                 alpha- 
                 Antibodies to this protein with 
                 Canine, Guinea Pig, 
               
               
                   
                 dystroglycan 
                 published epitope characteristics are 
                 Human, Mouse, Rat, 
               
               
                   
                   
                 not currently available from 
                 Rabbit 
               
               
                   
                   
                 commercial sources. An antibody 
               
               
                   
                   
                 might be available by collaboration 
               
               
                   
                   
                 with an academic group, wherein the 
               
               
                   
                   
                 epitope of the antibody would be 
               
               
                   
                   
                 known, or could be discovered. 
               
               
                   
                   
                 Alternatively a new antibody could 
               
               
                   
                   
                 be created to an immunogenic 
               
               
                   
                   
                 sequence peculiar to this protein, 
               
               
                   
                   
                 and this sequence could be included 
               
               
                   
                   
                 in the calibration product 
               
               
                   
               
               
                  3 
                 beta- 
                 IVVEWT NNTLPLEPCP KEQIAGLSRR 
                 Human 
               
               
                   
                 dystroglycan 
                 IAEDDGKPRP AFSNALEPDF 
               
               
                   
                   
                 KATSITVTGS GSCRHLQFIP 
               
               
                   
                   
                 VVPPRRVPSE APPTEVPDRD 
               
               
                   
                   
                 PEKSSEDDVY LHTVIPAVVV AAILLIA 
               
               
                   
                   
                 (SEQ ID NO:74) (from Human) 
               
               
                   
               
               
                  4 
                 Caveolin 
                 M(1)SGGKYVDSEGHLYTVP(17)C 
                 Human, Rat, Mouse, 
               
               
                   
                   
                 (SEQ ID NO:75) (1-17 from human 
                 Dog and Hamster 
               
               
                   
                   
                 caveolin-1) 
               
               
                   
               
               
                  5 
                 dystrobrevin 
                 ES IMGFRYRCQQ CHNYQLCQDC 
                 Mouse, Rabbit, Rat 
               
               
                   
                   
                 FWRGHAGGSH SNQHQMKEYT 
               
               
                   
                   
                 SWKSPAKKLT NALSKSLSCA 
               
               
                   
                   
                 SSREPLHPMF PDQPEKPLNL 
               
               
                   
                   
                 AHIVPPRPVT SMNDTLFSHS 
               
               
                   
                   
                 VPSSGSPFIT RSSPPKDSEV 
               
               
                   
                   
                 EQNKMLARAA PAFLKGRGIQ 
               
               
                   
                   
                 YSLNVADRLA DEH (SEQ ID NO:76) 
               
               
                   
               
               
                  6 
                 dystrophin 
                 Fusion protein, corresponding to 
                 Fish, Human, Mouse 
               
               
                   
                   
                 amino acids 3200-3684 of Human 
                 and Rat 
               
               
                   
                   
                 Dystrophin. 
               
               
                   
               
               
                  7 
                 Actin 
                 Synthetic peptide to the correct 
               
               
                   
                   
                 isoform of actin 
               
               
                   
               
               
                  8 
                 alpha- 
                 Antibodies to this protein with 
                 rabbit, mouse, 
               
               
                   
                 sarcoglycan 
                 published epitope characteristics are 
                 human 
               
               
                   
                   
                 not currently available from 
               
               
                   
                   
                 commercial sources. A new antibody 
               
               
                   
                   
                 could be created to an immunogenic 
               
               
                   
                   
                 sequence peculiar to this protein, 
               
               
                   
                   
                 and this sequence could be included 
               
               
                   
                   
                 in the calibration product 
               
               
                   
               
               
                  9 
                 beta- 
                 MAAAAAAAAEQQSSNGPVKKSMREKA 
                 Human 
               
               
                   
                 sarcoglycan 
                 VERRSVNKEHNSNFKAGYIPIDEDRLHK 
               
               
                   
                   
                 TGLRGRKGNLAICVIILLFILAVINLIITLV 
               
               
                   
