AU2012209004A1 - Improved immunoassay methods - Google Patents

Abstract

Abstract The invention relates to a method of detecting a disease state or disease susceptibility in a mammalian subject which 5 comprises detecting an antibody in a test sample comprising a bodily fluid from said mammalian subject wherein said antibody is a biological marker of a disease state or disease susceptibility the method comprising: (a) contacting said test sample with a plurality of different amounts of an antigen 0 specific for said antibody, (b) detecting the amount of specific binding between said antibody and said antigen, (c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each amount of antigen used in step (a) and (d) determining the presence or absence of 5 said disease state or disease susceptibility based upon the amount of specific binding between said antibody and said antigen at each different antigen concentration used. 'fSenameI

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: ONCIMMUNE LIMITED Actual Inventor(s): Andrea Murray, Tony Barnes, John Forsyth Russell Robertson, Caroline Chapman Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: IMPROVED IMMUNOASSAY METHODS Our Ref : 949533 POF Code: 45882/483680 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1la T)' present application is a divisional application from Australian patent application number 2006250923. The entire disclosure of which is incorporated herein by reference. 5 IMPROVED XIMNOASSAY MUTRODS Field of the invention - The invention generally relates to the field of diagnostic or prognosti, assays and in particular relates to optimisation of assays for the detection of antibodies in.a.,. sample .comprising patient bodily fluid, wherein such antibodies are used as biological markers of a disease state or disease susceptibility.

Background to the invention Many diagnostic, prognostic and/or monitoring assays rely on detection of a biological marker of a particular disease state or disease susceptibility. Such biological markers are commonly proteins or polypeptides that are characteristic of a particular disease or associated with susceptibility to disease. In recent years it has become apparent that antibodies, and in particular autoantibodies, can also. serve as biological markers of disease or disease susceptibility. Autoantibodies are naturally occurring antibodies directed to an antigen which an individual's immune system recognises as foreign even though that antigen actually originated in the individual. They may be present in the circulation as circulating free autoantibodies or in the form of circulating immune complexes consisting of autoantibodies bound to their target marker protein. Differences between a wild type protein expressed by "normal" cells and an altered form of the protein produced by a diseased cell or during a disease process may, in some instances, lead to the altered protein being recognised by an individual' s immune system as "non-self" and thus eliciting an immune response in that individual. This may be a humoral (i.e B cell-mediated) immune response leading to the production of autoantibodies immunologically specific to the altered protein. <filename> WO 2006/126008 PCT/GB20061001944 -2 WO 99/58978 describes methods for use in the detection/diagnosis of cancer which are based on evaluating the immune response of an individual to two or more distinct tumour markers. These methods generally involve contacting 5 a sample of bodily fluid taken.from the individual with 'a panel of..two or more. distinct tumour marker antigens, each derived from a separate tumour marker protein, and detecting the formation of complexes of the tumour marker antigens bound to circulating autoantibodies immunologically specific 10 for the tumour marker proteins. The presence of such circulating autoantibodies is taken as an indication -Qf the presence of cancer. Assays which measure the immune response of the 15 individual .to the presence of tumour marker protein in terms of autoantibody production provide an alternative to the direct measurement or detection of .tumour marker protein in bodily fluids. Such assays essentially constitute .indirect detection of the presence of tumour marker protein. Because 20 of the nature of the immune response, it is likely. that autoantibodies can be elicited by a very small amount of circulating tumour marker protein and indirect methods. which rely on detecting the immune response to tumour markers will consequently be more sensitive than methods for the direct 25 measurement of tumour markers in bodily fluids. Assay methods based on the detection of autoantibodies may therefore be of particular value early in the disease process and possibly also in relation to screening of asymptomatic patients, for example in screening to identify 30 individuals "at risk" of developing disease amongst a population of asymptomatic individuals, to identify individuals who have developed a disease amongst a population of asymptomatic individuals. In addition the method based on the detection of autoantibodies may be of 35 particular value early in the disease process and possibly also may be used to identify individuals -who have developed a disease amongst a population of symptomatic individuals. Furthermore, they may be useful for earlier detection of WO 2006/126008 PCT/GB2006/001944 -3 recurrent disease. The assay methods may also be a value in selecting or monitoring therapies for a disease. Antibodies and autoantibodies can also serve as 5 biological markers of other disease states or disease ' susceptibilities, of which