WO1999039210A1 - High density arrays for proteome analysis and methods and compositions therefor - Google Patents
High density arrays for proteome analysis and methods and compositions therefor Download PDFInfo
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- WO1999039210A1 WO1999039210A1 PCT/AU1999/000060 AU9900060W WO9939210A1 WO 1999039210 A1 WO1999039210 A1 WO 1999039210A1 AU 9900060 W AU9900060 W AU 9900060W WO 9939210 A1 WO9939210 A1 WO 9939210A1
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Definitions
- the present invention relates generally to high density protein arrays for proteome analysis. More particularly, the present invention provides high-density arrays comprising a primary protein array and a secondary antibody array, wherein the secondary array comprises monoclonal antibodies and/or antibody variants or derivatives that bind specifically or non-specifically to one or more proteins in the primary array, and wherein the latter information allows the secondary array to be used to determine the protein profile of the expression levels of a multiplicity of individual proteins in parallel, from biological extracts derived from a cell, tissue, organ or whole organism or a cellular extract, lysate or protein fraction derived therefrom.
- the profile that is obtained for any biological sample using the high-density arrays of the present invention comprises a sub-set of the total antigenic diversity of said biological sample that is also immunologically cross-reactive with one or more of the proteins in the primary protein array.
- the present invention also relates generally to methods of determining the epitope profile of cells, tissues, organs and whole organisms and cellular extracts, lysates or protein fractions derived therefrom, using the high density protein arrays described herein, in particular in relation to diagnostic and therapeutic applications.
- the term "derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
- the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
- nucleic acid-based arrays provide limited information on the cellular processes or disease associations in living organisms because, with the possible exception of DNA/protein interactions that regulate gene expression, a knowledge of protein expression, rather than nucleic acid expression, is of greatest utility in analysing cellular processes.
- Protein profiles of cells, tissues, organs or whole organisms provide important information on protein synthesis and turnover and a true indication of the health or otherwise of a cell, tissue, organ or organism. The phenotype of living cells is dependent upon the protein gene-products produced therein at any given time or developmental stage.
- Protein-based approaches for analysing cellular processes have been reviewed by Humphery-Smith and Blackstock (1997) and Humphery-Smith ef al., (1997).
- the technology is at present limited by several factors, including an absolute reliance upon conventional protein separation technologies, such as 2-dimensional gel electrophoresis, chromatographic procedures (including the more rapid technologies of FPLC and SMART, supplied by Pharmacia, Uppsala, Sweden), capillary electrophoretic techniques and mass spectrometry, to separate mixtures of proteins into individual analytes and/or to enrich individual analytes, a lack of sensitivity which prevents the detection of entire proteomes (i.e. the potential protein output of a cell, tissue, organ or organism), and an inability to analyse entire proteomes, rather than merely individual proteins, in a single assay.
- entire proteomes i.e. the potential protein output of a cell, tissue, organ or organism
- any protein may be produced in vitro without the use of a structural gene template or an expression gene constructs comprising same, and genetic manipulations to produce high-density arrays of expression gene construct matrices that are representative of the entire proteome of a cell, tissue, organ or organism, are not possible.
- the inventors sought to develop multiple array screening systems that could be readily applied to the analysis of entire proteomes.
- the inventor has developed a high density array comprising a primary and secondary array, and a screening system for use therewith, to provide unique tags in the secondary array, such as one or more animal-derived or phage- derived monoclonal antibodies, to each of the elements contained within a primary high-density array of proteins.
- the high density array developed by the inventor provides the means by which at least one tag is obtained per protein of the near-to- total proteome of any organism.
- many antibodies of the secondary array will be highly non-specific, a combination of one or many antibodies, for example hundreds - 5 -
- the present invention provides a method of substantially determining the proteome content of cells, tissues and organisms or fractions of any of the foregoing by the utilisation of a first or primary high density protein array and a secondary high density antibody array.
- one aspect of the invention provides a method of determining the protein profile of a biological sample comprising:
- Ab 1 ,Ab 2 ,Ab 3 , Ab n are monoclonal antibodies and/or antibody variants or derivatives that bind specifically or non-specifically to one or more proteins in the primary array;
- Xn is the coordinate of any particular monoclonal antibody and/or antibody variant or derivative along a first dimension of said array;
- Yn is the coordinate of any particular monoclonal antibody and/or antibody variant or derivative along a second dimension of said array; and wherein n is any positive finite number;
- the secondary high density antibody array is derived by means of a polyclonal immune response to a composite soup of the elements employed to generate the individual elements of the primary high density protein array.
- the primary high density protein array is an antigen array.
- this aspect of the invention may be used for comparative purposes, to determine whether the protein profile of the "test sample” possesses any differences in terms of expressed proteins, to a biological standard or reference.
- a further aspect of the invention provides a method of determining one or more proteins that are differentially-expressed between cells, tissues, organs, or organisms or biological samples derived therefrom comprising:
- a primary array of proteins a 1 (Xn Yn) , a 2 (Xn Yn) , a 3 (Xn Yn) ,a n (Xn Yn) comprising a multiplicity of protein elements designed to emulate a significant portion of the antigenic diversity of a cell, tissue, organ, organism from which the biological sample is derived, wherein a 1 ,a 2 ,a 3 ,....,a n are proteins;
- Xn is the coordinate of any particular protein along a first dimension of said array;
- Yn is the coordinate of any particular protein along a second dimension of said array; and wherein n is any positive finite number;
- Ab ⁇ Ab 2 ,Ab 3 , Ab n are monoclonal antibodies and/or antibody variants or derivatives that bind specifically or non-specifically to one or more proteins in the primary array;
- Xn is the coordinate of any particular monoclonal antibody and/or antibody variant or derivative along a first dimension of said array;
- Yn is the coordinate of any particular monoclonal antibody and/or antibody variant or derivative along a second dimension of said array; and wherein n is any positive finite number;
- one or more of the proteins in the primary array which form antigen-antibody complexes with elements of the secondary array as determined at (iv) may also be identified, by determining the coordinates (Xn.Yn) of said protein(s) which bind to the monoclonal antibodies and/or antibody variants or derivatives detected by screening the secondary array.
- the present invention clearly extends to any proteins isolated from the biological sample, once such proteins have been identified using the inventive method.
- This invention is predicated on the exploitation of the extraordinarily sensitivity of antibodies and antibody derivatives, in particular monoclonal antibodies, for the immunocapture 8
- the simultaneous action of screening high density antigen arrays simultaneously with monoclonal or phage-derived antibodies can be used to detect expression levels of cellular or excreted proteins.
- the essential use of two high-density arrays i.e. a primary array and a secondary array
- This process allows initial antibody screening and antibody specificity testing to be combined into a single step, with respect to the multiplicity of protein elements contained within the primary array.
- a further aspect of the invention provides an array for use in determining the protein profile of a cell, tissue, organ or organism or a biological sample derived therefrom, comprising:
- Ab ⁇ Ab 2 ,Ab 3 , Ab n are monoclonal antibodies and/or antibody variants or derivatives that bind specifically or non-specifically to one or more proteins in the primary array;
- Xn is the coordinate of any particular monoclonal antibody and/or antibody variant or derivative along a first dimension of said array;
- Yn is the coordinate of any particular monoclonal antibody and/or antibody variant or derivative along a second dimension of said array; and wherein n is any positive finite number.
- the present invention clearly extends to arrays of monoclonal antibodies or antibody variants or derivatives that are produced by screening the primary protein array described herein. Accordingly, a further aspect of the invention provides an array of monoclonal antibodies or antibody variants or derivatives comprising the antibodies
- Ab 1 ,Ab 2 ,Ab 3 ,....,Ab n are monoclonal antibodies and/or antibody variants or derivatives
- Xn is the coordinate of any particular monoclonal antibody and/or antibody variant or derivative along a first dimension of said array
- Yn is the coordinate of any particular monoclonal antibody and/or antibody variant or derivative along a second dimension of said array
- n is any positive finite number
- said array is produced by a method comprising:
- the secondary antibody array is prepared by selecting monoclonal antibodies or antibody variants that bind to proteins in a primary array which are derived from the same cell-type, tissue-type, organ-type, bodily fluid-type, blood-type, or serum-type as a biological sample which is to be used to screen the array.
- one or more of the monoclonal antibodies and/or antibody variants or derivatives are derived from hybridomas or other cells, or alternatively, from recombinant bacte ophage or viruses, which each express antibodies or antibody variants or derivatives which bind to one or more proteins in the primary array. More preferably, the hybridomas or other cells, or recombinant bacteriophage or viruses, each express antibodies or antibody variants or derivatives which bind to proteins in the primary array in a specific or non-specific manner .
- the present invention relates to the applications of the inventive secondary antibody array to the diagnosis and prophylactic and therapeutic treatment of humans and other animals for medical conditions.
- the secondary antibody array is prepared by selecting monoclonal antibodies or antibody variants that bind to proteins in a primary array which are - 11
- a further aspect of the invention provides a method of diagnosing a human or animal subject for a medical condition, ailment, illness or immune response or a predisposition for said medical condition, ailment or illness, said method comprising:
- a still further aspect of the invention provides a method of diagnosing a human or animal subject for a medical condition, ailment, illness or immune response or a predisposition for said medical condition, ailment or illness, said method comprising: (i) separately screening either or both the primary and/or secondary arrays of the present invention with:
- a biological sample derived from said subject comprising a cell, tissue, or organ sample, bodily fluid sample, blood or serum sample, or a fraction, derivative or protein extract of any one or more of said samples;
- the biological sample and the biological standard are derived from the same cell-type, tissue-type, organ-type, bodily fluid-type, blood-type, or serum-type.