                   
                 IWAVIRIGPNGCDSMEFHESGLLRFKQV 
               
               
                   
                   
                 SDMGVIHPLYKSTVGGRRNENLVITGN 
               
               
                   
                   
                 NQPIVFQQGTTKLSVENNKTSITSDIGM 
               
               
                   
                   
                 QFFDPRTQNILFSTDYETHEFHLPSGVK 
               
               
                   
                   
                 SLNVQKASTERITSNATSDLNIKVDGRA 
               
               
                   
                   
                 IVRGNEGVFIMGKTIEFHMGGNMELKAE 
               
               
                   
                   
                 NSIILNGSVMVSTTRLPSSSSGDQLGS 
               
               
                   
                   
                 GDWVRYKLCMCADGTLFKVQVTSQNM 
               
               
                   
                   
                 GCQISDNPCGNTH (SEQ ID NO:77) 
               
               
                   
               
               
                 10 
                 delta- 
                 Synthetic peptide containing amino 
                 Human. Does not 
               
               
                   
                 sarcoglycan 
                 acids 1-19 at the N terminus of 
                 react with chicken, 
               
               
                   
                   
                 human delta sacroglycan sequence 
                 canine, hamster, 
               
               
                   
                   
                   
                 mouse, rabbit, rat, 
               
               
                   
                   
                   
                 pi 
               
               
                   
               
               
                 11 
                 gamma- 
                 Synethetic peptide containing amino 
                 Human, chicken, 
               
               
                   
                 sarcoglycan 
                 acids 167-178 of rabbit gamma- 
                 canine, mouse 
               
               
                   
                   
                 sacroglycan sequence 
               
               
                   
               
               
                 12 
                 sarcospan 
                 Antibodies to this protein with 
               
               
                   
                   
                 published epitope characteristics are 
               
               
                   
                   
                 not currently available from 
               
               
                   
                   
                 commercial sources. A new antibody 
               
               
                   
                   
                 could be created to an immunogenic 
               
               
                   
                   
                 sequence peculiar to this protein, 
               
               
                   
                   
                 and this sequence could be included 
               
               
                   
                   
                 in the calibration product 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 17 
     RyR2Macromolecular Complex Collection for Humans and Mice 
       [0112]    The cardiac ryanodine receptor (RyR2), located in the sarcoplasmic reticulum (SR), is a calcium release channel which is centrally involved in the myocyte excitation-contraction (E-C). The ryanodine recpetor is also the center of a massive macromolecular complex, which includes numerous regulatory proteins that can modulate RyR2 function. This complex includes proteins that interact with the cytoplasmic part of the RyR2 directly or indirectly (e.g. calmodulin (CaM), FK-506-binding proteins, protein kinase A, Ca-CaM-dependent protein kinase, phosphatases 1 and 2A, mAKAP, spinophilin, PR130, sorcin, triadin, junctin, calsequestrin and Horner). Understanding both the physical/molecular nature of the protein-protein interactions between RyR and these other proteins is important since this complex and the modulation of the ryanodine receptor is believed to be involved in cardiac arrhythmias, pace-maker function of the heart and cardiac disease. 
         [0113]      FIG. 5  shows all of the known protein-protein interaction sites between RyR2 and the macromolecular complex components. The proteins labelled with red numbers are some of the proteins which are involved in the complex, and which have been used to produce a corresponding calibrant. Antibody epitopes for the proteins in this complex have been genetically encoded and expressed in bacteria to produce a single protein ( FIG. 6 ) that contains all of the antibody epitopes to each of the labelled proteins. The numerical notation of each protein corresponds to the notation in Table 14 below. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 14 
               
               
                   
               
               
                 Notation 
                 Epitope 
                 Amino Acid Sequence 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 A 
                 Histidine Tag 
                 HHHHHH 
                   
               
               
                   
                   
                 (SEQ ID NO: 78) 
               
               
                   
               
               