rheumatoid.arthritis, systemic lupus erythematous (SLE), primarY biliary cirrhosis (PBC), autoimmune thyroiditis (eg Hashimoto' s thyroiditis), autoimmune gastritis (eg pernicious anaemia), autoimmune 10 adrenalitis (eg Addison's disease), autoimmune .. hypoparathyriodism, autoimmune diabetes (eg Type 1 diabetes), myasthenia gravis are but examples. The present inventors have recognized that when assays 15 based on detection.of antibodies are used diagnostically or prognostically to assess the disease state, disease progression.or disease .susceptibility. of an individual within a population, difficulties can arise in devising a standardised assay methodology appropriate for the whole 20 population of subjects to be screened because the absolute amounts of antibody present vary dramatically from. individual to individual. This can produce a high incidence. of false negative results, for example amongst individuals having a low amount of antibody. Similarly there is a 25 difficulty in scoring true positive results because the variation in.absolute amounts of antibody from individual to individual means that it is difficult to set a threshold for a positive assay result that is appropriate for all individuals within the population screened. 30 The present inventors have now determined that the performance and more specifically the clinical utility and reliability of assays based on detection of antibodies, particularly autoantibodies, as biological markers of 35 disease can be improved dramatically by inclusion of an antigen titration step. By testing 'the sample suspected of containing antibodies against a series of different amounts of antigen and constructing a titration curve it is possible to reliably identify true positive screening results -4 independently of the absolute amount of antibody present in the sample. Such an approach is contrary to prior art methods which titrate antigen merely to construct a calibration curve to allow identification of the most appropriate antigen 5 concentration to be used for detecting antibodies in actual, patient samples. In these methods only a single point measurement is proposed for actual diagnosis. Thus, these methods will not allow for variation in amounts of the antibody to be detected from individual to individual resulting in the 10 incidence of false positives and false negatives as discussed. The present inventors have found that the antigen titration method of the invention discussed herein has greater specificity and sensitivity than measuring autoantibody reactivity at a single antigen concentration or methods in 15 which the serum sample is titrated rather than the antigen. The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general 20 knowledge in Australia as at the priority date of any of the claims. Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to 25 exclude other additives, components, integers or steps. Summary of the invention According to a first aspect of the invention there is provided a method of detecting a disease state or disease 30 susceptibility in a mammalian subject which, comprises detecting an antibody in a test sample comprising a bodily fluid from said mammalian subject wherein said antibody is a biological marker of a disease state or disease susceptibility, the method comprising: 35 (a) contacting said test sample with a plurality of different amounts of an antigen specific for said antibody, WlFQ.81631S816315 Spede 301107.doc - 4a (b) detecting the amount of specific binding between said antibody and said antigen, (c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each amount 5 of antigen used in step (a) and (d) determining the presence or absence of said disease state or disease susceptibility based upon the amount of specific binding between said antibody and said antigen at each different antigen concentration used. W'UFW.15315%a1315 Spode 301107.doc WO 2006/126008 PCT/GB2006/001944 -5 According to a second aspect of the invention there is provided a method of detecting an antibody in a test sample comprising a bodily fluid from a mammalian subject, wherein said antibody is directed to a foreign substance introduced 5 into said.mammalian subject, the method comprising: (a) contacting the test sample with a plurality of 'different amounts of an antigen specific for said antibody, (b) detecting the amount of specific binding between said antibody and said antigen, and 10 (c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each. amount of antigen used in step (a).

According to a third aspect of the invention there is 15 provided a method. of detecting an antibody. in a test sample comprising a bodily fluid from a mammalian subject wherein said antibody is a biological markere of a disease state or disease susceptibility, the method comprising: (a) contacting the test sample with a plurality of different 20 amounts of an antigen specific for.said antibody, (b) detecting the amount of specific binding between said antibody and said antigen, and (c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each 25 amount of antigen used in step (a). In all aspects of the invention the mammalian subject is preferably a human. 30 In all aspects of the invention the method is preferably carried out in vitro on a test sample comprising a bodily fluid obtained or prepared from the mammalian subject. 35 A particular feature of the invention in all its aspects is that the judgement as to whether the relevant antibody is or is not present in the test sample is based upon the amount of specific binding observed at each different antigen concentration tested, in other words the.