- the biological sample comprises blood or serum or a fraction or derivative of each thereof.
- the diagnostic methods described herein clearly extend to applications wherein the biological sample is obtained from the subject prior to screening or alternatively, wherein the is prepared array for screening with the biological sample and/or the biological standard.
- Such preparation may involve the selection of monoclonal antibodies or antibody variants that bind to proteins in a primary array which are derived from a healthy individual and from the same cell-type, tissue-type, organ-type, bodily fluid-type, blood-type, or serum-type as the biological sample.
- a still further aspect of the invention extends to compositions for the therapeutic or prophylactic treatment of a human or other animal subject, said compositions comprising a suite of protein elements and/or responsive antibody elements of relevance to disease genesis and/or disease susceptibility that have been identified by screening the primary and/or secondary array of the present invention and preferably subsequently isolated, in combination with a pharmaceutically-acceptable carrier or diluent.
- the active ingredient of such compositions is a composite of the multiplicity of elements employed in the construction of the primary array, used in approximately equimolar ratio at a sufficiently-high concentration of each individual protein component to produce an antibody response to each of said protein components, preferably a humoral and/or cellular immune response to each of said protein components, in a subject to which 13 -
- composition is administered, rather than merely producing a response to a few proteins in a naturally-occurring cellular soup.
- This aspect of the invention further extends to the use of such compositions in therapeutic, diagnostic and intervention protocols or for inclusion in drug screening programmes or molecular characterisation.
- a still further aspect of the invention contemplates a method of therapeutic treatment of a human or animal subject for a medical condition, ailment, or illness comprising administering the composition supra to said subject for a time and under conditions sufficient for the symptoms of said medical condition, ailment, or illness to abate.
- a still further aspect of the invention contemplates a method of prophylactic treatment of a human or animal subject for a predisposition to a medical condition, ailment, or illness comprising administering the composition supra to said subject for a time and under conditions sufficient for an antibody response or protective immune response to occur.
- Figure 1 is a schematic representation showing the dissection in parallel of the monoclonal elements from within a polyclonal response in animal-based or phage- based expression systems.
- Figure 2 is a graphical representation showing the populations of B cells within an immunised mouse (bell-shaped curve) and normally sampled during hybridoma production and screening procedures prior to serial dilution and usually in conjunction with a few mice.
- approximately 400-600 viable hybridoma post-fusion events ( — ) are obtained, as opposed to that population ( )of pre-clonally-diluted hybridomas which is intended to be sampled per mouse, comprising at least one order - 14 -
- the efficiency of the antibody production procedure is therefore dependent upon the efficiency of the immunisation protocol with respect to the antigens being presented to the mouse immune system, or the immune system of any other host, as the case may be.
- Figure 3-1 is a diagrammatic representation showing microtitre plate-sized PVDF or nitrocellulose membranes capable of containing 30,000 distinct antigens for a high throughput western blotting of IPTG-induced expression libraries or for gridding colonies for growth and induction on IPTG-containing agar, with ready-accessible current gridding technologies to array using sets of pins that dip one or more times into expression libraries.
- the former is preferred to reduce the likelihood of colony smearing or merging during growth.
- Figure 3-11 is a diagrammatic representation showing stacks of the microtitre plate- sized PVDF or nitrocellulose membranes presented in Figure 3-1.
- Figure 4 is a schematic representation showing the total potential antigenic diversity encoded by a given genome (rectangle) and the proportion of said total potential antigenic diversity exposed on a primary array in conjunction with an expression library for that genome (oval). Conformational epitopes and antigens that are simply not cloned as a result of poor gene library quality, or genes that encode toxic proteins or which are unstable when cloned, lie outside the oval field.
- Figure 5 is a schematic representation showing the dissection in parallel of the monoclonal elements from within a polyclonal response for the screening of viable B cell hybridoma culture supernatants, based upon a predetermined knowledge of monoclonal antibody specificity.
- Figure 6 is a copy of a photographic representation of a dot blot of mouse bleeds against 12 immunising antigens that were used as a mixture to immunise mice. - 15
- Antigens were dotted onto nitrocellulose and screened with antisera obtained from each of four immunised mice.
- the Figure indicates four panels of 12 antigens, each panel corresponding to a different mouse antisera used in the screening. Numbers at the side of each panel indicate the number of the mouse from which antisera were obtained. Numbers within each panel indicate the antigen (Table 1)
- One aspect of the present invention provides an array for use in determining the protein profile of a cell, tissue, organ or organism or a biological sample derived therefrom, comprising:
- primary and secondary arrays provides a unique signal or tag on a single or group of related antigens in the primary array with one or more monoclonal antibodies and/or antibody variants and/or derivatives in the secondary array. This is achieved by high- throughput western blotting of hybridoma supernatants and/or individual lots of one or more phage-derived antibody elements against whole primary arrays, at a rate of about 50,000 to 100,000 per annum.
- the primary array is a soup of elements that contains a significant portion of the antigenic diversity encoded by a genome. It is highly reproducible and may be used in the large-scale western blotting of patient sera, in the analysis of protein:protein interactions, in the synthesis of large quantities of recombinant proteins of interest for further study, the development of diagnostic kits and reagents and in the screening of cellular immune responses with respect to individual elements of the array.
- the soup of elements in the primary array may also be derived from an induced expression library and used as an inoculum for immunisation/vaccination.
- the primary array may be used, for example, as a subunit vaccine with Th1 and Th2 and Th1/Th2 inducing elements, based upon the statistical likelihood of occurrence of a particular array element within a whole gene expression library.
- the secondary high density protein array and positive responders to cellular extracts, represent an inexhaustible supply of antibodies of interest which may be stored frozen and provide for the affinity-enrichment from cellular extracts of native proteins of interest for further characterisation, molecular diagnosis and the production of therapeutic magic bullets.
- the secondary array described herein is not able to be constructed in a high-throughput and effective manner without access to high-density primary arrays.
- arrays shall be taken to mean any ordered arrangement of a plurality of specified integers, including both linear and non-linear arrangements of a plurality of proteins and/or monoclonal antibodies or antibody variants or derivatives.
- array includes any elements derived from a complex mixture of proteins resolved by 1 -dimensional or 2-dimensional gel electrophoresis or chromatography, or peptide or protein expression libraries and the ordered arrangement of proteins or antibodies, antibody variants or derivatives on a grid, such as in microtitre wells or on a membrane support or silicon chip or on a grid comprising a plurality of polymeric pins.
- the primary and secondary arrays described herein comprise a plurality of proteins and antibodies, respectively, ordered in two dimensions, wherein the number of proteins or antibodies in each dimension is at least one. Accordingly, the primary and/or secondary array may comprise a single row of a plurality of proteins or antibodies.
- plural is meant any large number. Single proteins or antibodies do not constitute an "array” within the context of the present invention, because these do not qualify as a plurality of proteins or antibodies. Nor do single proteins comprise a multiplicity of protein elements designed to emulate a significant portion of the antigenic diversity of a cell, tissue, organ or whole organism.
- Each protein within the primary array is assigned a coordinate (Xn,Yn) to facilitate its identification therein.
- each monoclonal antibody or antibody variant or derivative in the secondary array is assigned a coordinate (Xn.Yn) to facilitate its identification therein.
- the nature of specific antigenic relationships between proteins and antibodies may be determined by analysing the binding of monoclonal antibody or antibody variant or derivative having specific coordinates in the secondary array to - 18
- proteins having specific coordinates in the primary array and thereby allow the determination of the abundance of given antigens based on prior knowledge of the antigen-binding capacity of one or more antibodies prior to their exposure to native and/or denatured proteins from cellular extracts.
- Figure 1 demonstrates the functionality of parallel screening of a plurality of antigens and mixed abundances and binding affinities to a multiplicity of monoclonal antibodies. As will be apparent to those skilled in the art, such parallel screening represents a significant advance over the prior art, which relies exclusively upon 2-dimensional gel electrophoresis to analyse proteome expression.
- n will vary depending upon the complexity of the proteome of the species from which the array of the present invention is derived. For example, in the case of a bacterium, there may be approximately 4,000 to 20,000 gene products, compared to 100,000 to 300,000 gene products for the human proteome.
- n is not essential to the present invention, because technologies may be utilised which provide for a significant portion of the antigenic diversity of any proteome to be represented in array primary array and, as a consequence, for the appropriate number of monoclonal antibodies and/or antibody variants or derivatives to be contained in the secondary array of the present invention.
- the primary array comprises redundant proteins to increase the likelihood 19
- the secondary array comprises redundant monoclonal antibodies and/or antibody variants and/or antibody derivatives.
- redundancy increases the number of sites tagged per molecule, to provide increased experimental confidence in the response obtained and, in the case of comparative assays, such as between a biological sample and a biological standard or between a diseased and healthy individual or group of individuals, the potential for a differential response to be detected.
- redundancy in the secondary array increases the potential to uniquely identify a given antigen by a combination of one or more antibodies, to produce a unique signature for every sample or set of samples assayed.