                 B 
                 Glutathione S 
                 (whole protein) 
               
               
                   
                 Transferase 
               
               
                   
                 (GST) 
               
               
                   
               
               
                  1 
                 Calmodulin 
                 KRRWKKNFIAVSAANRFLLISSGAL 
               
               
                   
                 Binding Peptide 
                 (SEQ ID NO: 79) 
               
               
                   
                 (CBP) 
               
               
                   
               
               
                  2 
                 RyR2 
                 DQQEQVKEDMETK 
               
               
                   
                   
                 (SEQ ID NO: 80) 
               
               
                   
               
               
                  3 
                 Calmodulin 
                 DIDGDGQVNYEEFVQMMTAK 
               
               
                   
                   
                 (SEQ ID NO: 81) 
               
               
                   
               
               
                  4 
                 CamKII 
                 CTRFTDEYQLFEEL 
               
               
                   
                   
                 (SEQ ID NO: 82) 
               
               
                   
               
               
                  5 
                 Calstabin 2 
                 GVQVETISPGDGR 
               
               
                   
                 (FKBP12.6) 
                 (SEQ ID NO: 83) 
               
               
                   
               
               
                  6 
                 PKA 
                 KKGSEQESVKEFLAK 
               
               
                   
                   
                 (SEQ ID NO: 84) 
               
               
                   
               
               
                  7 
                 PP1/PP2A 
                 MSDSEKLNLDSIIGRLLE 
               
               
                   
                   
                 (SEQ ID NO: 85) 
               
               
                   
               
               
                  8 
                 Sorcin 
                 MAYPGHPGAGGGYYPGGYGGAPGGP 
               
               
                   
                   
                 AFPGQTQDPLYGYFAAVAGQDGQID 
               
               
                   
                   
                 (SEQ ID NO: 86) 
               
               
                   
               
               
                  9 
                 Calsequestrin 
                 EGLNFPTYDGKDRVVSLSEKNFKQVL 
               
               
                   
                   
                 KKYDLLCLYYHEPVSSDKVTQKQFQL 
               
               
                   
                   
                 KEIVLELV 
               
               
                   
                   
                 (SEQ ID NO: 87) 
               
               
                   
               
               
                 10 
                 Histidine Tag 
                 HHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 78) 
               
               
                   
               
             
          
         
       
     
         [0114]    Notations A and B can be cleaved and removed, which allows for the production of the true RyR2 macromolecular complex epitope calibrant. Notations 1 and 10 are tags which can be used in purification of the calibrant protein and also act as common and widely used antibody epitopes. 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO:88) 
                   
               
             
          
           
               
                 GGATCCAAACGCCGTTGGAAAAAGAACTTTATTGCAGTTAGTGCGGCCAA 
                   
               
               
                   
               
               
                 TCGCTTTCTGCTGATTTCTTCGGGAGCGCTGGATCAGCAAGAACAGGTTA 
               
               
                   
               
               
                 AAGAAGATATGGAAACCAAAGATATCGATGGTGATGGGCAGGTGAACTAT 
               
               
                   
               
               
                 GAAGAATTCGTACAGATGATGACCGCCAAATGCACCCGTTTTACGGACGA 
               
               
                   
               
               
                 ATACCAGCTGTTCGAAGAACTGGGCGTTCAAGTTGAAACAATCAGCCCGG 
               
               
                   
               
               
                 GAGATGGCCGCAAAAAAGGTAGTGAGCAGGAATCTGTTAAAGAGTTCCTG 
               
               
                   
               
               
                 GCGAAAATGTCAGATTCGGAGAAACTGAATCTGGATAGCATTATTGGTCG 
               
               
                   
               
               
                 CCTGCTGGAGATGGCTTACCCGGGTCATCCGGGCGCGGGCGGCGGCTATT 
               
               
                   
               
               
                 ACCCAGGCGGCTATGGCGGTGCCCCGGGTGGTCCGGCGTTTCCGGGCCAG 
               
               
                   