WO 2006/126008 PCT/GB2006/001944 -6 collective values, rather than just a reading at a single concentration. Thus, the determination of the presence or absence of disease state or disease susceptibility or antibodies to a foreign substance in a patient sample can 5 follow based directly on these collective values. Preferably, the judgement is made on the basis of the showing of a generally S-shaped'or sigmoidal curve when the.. amount of specific binding is plotted against the amount of antigen. As will be apparent from the Examples herein the 10 inventors have observed that the antigen titration methods of the invention have higher specificity and sensitivity than methods of diagnosis or detection based upon a single. reading and reduce incidence of false positive and false negative determinations. 15 Brief description of the drawings Figure 1: Measurement of autoantibodies in serum using .an antigen titration curve. Cancer patient serum 17766(C) 20 binds strongly.to the test antigen with a characteristic sigmoidal curve (-+-) but does not bind to the negative control, VOL (--a-) . In comparison, serum from a normal individual, 18052(N) does not bind to the test antigen (-*-) or to the negative control (--+-). 25 Figure 2: Comparison of p53 autoantibody levels in normal individuals and patients with primary breast cancer (PBC) as measured using the antigen titration assay. Autoantibody levels are expressed as the 0D 650 due to binding to the test 30 antigen (p53) minus that due to binding to the negative control. Normal cut-off (----- -) was calculated as the mean plus 2 standard deviations of the normal population. Figure 3 shows a comparison of p53 and NY-ESO autoantibody 35 levels in normal individuals and patients with lung cancer as measured using the antigen titration assay.. Autoantibody. levels are expressed as the OD 650 due to binding to the test antigen (p53 or NY-ESO) minus that due to binding to the negative control. Normal cut-off s (-----) were calculated WO 2006/126008 PCT/GB2006/001944 -7 as the mean plus 2 standard deviations of the normal population. Figure 4 shows a titration curve for detection of 5 autoantibodies. against p53 and.NY-ESO in a sample of ascites fluid taken from a patient with breast. cancer. This patient was tested but found not to produce autoantibodies against c-myc. 10 Figure 5 shows a titration-curve for detection of autoantibodies against BRCA1, BRCA2 and HER2 in a sample of serum from a patient with breast cancer (ductal carcinoma in situ). 15 Figure 6 shows a titration .curve for detection. of autoantibodies against NY-ESO in a sample of serum from a patient. with lung cancer. This patient was tested but found not to produce autoantibodies against p53 or c-myc. 20 Figure 7 shows a titration curve for detection of autoantibodies against NY-ESO and p53 in a sample. of serum from a patient with lung cancer. This patient .was tested but found not to produce autoantibodies against c-myc. 25 Figures 8(a) and 8(b) illustrate the results on two independent titration assays for autoantibodies against p 53 , c-myc and NY-ESO-1 in samples of serum from a "normal" subject (i.e. an individual with no evidence of cancer). 30 Figures 9(a) and 9(b) show the results of two independent titration assays carried out on samples of serum from a single patient with invasive breast cancer using a range of different antigens. 35 Figures 10(a) and 10(b) illustrate the results of titration assays in which samples of serum from .a clinically normal human subject were tested for the presence of autoantibodies using biotinylated antigens BRCA2, HER2, c-myc and NY-ESO-1, non-biotinylated BRCA1 and control expression products of WO 2006/126008 PCTGB2006/001944 -8 the "empty" vector VOL, which encodes the biotin tag but no. additional antigen. Figure 11 illustrates the results of auto-antibody analysis 5 from a patient with primary breast cancer using the antigen titration assay of the invention with .respect to antigens p53, c-myc and NY-ESO-1 and the' control expression product. of the "empty" vector VOL. 10 Figure 12 illustrates the results of a repetition of the ..assay shown in Figure 11 but with serum from a normal.. individual. Figure 13 illustrates further results obtained using the 15 antigen titration assay..of the invention on a..patient with. primary breast cancer but who also shows an antibody response to biotin. Figure 14 illustrates the results of experimental. comparison -20 on normal samples between an auto-antibody detection assay when the antigen is titrated in accordance with the invention and an auto-antibody .detection assay.in which.the antigen amount remains constant but the serum is titrated. 