- protein profile is meant the range of expressed proteins of a cell, tissue, organ or organism or any derivative fraction thereof, as distinct from the “proteome” of the cell, which includes the potential protein output of a cell, tissue, organ or organism.
- the present invention is particularly useful for the determination of specific proteins that are expressed in cell, tissues or organs of an organism, or whole organisms perse, at any time or developmental stage or disease state, by virtue of the provision of both an array that is representative of the proteome of the cell, tissue, organ or whole organism and a method of screening said array.
- the primary array is a redundant array that is representative of a significant portion of the antigenic diversity of a cell, tissue, organ or organism, as exemplified by Figure 2. - 20
- protein shall be taken to mean any molecule that comprises a sequence of naturally-occurring and/or glycosylated and/or acylated and/or non- naturally-occurring amino acid residues, including a fusion protein or a fusion molecule that comprises non-amino acid substituents, such as carbohydrates and/or lipids.
- polypeptides also included within the scope of the definition of a "protein” are recombinant polypeptides, chemically-synthesized peptides (such as produced by Fmoc chemistry), and peptides, oligopeptides and polypeptides derived from a full-length protein by chemical or enzymatic cleavage, using reagents such as CNBr, trypsin, or chymotrypsin, amongst others.
- reagents such as CNBr, trypsin, or chymotrypsin
- recombinant polypeptide and “recombinant peptide” as used herein shall be taken to refer to any polypeptide or peptide molecules that is produced in a virus particle or a cell by the expression therein of a genetic sequence encoding said polypeptide under the control of a suitable promoter, wherein a genetic manipulation has been performed in order to achieve said expression.
- Genetic manipulations which may be used in this context will be known to those skilled in the art and include, but are not limited to nucleic acid isolation, restriction endonuclease digestion, exonuclease digestion, end-filling using Klenow fragment of E.
- nucleic acid hybridisations may also be utilised in the preparation of recombinant polypeptide and peptide molecules, in confirming the identity of a nucleic acid molecule encoding said molecules and a genetic construct comprising the nucleic acid molecule.
- the proteins of the primary array are 21
- amino acids are particularly useful for coupling the protein to another peptide or polypeptide, to a large carrier protein or to a solid support.
- Amino acids that are useful for these purposes include but are not limited to tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof.
- Additional protein modification techniques can be used, e.g., NH 2 -acetylation or COOH-terminal amidation, to provide additional means for coupling the proteins to another polypeptide, protein, or peptide molecule, or a support. Procedures for coupling proteins to each other, or to carrier proteins or solid supports, are well known in the art. Proteins containing the above- mentioned extra amino acid residues at either the carboxyl- or amino-termini and either uncoupled or coupled to a carrier or solid support, are consequently within the scope of proteins used to produce the primary arrays of the present invention.
- the proteins of the primary array are modified by the addition of one or more reporter molecules, which are bound thereto to facilitate detection of said proteins.
- reporter molecule shall be taken to refer to any molecule which is capable of producing an identifiable or detectable result.
- Preferred reporter molecules include, but are not limited to, radiochemicals, fluorescent compounds such as rhodamine, biotin, DIG, immunologically-interactive peptides such as FLAG peptides, poly-His amino acid sequence or poly-Lys amino acid sequence or other known amino acid string and functional enzymes, such as alkaline phosphatase, horseradish peroxidase, the Escherichia coli ⁇ -galactosidase enzyme, the firefly luciferase protein (Ow ef al, 1986; Thompson et al, 1991) and the green fluorescent protein (Prasher ef al, 1992; Chalfie et al, 1994; Inouye and Tsuji, 1994; Cormack et al, 1996; Haas ef al, 1996; see also GenBank Accession No. U55762). 22
- the primary array will be a high-density protein array.
- high density protein array means an array comprising at least about 10 proteins per cm 2 , more preferably at least about 50 proteins per cm 2 , even more preferably at least about 100 protein per cm 2 , and still even more preferably at least about 500 proteins per cm 2 .
- approximately 30,000 protein elements can be transferred at a pitch of approximately 450 microns to solid supports that are positioned within or attached to the lid of a standard 96-well microtitre plate, at a rate of 90 per day, achieved by two gridding robots (KB Engineering, UK; Figure 3). Higher densities can be achieved but the daily production rate is reduced.
- hundreds, preferably thousands, more preferably tens-of-thousands and even more preferably hundreds-of-thousands of proteins may be contained in the primary array.
- Those skilled in the art will recognise the advantages in terms of time-saving and cost-effectiveness, of providing the primary array in as high a density as is technically- feasible.
- antigenic diversity is meant different epitopes (i.e. immunogenic regions) of proteins or peptide fragments or regions thereof.
- significant portion of the antigenic diversity shall be taken to mean a significant fraction or part of the different linear and conformational epitopes associated with all proteins contained within a cell, tissue, organ or whole organism that is representative of its proteome.
- the primary array of proteins comprises a plurality of B cell epitopes.
- the primary array comprises a plurality of B cell epitopes and T cell epitopes.
- Primary arrays comprising both B cell epitopes and T cell epitopes permit the identification of protein biological samples that are involved in both cellular and humeral immune responses, for example by using T cell mitogen assays and ⁇ -IFN assays (eg stimulation of nitric oxide production).
- B cell epitopes and T cell epitopes are determined by several intrinsic factors, including for example, accessibility, hydrophilicity and mobility of proteins and/or protein regions/domains.
- B cell epitopes are regions of proteins that are cross-reactive with immunoglobulin molecules on B cells. Regions of proteins need to be accessible to immunoglobulin molecules on B cells in order to be immunogenic. Preferably, B cell epitopes will be located on the surface of proteins. Protein folding tends to bury hydrophobic residues in the interior of the folded protein while leaving hydrophilic residues exposed to the aqueous environment. Therefore, hydrophilic regions of proteins are more likely to be immunogenic than hydrophobic ones. Segmental mobility of the polypeptide backbone in regions of proteins is associated with immunogenicity (Tainer et al., 1984), because more mobile regions of proteins are more likely to interact with Immunoglobulin molecules on B cells than less mobile regions.
- Predictive techniques include searches for either hydrophilic or mobile regions of proteins, for example by 24 -
- a non-predictive approach be taken, however a predictive approach can be employed in conjunction with toxic genes and genes corresponding to unstable clones in gene- libraries, for increased coverage or closure in fully-sequenced genomes and/or to increase the redundancy in primary arrays and/or to maximise the number of B cell epitopes represented in the primary array.
- animals are immunised with one or more proteins or whole cell, tissue or organ extracts, in order to generate polyclonal antiserum. This antiserum is then tested for binding to randomly-generated synthetic or recombinant peptides.
- peptide libraries containing peptides of various lengths such as pentapeptides or hexapeptides (Geysen et al., 1987; Scott and Smith, 1990; Houghton et al., 1991 ; Lam et al., 1991 ; du Plessis et al., 1994).
- These peptides can either contain overlapping sequences from the protein or random sequences.
- Synthetic or recombinant peptides that bind to the polyclonal serum represent B cell epitopes that are useful in the primary array of the present invention.
- T cell epitopes also comprise specific regions of proteins, determined primarily by the specificity of the major histocompatibility complex (MHC) molecules (Schaeffer et al.,
- Each different MHC molecule binds to a peptide with a different motif, of approximately 8 to 9 amino acid residues, in the case of MHC class I molecules, or 13 to 14 amino acid residues, in the case of MHC class II molecules (Falk et al., 1991; Rudensky et al., 1991).
- MHC class I molecules binds to a peptide with a different motif, of approximately 8 to 9 amino acid residues, in the case of MHC class I molecules, or 13 to 14 amino acid residues, in the case of MHC class II molecules (Falk et al., 1991; Rudensky et al., 1991).
- peptides oligopeptide, polypeptide or proteins selected from the list comprising synthetic peptides such as synthetic random amino acid sequences and/or synthetic peptides comprising signature amino acid sequences, and/or recombinant molecules such as those produced in peptide libraries and/or induced peptide expression libraries, such as by protein expression from within one or more cloned gene libraries, and/or naturally-occurring proteins, such as proteins derived from two-dimensional gel electrophoretograms of biological samples and/or naturally-occurring proteins produced in rapidly dividing cells such as by replication- induced protein synthesis (RIPS), amongst others.
- synthetic peptides such as synthetic random amino acid sequences and/or synthetic peptides comprising signature amino acid sequences
- recombinant molecules such as those produced in peptide libraries and/or induced peptide expression libraries, such as by protein expression from within one or more cloned gene libraries
- naturally-occurring proteins such as proteins derived from two-dimensional gel electro
- proteins used to construct the primary array are produced so as to be compatible with the MHC class I and MHC class II molecules, to improve their immunogenicity.
- Synthetic peptides are non-naturally-occurring proteins as hereinbefore defined that are produced by any method known to those skilled in the art, such as by using Fmoc chemistry.
- Synthetic peptides or recombinant peptides generated from expression of cloned nucleic acid inserts are particularly preferred, because they are more time-conserving - 26
- Preferred synthetic peptides for use in constructing the primary array will comprise at least 5 amino acid residues in length, more preferably about 5 amino acids to about 80 amino acids in length, and more preferably about 10 amino acid residues to about 20 amino acid residues in length.