               
               
                 ACCCAGGATCCGCTGTACGGCTATTTTGCAGCCGTGGCGGGCCAGGATGG 
               
               
                   
               
               
                 CCAAATCGATGAGGGCCTGAATTTCCCGACCTATGATGGGAAAGACCGTG 
               
               
                   
               
               
                 TTGTTTCACTGAGCGAAAAGAACTTTAAACAGGTACTGAAAAAATACGAT 
               
               
                   
               
               
                 CTGCTGTGCCTGTATTATCATGAACCAGTTTCCAGTGACAAAGTTACCCA 
               
               
                   
               
               
                 AAAACAGTTTCAGCTGAAGGAAATTGTACTGGAACTGGTGAAGCTT 
               
             
          
         
       
     
         [0115]    The optimised genetic sequence, which encodes for all of the proteins in Table 14 is shown above as SEQ ID NO:88. Below as SEQ ID NO:89 is the resulting amino acid sequence from the genetic code 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO:89) 
                   
               
             
          
           
               
                 KRRWKKNFIAVSAANRFLLISSGALDQQEQVKEDMETKDIDGDGQVNYEE 
                   
               
               
                   
               
               
                 FVQMMTAKCTRFTDEYQLFEELGVQVETISPGDGRKKGSEQESVKEFLAK 
               
               
                   
               
               
                 MSDSEKLNLDSIIGRLLEMAYPGHPGAGGGYYPGGYGGAPGGPAFPGQTQ 
               
               
                   
               
               
                 DPLYGYFAAVAGQDGQIDEGLNFPTYDGKDRVVSLSEKNFKQVLKKYDLL 
               
               
                   
               
               
                 CLYYHEPVSSDKVTQKQFQLKEIVLELV. 
               
             
          
         
       
     
         [0116]    The genetic sequence encoding the antibody epitopes that make up the calibrant (and purification tags) is synthesised and inserted into the  E. coli  expression vector pGS-21a ( FIG. 7 ). 
         [0117]    The pGS-21a plasmid, which now contains the genetic sequence to encode for the RyR2 macromolecular complex calibrant, is transformed into BL21 (DE3) pLysS  E. coli  cells. Transformed cells are selected and used to express the calibrant protein after the induction of gene synthesis with IPTG. After 3.5 hours of expression, the cells were harvested and re-suspended in sample buffer for analysis by SDS-PAGE and western blot. This was done in order to assess the purity of the calibrant product.  FIG. 8  shows a western blot result of a doubling dilution series of the expressed calibrant. 
         [0118]    Following successful expression of a considerably pure calibrant product, the calibrant expression was scaled up from 1 ml to 1.5 L ( FIGS. 9 and 11 ) and was used to produce a quantifiable calibration plot ( FIGS. 10 and 12 ). The calibration curve was performed in triplicate and each replica was probed with a different epitope antibody and quantified by measuring the densitometry of the expressed RyR2Macromolecular Complex Calibrant. 
       EXAMPLE 18 
     MAPKinase Pathway Collection for Humans and Mice 
       [0119]    Mitogen activated protein kinases (MAPK) are at the center of many signalling transduction pathways in eukaryotic cells. The study of MAPK pathways is important in the research of many disease areas such as inflammation, cancer and Parkinsons disease. We have used several proteins which are involved in one of the MAPK pathways to produce a corresponding calibrant. Antibody epitopes for the proteins on the in this pathway have been genetically encoded and expressed in bacteria to produce a single protein that contains all of the antibody epitopes to each of the labelled proteins. The numerical notation of each protein corresponds to the notation in Table 15 below. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 15 
               
               
                   
               
               
                 Notation 
                 Epitope 
                 Amino Acid Sequence 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 A 
                 Histidine Tag 
                 HHHHHH 
                   
               
               
                   
                   
                 (SEQ ID NO:78) 
               
               
                   
               
               
                 B 
                 Glutathione S 
                 (whole protein) 
               
               
                   