25 Figure 15 illustrates the results of an experimental comparison, on a sample from a patient with primary breast cancer, between an auto-antibody detection assay when the antigen is titrated in accordance with the invention and an auto-antibody detection assay when the antigen amount 30 remains constant but the serum is titrated. Detailed description of the invention The invention in general provides an immunoassay method 35 for detecting an antibody which serves as a biological marker for a disease state or disease susceptibility, characterised in that a sample to be tested for the presence of the antibody is tested for specific binding against different amounts of antigen specific for the antibody and a WO 2006/126008 PCT/GB2006/001944 -9 titration curve produced for antibody/antigen binding versus the amount of antigen tested. The general features of immunoassays, for example 5 ELISA, radioimmunoassays and the like, are Well. known to those skilled in the. art. (see Immunoas.say, E. Diamandis and T. Christopoulus, Academic Press,.Inc., San Diego, CA, 1996). Immunoassays for the detection of antibodies having a particular immunological specificity generally require the 10 use of a reagent (antigen) -that exhibits specific .immunological reactivity with the antibody under test... Depending on the format of the assay this antigen may be immobilised on a solid support. A sample to be tested for the presence of the antibody is brought into contact.with 15 the antigen and if antibodies of the required immunological specificity are present in the sample they will immunologically react with the.antigen. to form antibody antigen complexes which may then be detected or quantitatively measured. 20 The method of the invention is characterised in that a standardised sample to be tested for the presence.of the antibody is tested against a plurality of different amounts of antigen (also referred to herein as a titration series). 25 The sample is tested against at least two, and preferably at least three, four, five, six or seven different amounts of the antigen. Typical assays may also include a negative control which does not contain any antigen. 30 In this context the term "antigen" refers to a substance comprising at least one antigenic determinant or epitope capable of interacting specifically with the target antibody it is desired to detect or any capture agent interacting specifically with the variable region or 35 complementary determining regions of said antibody. The antigen will typically be a naturally occurring or synthetic biological macromolecule such as for example a protein or peptide, a polysaccharide or a nucleic acid and can include WO 2006/126008 PCT/GB2006/001944 - 10 antibodies or fragments thereof such as anti-idiotype antibodies. Skilled readers will appreciate that in the method of 5 the invention-the amount of antigenic determinants or ' epitopes. available for binding to the.target antibody is important for-establishing a titration series. In many assay formats the amount of antigenic determinants or epitopes available for binding is directly correlated with 10 the amount of antigen molecules present. However, in other embodiments, such as certain solid phase assay systems, the amount of exposed antigenic determinants or epitopes may not correlate directly with -the amount of antigen but may depend on other factors, such as attachment to the solid surface. 15 In these embodiments, references herein to."different amounts of antigen" in a titration series may be taken to refer to different amounts of the antigenic determinant or epitope. 