- the final length of peptide used will depend upon the cost of synthesis and immunogenicity of the shorter peptides. However, where the use of shorter peptides is highly desirable (eg.
- these may incorporate one or more N-terminal or C-terminal basic amino acid residues, in particular one or more lysine residues, to facilitate the formation of a peptide cluster of increased immunogenicity relative to the base peptide, or alternatively, to facilitate conjugation of the peptide to a hapten or other molecule.
- the synthetic peptides will comprise random amino acid sequences, to maximise the number of sequences represented.
- sequences may not be derived from the proteome of a cell, tissue, organ or organism, they may be useful for "describing" the proteome of the cell, tissue, organ or organism, by virtue of their immunological cross-reactivity to elements of the proteome.
- Random synthetic peptides are particularly useful as analogues of nonlinear (i.e.
- Random synthetic peptides that cross-react with a B cell and/or T cell epitope of the proteome may be identified by screening libraries of such sequences with polyclonal antibodies that bind to T cell and/or B cell epitopes of the proteome, said antibodies being generated, for example, by immunizing animals such as mice or rabbits with whole protein derived from rapidly-dividing cells and/or - 27
- the antibodies used to identify the random synthetic peptides may be monoclonal or recombinant antibodies, in crude or purified form. Those random synthetic peptides that comprise T cell epitopes may be identified by their ability to stimulate T cell cytotoxic or proliferative responses in vitro.
- the immunogenic and/or antigenic activity of proteins in the proteome may be increased by their conjugation to haptens, such as KLH, or to substituents that make them less sensitive to enzymatic degradation, and which are more selective.
- haptens such as KLH
- substituents that make them less sensitive to enzymatic degradation and which are more selective.
- the proline analogue, 2-aminocyclopentane carboxylic acid ( ⁇ AC 5 c) has been shown to increase the immunogenic activity of a naturally-occurring polypeptide more than 20 times (Mierke et al, 1990; Portoghese ef al, 1990; Goodman et al, 1987).
- the synthetic peptides will comprise signature amino acid sequences.
- signature amino acid sequence is meant an amino acid sequence that is representative of a particular class of protein or enzyme (eg. a phosphatase, kinase, helix-loop-helix protein, zinc-finger(lll) protein, etc) or an amino acid sequence that is representative of a particular region or domain of a protein (eg. DNA-binding domain, activation region of a transcription factor, ATP-binding site, etc).
- signature amino acid sequences may be determined empirically, using nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography to identify specific secondary, tertiary or quaternary structures in proteins that comprise substrate-binding domains, B cell epitopes, T cell epitopes, or other functional regions or domains, and then determining the specific amino acid sequences which form such structures.
- NMR nuclear magnetic resonance spectroscopy
- X-ray crystallography to identify specific secondary, tertiary or quaternary structures in proteins that comprise substrate-binding domains, B cell epitopes, T cell epitopes, or other functional regions or domains
- B cell epitopes may be identified by NMR and/or X-ray crystallographic analysis of antigen:antibody complexes, wherein X-ray techniques require the complex to be crystallized, whereas NMR allows analysis of the complex in a liquid state.
- a second generation synthetic peptide that mimics a B cell or T cell epitope of a protein may be produced by screening synthetic random peptides for antibody- binding activity or T cell proliferative responses, respectively.
- peptides comprising signature amino acid sequences do not include hydrophobic regions, regions that are likely to be internal to the protein, or regions comprising proline-rich and/or cysteine-rich amino acid sequences.
- Peptides comprising signature amino acid sequences may also be produced as recombinant peptides.
- Recombinant proteins such as those derived from the expression of cDNA inserts, are capable of emulating many conformational epitopes, however not all as in the native protein.
- Recombinant peptides including those comprising signature amino acid sequences, will preferably be derived from isolated nucleic acid molecules that comprise nucleotide sequences derived from one or more Expressed Sequence Tags (ESTs), amplification products, including PCR products or isothermic amplification products, cDNA sequences, exon regions of isolated genes or random sheared genomic DNA.
- ESTs Expressed Sequence Tags
- amplification products including PCR products or isothermic amplification products
- cDNA sequences exon regions of isolated genes or random sheared genomic DNA.
- Recombinant peptides may be produced by standard means known to those skilled in the art, the only requirement being that the nucleotide sequence of the nucleic acid molecule encoding the peptide is presented in an expressible format.
- expressible format shall be taken to indicate that a protein-encoding region of a nucleic acid molecule placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in a cellular or cell-free system.
- nucleic acid molecule placed in operable connection with a promoter comprises a nucleotide sequence encoding the proteome protein of interest (i.e. the protein to be included in the primary array), in addition to one or more nucleotide 30
- intein shall be taken to mean an excisable protein element or any number of protein purification/enrichment tags linked to a cleavage site for recovery of all or a part of a fusion protein from an affinity column and encoded in or near a vector cloning site.
- the inclusion of inteins in the recombinant peptide of the present invention is to facilitate the excision of the recombinant proteome protein of interest or a fragment thereof from any other recombinant amino acid sequences that have been co-expressed therewith. More preferably, the nucleotide sequence encoding the recombinant proteome protein of interest or fragment thereof is flanked by nucleotide sequences encoding inteins. This ensures that the recombinant proteome protein of interest or fragment thereof is obtainable as a protein of predictable length following its expression and subsequent purification.
- Preferred nucleotide sequences encoding highly immunogenic proteins include those encoding proteins that facilitate Th1 ;Th2-type and/or Th1/Th2-type responses obtained by cholera toxin, interleukins, or interferon molecules, amongst others, the purpose of including such sequences is to facilitate the primary and secondary antibody responses obtained by immunising animals with the proteome protein of interest, in applications where specific antibodies against said protein are required, and particularly where the ensemble or a multiplicity of the constituent elements used in the construction of primary arrays are employed to engender an immune response.
- promoter includes the transcnptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
- promoter is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule which encodes a protein.
- Preferred promoters can contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression of the said nucleic acid molecule.
- Placing a nucleic acid molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence.
- Promoters are generally, but not necessarily, positioned 5' (upstream) to the genes that they control.
- the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
- the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
- the prerequisite for producing intact polypeptides in bacteria such as E. coli is the use of a strong promoter with an effective ribosome binding site.
- Typical promoters suitable for expression in bacterial cells such as E. coli include, but are not limited to, the lacz promoter, temperature-sensitive ⁇ L or ⁇ R promoters, T7 promoter or the IPTG- inducible tac promoter.
- a number of other vector systems for expressing the nucleic acid molecule of the invention in E. coli are well-known in the art and are described for example in Ausubel ef al (1987) or Sambrook ef al (1989). Numerous plasmids with suitable promoter sequences for expression in bacteria and efficient ribosome - 32
- Typical promoters suitable for expression in viruses of eukaryotic cells and eukaryotic cells include the SV40 late promoter, SV40 early promoter and cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter amongst others.
- Means for introducing the isolated nucleic acid molecule or a genetic construct comprising same into a cell for expression are well-known to those skilled in the art. The technique used for a given organism depends on the known successful techniques. Means for introducing recombinant DNA into animal cells includes microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., Wl, USA) amongst others.
- proteins for inclusion in the primary array are produced in the form of a recombinant peptide library.
- peptide library shall be taken to mean the inducible protein products of any set of diverse nucleotide sequences encoding a set of diverse amino acid sequences, wherein said nucleotide sequences are preferably contained within a suitable plasmid, cosmid, bacteriophage or virus vector molecule which is suitable for maintenance and/or replication in a cellular host.
- peptide library includes a random peptide library, in which the extent of diversity between the amino acid sequences or nucleotide sequences is numerous, and a limited peptide library in which there is a lesser degree of diversity between said sequences.
- peptide library further encompasses random amino acid sequences derived from a cellular source, wherein the amino acid sequences are encoded by a second nucleotide 33
- a "peptide library” further includes cells, virus particles and bacteriophage particles comprising the individual amino acid sequences or nucleotide sequences of the diverse set.
- the peptide library is produced as a display library, wherein the protein for inclusion in the primary array is expressed on the surface of a filamentous phage (i.e. a phage display library) which has been introduced into a suitable bacterial host cell, or on polysomes or other suitable display system.
- a filamentous phage i.e. a phage display library
- Phage display has rapidly matured as a widespread technology for harnessing the chemical and structural diversity of peptide libraries.
- phage display technology it is possible to express one or more recombinant proteins from peptide libraries on the surface of a filamentous bacteriophage, such as M13, as biologically-active or immunologically interactive molecules (for reviews, see Burritt ef al., 1996; Burton, 1995; Clackson and Wells, 1994; Cortese et al., 1995; Daniels et al., 1995; Lowman, 1997; O'Neil and Hoess, 1995; and Sternberg and Hoess, 1995). Display of peptides and proteins on the surface of bacteriophage. Alternative systems for the display of proteins from combinatorial libraries have also been described (Mattheakis et a/., 1994; Winter, 1994).
- Preferred peptide libraries according to this embodiment of the invention are induced peptide expression libraries.
- preferred peptide libraries are "representative libraries", comprising a set of amino acid sequences or nucleotide sequences encoding same, which includes all possible combinations of nucleotide sequences encoding a specified length of peptide. 34 -
- the peptide library comprises cells, virus particles or bacteriophage particles comprising a diverse set of nucleotide sequences which encode the diverse set of amino acid sequences (i.e. the proteome proteins), placed operably under the control of a promoter sequence which is capable of directing the expression of said nucleotide sequence in said cell, virus particle or bacteriophage particle.