                 Transferase 
               
               
                   
                 (GST) 
               
               
                   
               
               
                  1 
                 Calmodulin 
                 KRRWKKNFIAVSAANRFLLISSGAL 
               
               
                   
                 Binding Peptide 
                 (SEQ ID NO:90) 
               
               
                   
                 (CBP) 
               
               
                   
               
               
                  2 
                 EGF Receptor 
                 ETKPNGIFKGPTAENAEYLRVAPPSS 
               
               
                   
                   
                 EFIGA 
               
               
                   
                   
                 (SEQ ID NO:91) 
               
               
                   
               
               
                  3 
                 Grb2 (Gab1) 
                 KGACHGQTGMFPRNYVTPVNRNV 
               
               
                   
                   
                 (SEQ ID NO:92) 
               
               
                   
               
               
                  4 
                 SOS1 
                 KKSDHGNAFFPNSPSP 
               
               
                   
                   
                 (SEQ ID NO:93) 
               
               
                   
               
               
                  5 
                 Ras 
                 EYDPTIEDSYRKQ 
               
               
                   
                   
                 (SEQ ID NO:94) 
               
               
                   
               
               
                  6 
                 A-Raf 
                 QGPSPFTQQRDQEHFSF 
               
               
                   
                   
                 (SEQ ID NO:95) 
               
               
                   
               
               
                  7 
                 MEK1 
                 PKKKPTPIQLNP 
               
               
                   
                   
                 (SEQ ID NO:96) 
               
               
                   
               
               
                  8 
                 MEK2 
                 MLARRKPVLPALT 
               
               
                   
                   
                 (SEQ ID NO:97) 
               
               
                   
               
               
                  9 
                 ERK1 and ERK2 
                 PFTFDMELDDLPKERLKELIFQETAR 
               
               
                   
                   
                 FQPGAPEAP 
               
               
                   
                   
                 (SEQ ID NO:98) 
               
               
                   
               
               
                 10 
                 RSK 1,2 and 3 
                 VSKMLHVDPHQRLTA 
               
               
                   
                   
                 (SEQ ID NO:99) 
               
               
                   
               
               
                 11 
                 Histidine Tag 
                 HHHHHH 
               
               
                   
                   
                 (SEQ ID NO:78) 
               
               
                   
               
             
          
         
       
     
         [0120]    Notations A and B can be cleaved and removed, which allows for the production of the true MAPKinase pathway epitope calibrant. Notations 1 and 11 are tags which can be used in purification of the calibrant protein and also act as common and widely used antibody epitopes. 
         [0121]    The optimised genetic sequence, which encodes for all of the proteins in Table 15 is shown below as SEQ ID NO:100. 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO 100) 
                   
               
             
          
           
               
                 GGATCCAAACGTCGCTGGAAGAAAAATTTCATTGCGGTGTCGGCAGCGAA 
                   
               
               
                   
               
               
                 TCGTTTTCTGCTGATCAGCTCTGGCGCTCTGGAGACAAAACCGAACGGTA 
               
               
                   
               
               
                 TCTTTAAAGGGCCAACAGCGGAAAATGCCGAATACCTGCGCGTGGCACCT 
               
               
                   
               
               
                 CCGTCTTCGGAGTTTATTGGCGCCAAGGGGGCATGCCACGGTCAGACCGG 
               
               
                   
               
               
                 TATGTTTCCTCGTAATTATGTGACCCCTGTTAACCGTAACGTCAAAAAAT 
               
               
                   
               
               
                 CCGATCACGGGAACGCATTCTTTCCGAATTCTCCGTCCCCGGAGTACGAT 
               
               
                   
               
               
                 CCGACTATTGAAGACAGTTACCGTAAACAGCAGGGACCTAGCCCGTTTAC 
               
               
                   
               
               
                 CCAACAGCGCGACCAGGAACATTTTAGTTTTCCGAAAAAGAAACCGACCC 
               
               
                   
               
               