20 The relative or absolute amount of .specific .binding. between antibody and antigen is determined for each different .amount ,of antigen (antigenic..determinant .or epitope) used and used to plot or calculate a curve of the (relative or absolute) amount of specific binding versus the 25 amount of antigen for each amount of antigen tested. Typical results are illustrated, by way of example only, in the accompanying Figures for detection of a number of different antibodies. The presence in the test sample of antibody reactive with the antigen used in the assay is 30 determined based upon the amount of specific binding observed at each antigen amount and is generally indicated by a generally S-shaped or sigmoidal curve. The absolute amounts of specific binding between antibody and antigen are generally not material, unless it is desired to produce a 35 quantitative measurement. For a simple yes/no determination of the presence or absence of antibodies it is sufficient only that a curve of the correct shape is produced. If there is no variation in detectable binding over the different amounts of antigen tested then this can be scored WO 2006/126008 PCT/GB20061001944 - 11 as an absence of a detectable amount of the antibody. In preferred embodiments of the invention the method is non quantitative. It can thus give a yes/no determination of presence or absence of antibody using a dimensionless 5 proportional relationship which is independent of signal' strength. A measure of the amount of antibody present in a particular sample can, if desired, be derived from the 10 results of antigen titration assays. The method of the invention is advantageous for use in clinical diagnostic, prognostic, predictive and/or monitoring assays where the absolute amounts of target 15 antibody present.can vary enormously from patient-to patient. The inventors have observed that if such assays are based on detection of antibody binding.using a.single amount/concentration of test antigen, patient..samples containing .an amount of antibody which is at the very low or 20 the very high end of the normal physiological range of amount of antibody across the population can be missed due to limitations of the assay methodology; samples with a low amount of antibody may be scored as false negative results, whereas those with very high levels of antibody may be off 25 the scale for accurate detection within the chosen assay methodology. The titration assay method of the invention is also particularly suitable for the detection of 30 antibodies/autoantibodies as biological markers of disease state or susceptibility where there is considerable patient to-patient variation in the specificity and affinity of the antibodies/autoantibodies for target antigen. Autoantibody responses by their very nature can vary significantly from 35 patient-to-patient, with variation occurring both in the absolute amounts of autoantibody present and in the specificity/affinity of the autoantibodies. The method of the invention can take account of this patient-to-patient WO 2006/126008 PCT/GB2006/001944 - 12 variation, thus enabling a standard assay format to be developed for any given antibody/autoantibody. Interactions between autoantibodies and their target 5 antigens are generally of low affinity but the strength bf binding- may vary from pa.tient-to-patiexit, as outlined above. The method of the invention is particularly suited to detection of low affinity binding, as a positive result can be inferred from the shape of the titration curve. 10 The method of the invention also provides a safeguard against day-to-day variation in the performance of immunoassays used for detection of autoantibodies/antibodies for diagnostic, prognostic and/or monitoring (disease state 15 or therapy) purposes. It is often observed that.there can be considerable day-to-day variation in signal strength when carrying out immunoassays for detection of antibodies. in samples comprising patient bodily fluids. Such variation might arise, for example, because of differences in the way 20 in which the samples were obtained and stored prior to testing. Such factors make -it difficult to score the results of clinical assays with certainty, for example on the basis of a simple threshold value of antibody/antigen binding. The present invention minimises the effects of 25 such day-to-day variation since a positive result for the presence of antibody is clearly evident from the shape of the titration curve, independent of signal strength. Still further advantages of the method of the invention 30 are that it allows dilution of the patient sample, yet still produces consistent results, and also that it will generally produce the same qualitative screening result (positive/negative) using bodily fluids from different sources in one individual (e.g. blood or serum versus 35 ascites fluid or pleural effusion), even though the absolute concentration of antibodies may be different in the different fluids'.