- the nucleotide sequences which encode the proteome proteins are derived from randomly-synthesized oligonucleotides, and more preferably, from randomly-sheared genomic DNA.
- peptide libraries such as those wherein protein expression is from one or more cloned gene libraries, including phage display libraries, may be generated by "shotgun" cloning of pools of said nucleotide sequences into a suitable plasmid vector or other expression vector, such as the ⁇ ZAP series of vectors (Stratagene, Inc., CA., USA), thereby facilitating the screening of large numbers of peptide-or polypeptide encoding clones in yeast and/or bacterial cells.
- a suitable plasmid vector or other expression vector such as the ⁇ ZAP series of vectors (Stratagene, Inc., CA., USA)
- the nucleotide sequence encoding the proteome protein in an expressible format further includes a sequence targeting the protein to the surface of a cell in which it is expressed (eg. a trans-membrane domain), or alternatively, in the case of virus-mediated or bacteriophage-mediated expression, the proteome protein may be produced as a fusion protein with the coat protein of the virus or bacteriophage, to facilitate immunoreactivity of the expressed proteins using intact cells, virus particles or bacteriophage.
- the primary array is constructed using naturally-occurring proteins.
- naturally-occurring protein is meant a protein derived from a virus particle, bacteriophage, cell, tissue, organ or organism, including a partially-purified or purified product. 35 -
- array elements derived from protein spots are electro-eluted from two-dimensional gel electrophoretograms of naturally-occurring protein mixtures.
- two-dimensional gel electrophoretograms of naturally-occurring protein mixtures are transferred to nitrocellulose or PVDF membranes and individual proteins are excised from the membranes as spots. More preferably, the proteins are transferred with the assistance of robotics ( Figure 3), onto grids as high-density arrays.
- Proteins initially resolved by the use of "proteomic contigs” may also be used in the construction of the primary array, each of which is preferably transferred, more preferably with the assistance of robotics ( Figure 3), onto grids, as high-density arrays.
- a "proteomic contig” is a window of protein expression. Proteomic contigs produced as described herein are collated to represent the entire proteome of an organism or a significant fraction thereof.
- the proteins of the primary array are preferably bound to a solid support or matrix to facilitate screening with monoclonal antibodies and/or antibody variants and/or derivatives.
- the solid support is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
- the solid supports may be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane (eg.
- binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the protein molecule to the solid support.
- the secondary array comprises monoclonal antibodies and/or antibody variants and/or antibody derivatives, optionally bound to a solid porous or non-porous support or matrix and optionally labelled with one or more reporter molecules to facilitate their quantitation and/or detection.
- the secondary array is an array of monoclonal antibodies and/or hybridomas producing same and/or antibody variants and/or antibody derivatives, that bind to multiple antigenic determinants in the primary array. More preferably, the monoclonal antibodies and/or hybridomas producing same and/or antibody variants and/or antibody derivatives each bind to one or more antigenic determinants on the primary array.
- the pattern of antigen recognition of such molecules may be determined from data on the coordinates of the proteins in the primary array and of the coordinates of the monoclonal antibodies in the secondary array, and used to elaborate on the nature of shared epitopes in the proteins of the primary array.
- the secondary array will be a high-density antibody array.
- high density antibody array means an array comprising at least about 10 monoclonal antibodies and/or antibody variants and/or antibody derivatives per cm 2 , more preferably at least about 50 monoclonal antibodies and/or antibody variants and/or antibody derivatives per cm 2 , even more preferably at least about 100 monoclonal antibodies and/or antibody variants and/or antibody derivatives per cm 2 , and still even more preferably at least about 500 monoclonal antibodies and/or 37 -
- the secondary array comprises about 100,000 monoclonal antibodies and/or antibody variants and/or antibody derivatives per cm 2 .
- hundreds, preferably thousands, more preferably tens-of-thousands and even more preferably hundreds-of-thousands of antibodies may be contained in the secondary array.
- the term "monoclonal antibody” shall be taken to include both an immunoglobulin molecule produced by a hybridoma, and a hybridoma producing one or more immunoglobulin molecules, irrespective of whether or not the specificity of said immunoglobulin molecules is the same.
- Monoclonal antibodies are obtainable by immunisation with an appropriate gene product, epitope, peptide, or fragment of a gene product, or alternatively, a mixture comprising a plurality of same.
- Monoclonal antibodies may be selected from naturally occurring polyclonal antibodies raised against one or more epitopes, peptides, or protein fragments, derived from recombinant or naturally-occurring sources.
- antibody producing cells can be harvested from an animal immunised with the protein and fused with myeloma cells by standard somatic cell fusion procedures, thus immortalizing these cells and yielding hybridoma cells.
- somatic cell fusion procedures Such techniques are well known in the art (see, for example Douillard and Hoffman, 1981).
- hybridoma technique originally developed by Kohler and Milstein (1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., 1983), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole ef al., 1985), and screening of combinatorial antibody libraries (Huse et al., 1989).
- Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the peptide and monoclonal antibodies isolated therefrom. 38
- antibody variant shall be taken to refer to any synthetic antibodies, recombinant antibodies or antibody hybrids, such as but not limited to, a single-chain antibody molecule produced by phage-display of immunoglobulin light and/or heavy 5 chain variable and/or constant regions, or other immunointeractive molecule capable of binding to an antigen in an immunoassay format that is known to those skilled in the art.
- Recombinant antibodies comprising immunoglobulin light and heavy chain variable 10 and constant regions, expressed on the surface of a bacteriophage or virus particle, such as in a phage display library, are preferred.
- Optimization of high-affinity antibodies by phage display of combinatorial antibody libraries is a robust mimic of immune selection for natural antibody diversity, as an alternative to traditional hybridoma and immunisation technologies.
- cell sub-population-specific monoclonal antibodies may also be derived from synthetic phage antibody libraries (de Kruif ef al., 1995). Techniques for the production of recombinant antibodies in phage display libraries are well-known in the art (Crosby and Schorr, 1995; Winter et al., 1994).
- bacteriophage-based vector systems are available for expressing immunoglobulins in phage display libraries, for example the ⁇ lmmunoZAP vector series including ⁇ lmmunoZAP L , ⁇ lmmunoZAP H, ⁇ lmmunoZAP H/L and ⁇ SurfZAP, for expressing immunoglobulin Fab fragments, light chains and heavy chains on the surface of a filamentous phage (Hogrefe ef a/., 1993; Mullinax ef a/., 1990; Shopes,
- the primary arrays of protein can be constructed as described supra, and used to dissect in parallel the monoclonal elements from within a polyclonal response in animal-based or phage-based systems, concomitantly with determining the specificity of the antibodies produced from said animal-based or phage-based systems.
- biopanning of phage-based antibody libraries may be performed, essentially as shown in Figure 1.
- antibody derivative refers to any modified antibody molecule that is capable of binding to an antigen in an immunoassay format that is known to those skilled in the art, such as a fragment of an antibody (Fab fragment), or an antibody molecule that is modified by the addition of one or more amino acids or other molecules to facilitate coupling the antibodies to another peptide or polypeptide, to a large carrier protein or to a solid support (eg. the amino acids tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof, NH 2 -acetyl groups or COOH-terminal amido groups, amongst others).
- Fab fragment fragment of an antibody
- an antibody molecule that is modified by the addition of one or more amino acids or other molecules to facilitate coupling the antibodies to another peptide or polypeptide, to a large carrier protein or to a solid support (eg. the amino acids tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof, NH 2
- the antibody molecules or antibody variants or derivatives of the secondary array are modified by the addition of one or more reporter molecules, which are bound thereto to facilitate their detection.
- the present embodiment it is possible to detect one or more antigenic determinants in a labelled mixture of antigens, by a variety of means including fluorescence, chemiluminescence, isotopic determination, enzymatic labelling, amongst others.
- Preferred reporter molecules for binding covalently or non-covalently to antibodies include, but are not limited to, radiochemicals, fluorescent compounds such as rhodamine, biotin, DIG, immunologically-interactive peptides such as FLAG peptides, poly-His or poly-Lys amino acid sequences or other known amino acid string, protein 40
- lectins eg. phytohemagglutinin A
- secondary antibodies and functional enzymes such as alkaline phosphatase, horseradish peroxidase, the Escherichia coli ⁇ - galactosidase enzyme, the firefly luciferase protein (Ow et al, 1986; Thompson ef al, 1991) and the green fluorescent protein (Prasher ef al, 1992; Chalfie ef al, 1994; Inouye and Tsuji, 1994; Cormack et al, 1996; Haas ef al, 1996; see also GenBank Accession No. U55762).
- reporter molecules comprising immunologically-interactive peptides or functional enzymes
- these are preferably linked to recombinant antibodies by producing fusion proteins comprising both the immunoglobulin and reporter molecule moieties, wherein the corresponding gene regions encoding said moieties are spliced together in-frame and the recombinant nucleic acid molecule is expressed in a suitable cellular, viral or bacteriophage expression system.
- Techniques for producing such fusions molecules are well-known in the art.
- the monoclonal antibodies and/or antibody variants and/or antibody derivatives used in the construction of the secondary array may be derived from monoclonal or polyclonal antibody sources.