                 CTATCCAGCTGAATCCGATGCTGGCTCGCCGCAAACCGGTCCTGCCAGCT 
               
               
                   
               
               
                 CTGACCCCTTTTACGTTCGATATGGAACTGGATGATCTGCCGAAGGAACG 
               
               
                   
               
               
                 CCTGAAGGAACTGATCTTTCAGGAAACCGCTCGTTTTCAGCCGGGTGCGC 
               
               
                   
               
               
                 CGGAAGCCCCGGTAAGCAAAATGCTGCACGTGGACCCGCATCAGCGCCTG 
               
               
                   
               
               
                 ACGGCAAAGCTT 
               
             
          
         
       
     
         [0122]    The resulting amino acid sequence from the genetic sequence of SEQ ID NO:100 is given below as SEQ ID NO:101. 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO: 101) 
                   
               
             
          
           
               
                 KRRWKKNFIAVSAANRFLLISSGALETKPNGIFKGPTAENAEYLRVAPPS 
                   
               
               
                   
               
               
                 SEFIGAKGACHGQTGMFPRNYVTPVNRNVKKSDHGNAFFPNSPSPEYDPT 
               
               
                   
               
               
                 IEDSYRKQQGPSPFTQQRDQEHFSFPKKKPTPIQLNPMLARRKPVLPALT 
               
               
                   
               
               
                 PFTFDMELDDLPKERLKELIFQETARFQPGAPEAPVSKMLHVDPHQRL 
               
               
                   
               
               
                 TA. 
               
             
          
         
       
     
         [0123]    The genetic sequence encoding the antibody epitopes that make up the calibrant (and purification tags) is synthesised and inserted into the  E. coli  expression vector pGS-21a ( FIG. 14 ). 
         [0124]    The pGS-21a plasmid, which now contains the genetic sequence to encode for the MAPK pathway calibrant, is transformed into BL21 (DE3) pLysS  E. coli  cells. Transformed cells are selected and used to express the calibrant protein after the induction of gene synthesis with IPTG. After 3.5 hours of expression, the cells were harvested and re-suspended in sample buffer for analysis by SDS-PAGE and western blot. This was done in order to assess the purity of the calibrant product.  FIG. 15  shows a western blot result of a doubling dilution series of the expressed calibrant. Following successful expression of a considerably pure calibrant product, the calibrant expression was scaled up from 1 ml to 1.5 L ( FIGS. 16 ,  18  and  20 ) and was used to produce a quantifiable calibration plots ( FIGS. 17 ,  19  and  21 ). The calibration curve was performed in triplicate and each replica was probed with a different epitope antibody and quantified by measuring the densitometry of the expressed MAPKinase Pathway Calibrant. 
       REFERENCES 
       [0000]    
       
         1. Stamm S., Ben-Ari, S., Rafalska, I., Tang, Y., Zhang, Z., Toiber, D., Thanaraj, T. A., &amp; Soreq, H. (2005) Function of alternative splicing. Gene 344, 1-20, 2005.) 
         2. Brandl C J., DeLoon S., Martin, D R., &amp; MacLennan D H (1987) J. Biol. Chem. 262, 3768-74 
         3. Brandl C J., Green N M., Korczak B., &amp; MacLennan D H (1986) Cell 44, 597-607 
         4. MacLennan D H., Brandl C J., Korczak B., &amp; Green N M (1985) Nature 316, 696-700 
         5. Campbell A M., Kessler P D., &amp; Fambrough D M. (1992) J. Biol. Chem. 267, 9321-25 
         6. Burk S E., Lytton J., MacLennan D H., &amp; Shull G E. (1989) J. Biol. Chem. 264, 18561-8 
         7. Priori S G., Napolitano C., Memmi M., Colombi B., Drago F., Gasparini M., DeSimone L., Coltorti F., Bloise R., Keegan R., Cruz Filho F E S., Vignati G., Benatar A., &amp; DeLogu A. (2002) Circulation 106, 69-74.