WO 2006/126008 PCT/GB2006/00194 - 13 The method of the invention may be carried out in any suitable format which enables contact between. a sample suspected of containing the antibody and multiple different amounts of an antigen. Conveniently, contact between the 5 sample and different amounts of the antigen may take place in separate but parallel reaction chambers such as'the wells of a microtitre plate. Varying' amounts of the antigen can-. be coated onto the wells of the microtitre plate by preparing serial dilutions from a stock of antigen across 10 the wells of the microtitre plate. The stock of antigen may .. be of known or unknown concentration. Aliquots of the .test sample may then be added to the-wells of the plate, with the volume and dilution 9f the test sample kept constant in each well. The absolute amounts of antigen added to the wells of 15 the microtitre plate may vary depending on such factors as the nature of the target antibody, the nature of the sample under test, dilution..of the test sample,.etc, as will be appreciated by those skilled in the art. Generally the amounts of antigen and the dilution of the test sample will 20 be selected so as.to produce a range of signal strength which falls within the acceptable detection range of the read-out chosen for detection.of antibody/antigen.binding.. in the method. Typical amounts and dilutions for testing of human serum samples suspected of containing anti-tumour 25 marker autoantibodies are given in the accompanying examples. Conveniently the tested amounts of antigen may . vary in the range of from 0.01p4g/ml to 10pg/ml. . As aforesaid, it is also possible to construct a titration curve starting with a single stock of antigen even 30 when the absolute concentration of antigen in the stock is unknown. Provided that a same single stock solution is used and serially diluted in the same manner, it is possible to compare the results of separatetitration assays for this antigen run on different starting test samples. 35 In a further embodiment different amounts of the antigen (antigenic determinants or epitopes) may be immobilised at discrete locations or reaction sites on a solid support. The entire support may then be brought into WO 2006/126008 PCT/GB2006/001944 - 14 contact with the test sample and binding of antibody to( antigen detected or measured separately at each of the discrete locations or-reaction sites. Suitable solid supports also include microarrays, for example arrays 5 wherein discrete sites or spots on the array comprise different amounts of. the. antigen. Microarrays can be prepared by immobilising different amounts of a particular antigen at discrete, resolvable reaction sites on the array. In other embodiments the actual amount of immobilised 10 antigen molecules may be kept substantially constant but the .. size of the sites or spots on the array varied in order to alter the amount of binding epitope available, .providing a titration series of sites or spots with different amounts of available binding epitope. In such embodiments the two 15 dimensional surface concentration.of the binding epitope(s) on the antigen is important in preparing the titration series, rather.then the absolute amount of antigen.. Techniques for the preparation and interrogation of protein/peptide microarrays are generally known in.the art. 20 It will be understood from the above discussion.that in all of the embodiments of the invention variation.in.the amount of antigen may be achieved by changing the antigen or epitope density against which the sample is tested, or by 25 maintaining antigen or epitope density but increasing the surface area over which antigen is immobilized, or both. Microarrays may be used to perform multiple assays for antibodies of different specificity on a single sample in 30 parallel. This can be done using arrays comprising multiple sets of different antigens, each set comprising a particular antigen at multiple different amounts or concentrations. The term "different antigens" encompasses antigens derived from different proteins or polypeptides (such as antigens 35 derived from unrelated proteins encoded by different genes) and also antigens which are derived from different peptide epitopes of a single protein or polypeptide. A given microarray may include exclusively sets of different antigens derived from different proteins or pqlypeptides, or WO 2006/126008 PCT/GB2006/001944 exclusively sets of different antigens derived from different peptide epitopes of a single protein or polypeptide, or a mixture of the two in any proportion. It. should be noted that each individual antigen set of 5 different-amounts or concentrations in any embodiment of the invention will generally comprise just one antigen and not mixtures thereof. As used herein the term "bodily fluid", when referring 10 to the material to be-tested for the presence of antibodies .. using the method of the invention, includes inter alia. plasma, serum whole blood, urine, sweat, lymph, faeces, cerebrospinal fluid,.ascites, pleural effusion, seminal fluid, sputum, nipple aspirate, post-operative seroma or 15 wound diainage-fluid... As aforesaid,.the methods.of the. invention are preferably carried out in vitro on a test sample comprising.bodily fluid removed from the test subject. The type of bodily fluid used may vary depending upon the identity of the antibody to be tested and the 20 clinical situation in which the assay is used. In general, it is preferred to perform the assays on samples of serum or plasma. The test sample may includefurther.components. in addition to the bodily fluid, such as for example diluents, preservatives, stabilising agents, buffers etc. 25 The term "antigen" is used herein in a broad sense to refer to any substance which exhibits specific immunological reactivity with a target antibody to be detected. Suitable antigens may include, but are not limited to, naturally 30 occurring proteins, recombinant or synthetic proteins or polypeptides, synthetic peptides, peptide mimetics, etc, also polysaccharides and nucleic acids. Specifically, where "antigen" is used herein it is intended to encompass any capture agent, whether of human origin, mammalian origin or 35 otherwise, capable of specific immunological interaction with the variable or complementary determining regions of the antibody to be detected. For example anti-idiotypic antibodies may be regarded as an antigen for this purpose as may antigens generated by phage display.