- nucleotide sequences encoding immunoglobulin variable light and heavy chains may be derived from hybridomas or lymphocytes derived from animals that have been previously immunised with antigen, as for the production of monoclonal or polyclonal antibodies.
- Conventional immunisation strategies may be utilised to facilitate polyclonal or monoclonal antibody production, or the production of antibody derivatives, including the immunisation of animals, in particular rabbits or mice, with: (i) whole cells, tissues, organs or whole organisms or sub-cellular fractions or lysates, extracts or other derivatives thereof; and/or (ii) DNA derived from biological samples corresponding to the whole or fractions of cells, tissues, or organisms and lysates thereof; (i.e. DNA vaccination); and/or (iii) proteins derived from a biological sample that has been subjected to a 41
- a "soup" comprising the elements used to produce the primary array or a multiplicity or a part thereof.
- mice will be required to produce sufficient monoclonal antibodies against complex protein mixtures to represent at least one epitope per protein, for 25-75% of the total proteome.
- the remaining monoclonal antibodies or antibody variants or derivatives will be derived from antibodies against single proteins.
- additional mice may be immunised.
- more complex proteomes such as the human proteome, more than 12-20 mice, and in particular, up to 100 mice, more preferably up to about 500 42
- mice may be used to produce sufficient monoclonal antibodies against complex protein mixtures to represent 25-75% of the total proteome.
- primary arrays are exposed to ever-increasing numbers of antibody derivatives by high-throughput western blotting, so as to increase redundancy within the primary array and thereby better emulate the antigenic diversity seen in the proteome of the cell, tissue, organ or whole organism.
- the monoclonal or polyclonal antibodies for use in constructing the secondary array a good immunogenic response is required to ensure that said antibodies have the ability to bind specifically (i.e. uniquely) or non-specifically (i.e. not necessarily uniquely), to individual epitopes in proteins of the primary array.
- the specific or non-specific binding of the anfibodies to one or more proteins of the primary array provides combined information or binding attributes in the determination of the protein profile of a cell, tissue, organ, or organism.
- the progress of immunization can be monitored by detection of antibody titres in plasma or serum.
- Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.
- antisera can be obtained and, if desired, IgG molecules corresponding to the polyclonal antibodies can be isolated from the antisera.
- the immunogenically effective amounts of the immunising protein must be determined empirically. Factors to be considered include the immunogenicity of the native peptide, whether or not the peptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier, the route of administration for the composition, (i.e. intravenous, intramuscular, subcutaneous, efc), and the number of immunizing doses to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation. - 43
- mice In preparing the anfibodies, it is particularly preferred to immunize mice with mixtures of antigens, more preferably at least about 10 to about 100 antigens simultaneously.
- antigens associated with improved immunogenicity in such antigen mixtures are expressed at approximately equimolar ratios.
- most low-abundance antigens in these mixtures which correspond to poorly-expressed proteins in naturally-occurring mixtures of cellular antigens, are better-expressed ( As will be known to those skilled in the art, because of their low intra-cellular abundance, these antigens are often poorly immunogenic.)
- Any convenfional immunoassay format may be used to screen the primary arrays (eg. ELISA, RIA, western blot and the like).
- Those hybridomas that produce antibodies binding to one or more antigens can be employed to generate a unique and/or desirable recognition for use in screening biological material of interest.
- the screening of hybridomas or other anfibody-producing cells or virus or phage particles is completed when monoclonal antibodies or antibody variants or derivatives have been identified that bind to a useful portion of the primary array or uniquely to facilitate identification of any protein derived from a multiplicity of open reading frames, or uniquely identify all protein isoforms encoded or produced by a genome. As the number of elements increases, then so too does the utility of the secondary array.
- the monoclonal antibodies or antibody variants or derivatives are ordered into a secondary array.
- the secondary array may be constructed methodically, adding batches of anfibodies to pre-existing arrays, to form new, more complete and/or more redundant secondary arrays as each new or monoclonal antibody or antibody variant or derivative is detected. .44.
- the monoclonal anfibodies or antibody variants or derivatives of the secondary array prefferably be coupled to a solid support.
- the nature of the solid support used and the mode of binding monoclonal antibodies or antibody variants or derivatives of the secondary array to the solid support is the same as for the proteins of the primary array, as described supra.
- the antibodies of the secondary arrays are also useful for the affinity-purification or enrichment of proteins to which it binds, either as a single purification step or alternatively, in combination with other known procedures for the purification of proteins.
- proteins may be purified based upon their size, charge or ability to bind specifically to anfibodies against the intact polypeptide, using one or a combination of gel electrophoresis, size-exclusion chromatography, reverse phase chromatography, ion-exchange chromatography or affinity chromatography. After purification/enrichment, a reduced number of protein bands or spots should be detectable with one-dimensional or two-dimensional gel electrophoresis.
- Methods for the affinity purification of proteins using antibodies are well-known to those skilled in the art.
- the present invention will provide for the purification/enrichment of native proteins from a cellular soup, to facilitate further protein characterisation by any number of analytical and/or structural methodologies well-known to those skilled in the art.
- the N-terminal or total sequencing of the isolated protein may also be carried out. This provides the possibility to compare the sequence of the protein with known proteins in databases and may also be arrived at by analysis of cDNA clones encoding elements used in the construction of primary arrays (eg. by analysis of cells expressing induced proteins in solution or on membranes overlaid on agar and lysis of said cells to allow access to the antigens therein).
- a second aspect of the present invention clearly encompasses the above-mentioned - 45
- Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay as applied to the present invention, an unlabelled protein of the primary array (the primary array per se), immobilised on a solid substrate, is brought into contact with the monoclonal antibody or antibody variant or antibody derivative to be tested.
- a second antibody specific to the first-mentioned antibody labelled with one or more reporter molecules capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of another complex of antigen- antibody-labelled antibody. Any unreacted material is washed away, and binding of the monoclonal antibody or anfibody variant or antibody derivative to the protein of the primary array is determined by observation of a signal produced by the reporter molecule.
- Variations on the forward assay include a simultaneous assay, in which both the protein of the monoclonal antibody or antibody variant or antibody derivative and the labelled antibody are added simultaneously to the bound protein.
- reporter molecules are enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes), bioluminescent and chemiluminescent molecules.
- an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
- glutaraldehyde or periodate As will be readily recognised, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include 47
- the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.
- suitable enzymes include alkaline phosphatase and peroxidase.
- fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
- the enzyme-labelled antibody is added to the first protein- antibody complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the tertiary antigen- antibody-antibody complex.
- the substrate will react with the enzyme linked to the second antibody, producing a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
- fluorescent compounds such as fluorescein and rhodamine
- fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
- the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
- enzyme immunoassays EIA
- the fluorescent labelled antibody is allowed to bind to the first protein-antibody complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
- Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method.
- the results may either be qualitative, by simple observation of the visible signal produced by the reporter molecule, or may be quantitated by comparing with a control sample containing known amounts of protein.
- the solid surface to which the protein or monoclonal antibody or antibody variant or anfibody derivative is bound is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
- the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
- the binding processes are well-known in the art and generally consist of cross-linking, covalent binding or physical adsorption.
- a further embodiment of the present invention provides for the correction of concentration-dependent variation in signal intensity at the stage of screening the primary array with antibodies to construct the secondary array.
- this is achieved by labelling the proteins of said array with one or more reporter molecules, for example prior to their attachment to the solid support or matrix, and determining the signal obtained, which is representative of the amount of protein bound to said solid support or matrix.
- the signal obtained from the reporter molecule and the signal obtained in the immunoassay are compared.
- the signal intensity obtained for the immunoassay is then adjusted to account for the concentration of protein, as determined by the signal intensity obtained using the reporter molecule.
- Suitable reporter molecules for detecting the amount of protein bound to the solid support include immunogenic peptides or protein regions, such as but not limited to a FLAG peptide, poly-His or poly-Lys amino acid sequence or other known amino acid string.
- immunogenic peptides or protein regions such as but not limited to a FLAG peptide, poly-His or poly-Lys amino acid sequence or other known amino acid string.
- the use of such amino acid sequences is particularly preferred wherein the protein of the primary array is a recombinant or synthetic protein, because the immunogenic peptide or protein region can be produced as an in-frame fusion with said recombinant or synthetic protein.
- the preferred mode of detection of immunogenic peptide reporter molecules is the use of an anfibody molecule that specifically binds to such sequences, in which case a standard immunoassay format may be employed.
- the secondary array is used to screen complex mixtures of cellular proteins, such as those labelled fluorescently, isotopically, enzymatically, or by other means known to those skilled in the art, to determine the level of binding specificity and/or cross- reactivity to antigens on the solid support, in a standard immunoassay format.
- Such immunoassays can be conducted simultaneously with an immunoassay to screen the primary array with the monoclonal antibody or antibody variant or derivative, as the case may be.
- two distinct second antibodies are used, which bind specifically to either the anfibody against the reporter molecule or to the antibody being screened, but not to both, and that each second anfibody is labelled with a different reporter molecule, to facilitate separation of the signals obtained.
- any other suitable reporter molecule described herein may be used to label the proteins of the primary array, subject to the proviso that said reporter molecule is distinct from that used to detect the binding of the monoclonal antibody or antibody variant or antibody derivative to said protein.
- the screening of the primary array may also be normalised to reduce or remove variation arising from differential association and dissociation constants of antibodies - 50
- the array of the present invention is particularly useful for the purpose of determining the protein profile of a biological sample derived from an organism which is the same as, or closely-related to, the organism in respect of which said array is representative of the proteome.
- an array of the present invenfion prepared with a specific organism in mind is representative of a significant portion of the antigenic diversity of that organism and, as a consequence, may be used to ascertain any subset of that organism's proteome, such as the protein profile of a particular cell, tissue, or organ, either in a healthy or diseased state, or following exposure of a particular cell, tissue, organ or whole organism to an external stimulus, such as a chemical compound or a biological agent (eg. a fungal or viral pathogen).
- the present invenfion has particular utility in the medical and pharmacological fields, for determining alterations in protein profiles of patients suffering from particular diseases or infections or alternatively, to monitor changes in protein profiles during treatment with drugs.
- the secondary array described supra is screened with said biological sample, to determine those proteins therein which bind to one or more monoclonal antibodies and/or antibody variants or derivatives in said secondary array in a uniquely-definable manner.
- the biological sample may be any lysate or extract of a cell, tissue, organ, or whole organism, or a derivative fraction thereof, the only requirements being that said biological sample is in a form suitable for conducting an immunoassay (eg. correct pH and buffer composifion) and further, that said bioioigcal sample is derived from an - 51
- Particularly preferred biological samples include bodily fluids such as tears, saliva, urine, semen or other exudate, blood, serum, or tissue biopsies comprising skin, heart, liver, kidney, lung, intestine, colon, or foetal cells, or protein extracts derived from of any one or more of said samples.
- Other biological samples are also encompassed by the present invention, which is generally applicable and not to be limited by the source of biological sample being assayed, or the purpose for which the subject assay is being performed.
- Screening of the secondary array may be performed using a standard immunoassay format, similar to that described supra.
- the direct binding assay is particularly preferred, wherein the biological sample is brought into contact with the secondary array for a time and under conditions sufficient for an antibody-protein complex to occur and the complex is then washed to remove unbound components.
- the proteins in the biological sample are labelled with a suitable reporter molecule to facilitate their detection following binding.
- the reporter molecule may be any reporter molecule that can be routinely conjugated to proteins in complex protein mixtures or alternatively, the proteins may be labelled after the unbound molecules have been washed away.
- fluorescent compounds such as fluorescein and rhodamine, or radioisotopes are preferred.
- NMR nuclear magnetic resonance
- circular dichroism changes in electric current
- X-ray diffraction or laser technology may be used to detect binding of the proteins to the antibody molecules.
- interesting proteins that are detected may be purified from biological samples by standard procedures and optionally, the amino acid sequences of such purified proteins and/or post-translational modifications of the isolated proteins may be determined. Additionally, the amino acid sequence of one or more proteins identified in the primary array may be determined if it is not already known.
- the nucleotide sequence of DNA encoding said proteins may be determined using standard procedures.
- the isolated DNA may then be used to produce recombinant proteins that were employed in the primary array, again using standard procedures known to those skilled in the art.
- the screening of the secondary array with a biological sample can be susceptible to variation from differential amounts of antibody bound to the solid support in the immunoassay or from differential association and dissociation constants of anfibodies for their specific antigens in the binding reaction.
- a further embodiment of the present invenfion provides for the correction of concentration-dependent variation in signal intensity at the stage of screening the screening the secondary array with the biological sample. - 53 -
- said normalisation is achieved by screening the secondary array with one or more reporter molecules that bind to all monoclonal anfibodies and/or antibody variants or derivatives in said array, comparing the signal obtained using said reporter molecule to the signal obtained in the immunoassay using the biological sample, and finally, adjusting the signal intensity obtained using the biological sample to account for the amount of anfibody bound as determined by the signal intensity obtained using the reporter molecule.
- Molecules that bind generally to antibodies are well-known in the art, and include protein A, lectins and secondary anfibodies. Wherein second antibodies are used to detect both the amount of antibody bound to the support and the binding of the proteins in the biological extract to the secondary array anfibodies, it is preferred that each second antibody is labelled with a different reporter molecule, to facilitate separation of the signals obtained.
- the screening of the secondary array may be normalised by averaging the signal intensity obtained for binding of the same protein to several monoclonal antibodies and/or anfibody variants or derivatives that bind to different epitopes on the same protein, which binding would become apparent from "keying" the coordinates of the antibodies bound by the sample back to the primary array.
- a variation of the screening strategy described supra may be applied to the comparison of biological samples, for example in applications wherein it is important to ascertain the differential expression of specific proteins of the proteome.
- a significant application of the present invention is the comparison of healthy and diseased tissues from humans and other animals and organisms, or to monitor the effects of any chemicals administered to humans or other organisms, such as during treatment of a specific disease to determine the efficacy of treatment.
- invention provides a significant contribution to the characterisation of multigenic traits and other multiprotein phenomena that are aetiologically-associated with disease, such as the major diseases of humans or other organisms (eg. one or more of the following: cancer, genetically-inherited disorders, autoimmune disorders, infections and environmental tissue damage heart disease, amongst others).
- diseases of humans or other organisms eg. one or more of the following: cancer, genetically-inherited disorders, autoimmune disorders, infections and environmental tissue damage heart disease, amongst others.
- the inventive method may be applied to the diagnosis or determination of an immune response in a human or animal subject, wherein the biological sample assayed using the array comprises blood or serum or a fraction or derivative of each thereof.
- Such information may be particularly important in the casse of diagnosing autoimmune diseases or alternatively, past or present infections of host animals by pathogens.
- the same organism-specific secondary array is screened separately as described herein, with two or more biological samples and the signals obtained using each of said biological samples is compared, to determine those proteins which are differentially expressed.
- the determined proteins may be further characterised for their association with the specific phenotype being analysed.
- the phenotype being assayed may be any multiprotein- based phenotype, however the present invention can, of course, be applied to the analysis of single-protein-based phenotypes/phenomena in biological systems.
- a further aspect of the present invention provides a method of diagnosing a human or animal subject for a medical condifion, ailment, illness or immune response or a predisposition for said medical condifion, ailment or illness, said method comprising screening the array of the present invenfion with a biological sample derived from said subject and comparing the protein profile thereof with the protein profile of a biological standard derived from a healthy individual, wherein differences between the biological sample the biological 55
- the biological standard may pre-determined or alternatively, screening of the array with the test sample and the standard may be conducted simultaneously.
- the biological standard is derived from the same cell-type, tissue-type, organ-type, bodily fluid-type, blood-type, or serum-type as the biological sample being tested.
- the present invention clearly encompasses the steps of obtaining the biological sample prior to screening and/or preparing the array for screening with the biological sample and/or the biological standard.
- the array can be prepared for screening, by selecting a sub-set of monoclonal antibodies or anfibody variants that bind to proteins in the primary array derived from a healthy individual and from the same cell-type, tissue-type, organ-type, bodily fluid-type, blood-type, or serum-type as the biological sample. In this manner, any differences in binding that are observed when the sub-set is screened with the biological test sample will become evident immediately.
- compositions for the therapeutic or prophylactic treatment of a human or other animal subject comprising a suite of protein elements and/or responsive antibody elements of relevance to disease genesis and/or disease susceptibility that have been identified by screening the primary and/or secondary array of the present invenfion and preferably subsequently isolated, in combination with a pharmaceutically-acceptable carrier or diluent.
- the active ingredient of such composifions is a composite of the multiplicity of elements employed in the construction of the primary array, used in approximately equimolar ratio at a sufficiently-high concentration of each individual protein component to produce an anfibody response to each of said protein components.
- the immune response is a humoral and/or cellular immune response to each of the protein components forming the active ingredient, in a subject to which said composition is administered, rather than merely a response to a few proteins in a naturally-occurring cellular soup.
- pharmaceutically acceptable carrier and/or diluent includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
- the use of such media and agents for pharmaceufical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- composifions are used for prophylaxis, it is particularly preferred that they produce an antibody response or protective immune response when administered.
- the composition elicits or stimulates an immune response when administered to a subject in need of treatment.
- the immune response is a primary or secondary anfibody response.
- the immune response against the protein or antibody is a protective immune response.
- compositions are intended for therapeutic treatment, they are administered to the subject for a time and under conditions sufficient for the symptoms of said medical condifion, ailment, or illness to abate. 57
- compositions for eliciting immune responses may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules).
- the immunogenic proteins contained therein may be required to be coated in a material to protect them from the action of enzymes, acids and other natural conditions which otherwise might inactivate said immunogen.
- the immunogenic protein may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes.
- an “adjuvant” as used herein is to be taken in its broadest sense and includes any immune-stimulating compound, including a cytokine molecule , resorcinol, a non-ionic surfactant such as polyoxyethylene oleyl ether or n-hexadecyl polyethylene ether, amongst others.
- Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
- Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.
- compositions of the present invention may also be administered parenterally or intraperitoneally.
- Dispersions of the immunogenic protein component can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparafions can contain a preservative to prevent the growth of microorganisms.
- the forms suitable for injectable use include sterile aqueous solufions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must 58
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
- Sterile injectable solufions are prepared by incorporating the immunogenic protein component in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by heat-sterilisation, irradiation or other suitable sterilisafion means.
- dispersions are prepared by incorporating the various sterilised active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
- compositions When the compositions are suitably protected as described above, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or enclosed in hard or soft shell gelatin capsule, or compressed into tablets, or - 59
- compositions may be admixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
- composifions and preparations should contain at least 1% by weight of active compound.
- the percentage of the composifions and preparafions may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
- the amount of immunogenic protein in such vaccine preparafions is such that effective immunisation will be achieved with between one and five doses of said vaccine.
- the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
- a binder such as gum tragacanth, acacia, corn starch or gelatin
- excipients such as dicalcium phosphate
- a disintegrating agent such as corn starch, potato starch, alginic acid and the like
- a lubricant such as magnesium stearate
- a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint
- tablets, pills, or capsules may be coated with shellac, sugar or both.
- a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- the compositions may be incorporated into sustained-release preparations and formulations.
- Dosage unit form refers to physically discrete units suited as unitary dosages for the human or animal 60 -
- each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable carrier.
- Figure 5 is a schematic representation showing the dissection in parallel of the monoclonal elements from within a polyclonal response, for the screening of viable B cell hybridoma culture supernatants.
- the scheme presented therein which depends upon a predetermined knowledge of monoclonal anfibody specificity, is also applicable to phage-based antibody screening and biopanning as described herein.
- This example demonstrates this principle of the present invention, in particular the potential for the invenfion to generate in parallel a good immune response sufficient to allow the generation of multiple useful specific anfibodies and to provide for the screening of at least one order of magnitude of antigens in parallel, thereby facilitating a higher throughput and reduced time and costs for the generation of monoclonal anfibodies and costs associated with screening antigens and/or anfibodies in parallel, than is provided by the prior art.
- the examples described herein which produce antibodies in a high throughput setting, demonstrate the general applicability of the invenfion to the screening of the output of the human genome.
- the reduced time and cost associated with the present invention is greatly facilitated by the combination of steps described herein, that test initially for specificity and subsequently, for cross-reactivity or degree of specificity, the latter often being the greatest cost element in monoclonal antibody production by methods which involve either experimental animals or phage-derived molecular procedures.
- hybridomas or phage-derived antibody derivatives are not currently screened against antigens that are representative of significant - 62 -
- mice An immunogen comprising 50 ⁇ g of a mixture of 12 antigens in equimolar concentration was used to immunise four (4) mice (2 x Balb/C, 2 CBA), in the presence of adjuvant. Approximately one month later, mice were boosted with 25 ⁇ g antigen mixture plus adjuvant. Nine days later, mice were bled to obtain serum. The composifion of the immunisation mixture used is listed in Table 1.
- Bleeds obtained from the immunised mice were screened by ELISA against each of the 12 individual antigens contained in immunisation mixture. Briefly, ELISA plates were coated with individual antigens at levels of 10 ⁇ g/ml and 1 ⁇ g/ml. The antigens were diluted in a standard carbonate coating buffer at pH 9.6 prior to coating and 50 ⁇ l of antigen solution at the appropriate concentration was added to each microtitre well, and incubated overnight at 4°C. The microtitre wells were then blocked for 1 hour at ambient temperature with 0.2%(w/v) casein in phosphate-buffered saline solution (PBS).
- PBS phosphate-buffered saline solution
- Table 3 emulate a more complex primary array that is employed to screen a far greater number of hybridoma supernatants and/or phage-derived anfibody elements than exemplified herein.
- the only difference between the subject matter exemplified herein and such a large-scale screening is that the latter depends heavily upon automation and robotics to handle the larger volume of samples, and a greater reliance upon computing to handle data-processing.
- a dotblot of mouse bleeds against these 12 immunising antigens was also performed. Anfigens were dotted onto nitrocellulose, such that 10 ug/ of each antigen in 10 ug/ml was spotted onto 5 sheets of nitrocellulose in a grid pattern. The nitrocellulose sheets were dried for 2 hours at ambient temperature and blocked for 1 hour at ambient temperature, using 5% (w/v) Blotto. Mouse antisera obtained from each of immunised mice (mouse #1 -mouse #4), and a non-immunised control (i.e. mouse #5) were diluted 1 :400 in Blotto and incubated with the nitrocellulose sheets for 1 hour at room temperature.
- the data obtained were normalised by subtracting the background (i.e. mouse #5) for each antigen in the mixture from the signal obtained for that antigen, in each test mouse.
- the normalised data are presented in Table 5.
- the primary array of 12 anfigens described in the preceding Example was screened with monoclonal anfibodies prepared from the two highest-responding mice that had been immunised with a mixture of these 12 anfigens (i.e. mouse #3 and mouse #4 15 described in Example 1).
- mice were boosted intraperitoneally with 15 ⁇ g of the 12-antigen mixture in PBS, three days prior to fusion.
- spleens were removed from mouse #3 and mouse #4 (CBA), and a single cell suspension made 0 from each. Spleen cells from each mouse were then centrifuged, together with 10 8 SP2/O myeloma cells. Fusions were performed by adding 1ml of PEG (1600) to each spleen cell/myeloma cell pellet, over a period of 1 min duration with gentle mixing, and the cell suspensions were then slowly diluted with DME, over a period of 10 minutes.
- Fused cells were centrifuged gently and resuspended in Hybridoma Serum Free Medium (HSFM), containing 10% (v/v) Foetal Calf Serum (FCS), 10% (v/v) IL-6- conditioned medium and HAT medium, (i.e. H/10/10/HAT medium). Each fusion was plated out over approximately 12 microtitre plates (i.e.12 hybridoma plates), using
- ELISA plates (12 x 96 microtitre plates) were each coated with 50 ⁇ l of each of the 12 individual anfigens from the immunisation mixture referred to in the preceding Example (i.e. the primary array). Each antigen was diluted to 10 ⁇ g/ml in carbonate buffer, pH 9.6, prior to coating. Plates were incubated for 20 hours at 4°C. The layout of the primary array is presented in Table 6.
- the primary array plates were blocked for 1 hour at room temperature with 50 ⁇ l 0.2% casein/PBS per well. Plates were washed three times using PBS/tween solution and hybridoma supernatants, diluted 1 :2 with PBS, added to each well.
- each hybridoma was used to screen each antigen, by transferring 50 ⁇ l of each of diluted fusion cell culture supernatant from hybridoma plates 1-12 to antigen-coated plates 1-72 (i.e. supernatants from hybridoma plate 1 were transferred to antigen- coated plates 1 , 7, 13, 19, 25, 31 ; supernatants from hybridoma plate 2 were transferred to antigen-coated plates 2, 8, 14, 20, 26, 32; supernatants from hybridoma plate 3 were transferred to antigen-coated plates 3, 9, 15, 21 , 27, 33; supernatants 76 -
- Plates 1-72 were incubated for 1 hour at 73°C in humidified box, washed three times using PBS/tween, and 50 ⁇ l of a 1 :4,000 dilufion of anti-mouse serum conjugated to HRP (Silenus product code DAH),in TBS/BSA/tween solution, was added to each well. The plates were then incubated for 1 hour at 37°C, washed three times as before in PBS/tween and 50 ⁇ l of OPD substrate was added to each well. P were incubated for 30 minutes at room temperature, and the absorbance at 492 nm, with a reference wavelength of 620 nm, was determined.
- Results are presented in Table 7. Positive samples were retained for storage by freezing. Positive samples were defined as those having an absorbance value at 492 nm of 0.2 units, or greater. For all anfigens except for antigen #1 (Aprotinin), it was possible to select positive hybridoma samples for storage producing absorbance values at 492 nm of 0.3 units or greater, based upon the higher antibody titre produced by those hybridomas. Only a few low-level reactor hybridomas were obtained to antigen #1 , which were selected and are tested in a confirmatory screen.
- Hybridomas selected for . storage were transferred to 1ml microtitre wells in H/10/10/HAT medium. As cells became confluent, they were expanded to 6 ml wells - 77
- Hybridoma cell culture supernatants from approximately 100 positive hybridomas obtained in the primary screening were re-screened by ELISA using similar conditions as states supra, except that supernatants were used undiluted and each supernatant was screened against each antigen.
- Plate #13 column 1 ; antigen 5; column 3, antigen 8; column 5, antigen 7; column 7, antigen 12; column 9, antigen 11 ; and column 11 , antigen 2.
- Plate #14 column 1; antigen 6; column 3, antigen 9; column 5, antigen 10; column 7, antigen 1 ; column 9, antigen 4; and column 11 , antigen 3.
- the layout of hybridoma supernatants on each of plates 1-12 is shown in Table 8.
- the layout of hybridoma supernatants on each of plates 13 and 14 is shown in Table 9.
- Table 10 Data presented in Table 10 are representative of the secondary array of the invention and exemplify the ability of the present invenfion to produce a fingerprint or unique tag to each of the 12 target anfigens tested, through the recognition patterns of one or more antibodies at greater than two standard deviations above a background response (i.e. a positive response).
- the data provided herein indicate the general applicability 78 -
Abstract
Description
Claims
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CA (1) | CA2319828A1 (en) |
WO (1) | WO1999039210A1 (en) |
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EP1051624A4 (en) | 2002-05-02 |
EP1051624A1 (en) | 2000-11-15 |
CA2319828A1 (en) | 1999-08-05 |
AU2259799A (en) | 1999-08-16 |
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