WO 2006/126008 PCT/GB2006/001944 - 16 Certain antigens may comprise or be derived from proteins or polypeptides isolated from natural sources, including but not limited to proteins or polypeptides 5 isolated from patient tissues or bodily fluids. In sucli embodiments the antigen .. may comprise substantially all of the naturally-occurring protein-, i.e. protein substantially:in the form in which it is isolated from the natural source, or it may comprise a fragment of the naturally occurring 10 protein. To be effective as an- antigen in the method of the ..invention any such "fragment" must retain immunological reactivity with the antibodies for which it will be used to test. Suitable fragments might, for example, be prepared by chemical or enzymatic cleavage of the isolated protein. 15 Depending on the precise nature of the assay in which it will.be used, the.antigen. may comprise a naturally.. occurring protein, or fragment thereof, linked to one or more further molecules which impart some desirable 20 characteristic not. naturally present in the protein. .For example., the protein or fragment may be conjugated to a revealing label, .such as for example a fluorescent .label, coloured label, luminescent label, radiolabel or heavy metal such as colloidal gold. In other embodiments the protein or 25 fragment may be expressed as a fusion protein. By way of example, fusion proteins may include a tag peptide at the N or C- terminus to assist in purification of the recombinantly expressed antigen. 30 Depending on the format of.the assay in which it is to be used the antigen may be immobilised on a solid support such as, for example, the wells of a microtitre plate, microarray beads or chips or magnetic beads. Immobilization may be effected via non-covalent adsorption or covalent 35 attachment. Any suitable attachment means may be used provided this does not adversely affect the ability of the antigen to WO 2006/126008 PCT/GB206/001944 - 17 immunologically react with the target antibody to a significant extent. The invention -is not limited to solid phase assays, but 5 also encompasses assays which, -in whole or in part, are carried out in liquid phase, for example solution phase bead assays. In one embodiment, antigens may be labelled with a 10 ligand that would facilitate immobilisation, such as biotin. .The antigen can then be diluted to a suitable titration range and then allowed to react with autoantibodies-i-n patient samples in solution. The resulting immune complexes can then be immobilised on to a solid support via a ligand 15 receptor interaction..(e.g. biotin-streptavidin) and the remainder of -the assay performed as described below.. To facilitate the production.of biotinylated antigens for use in the assay methods of the invention, .cDNAs 20 encoding a full.length antigen, a truncated version .thereof or an antigenic fragment thereof may be expressed as a fusion protein labelled with a protein or polypeptide tag to which the biotin co-factor may be attached via an enzymatic reaction. Vectors for the production of recombinant 25 biotinylated antigens are commercially available from a number of sources. - As illustrated in the accompanying examples, an additional advantage of the use of the titration curve 30 approach with biotinylated antigens is that the assay is able to distinguish between binding of the biotin component to anti-biotin antibodies and true binding of the antigen to its cognate antibody. The inventors have observed that a significant number of the human population naturally produce 35 anti-biotin antibodies which might lead to the production of false positive results in assays based on the use of biotinylated antigen.

WO 2006/126008 PCT/GB2006/001944 - 18 As aforesaid, the "immunoassay" used to detect antibodies according to the invention may be based on standard techniques known in the art, with the exception that multiple amounts of antigen are used to create a 5 titration; curve. In a most preferred embodiment the immunoassay may be an EnISA. ELISAs are generally well known in the art. In a typical -"indirect" ELISA an antigen. having specificity for the antibodies under test is immobilised on a solid surface (e.g. the wells of a standard 10 microtiter assay plate, or- the surface of a microbead or a ..microarray) and a sample comprising bodily fluid to be tested for the presence of antibodies is brought intocontact with the immobilised antigen. Any antibodies of the desired specificity present in the sample will bind to the 15 immobilised.antigen. The bound antibody/antigen. complexes. may then be detected using any suitable method. In one preferred.embodiment. a labelled secondary anti-human immunoglobulin antibody, which specifically recognises an epitope common to' one or more classes of human 20 immunoglobulins, is used to detect the antibody/antigen complexes. Typically the secondary antibody. will.be anti IgG or anti-IgM. The secondary antibody .is usually labelled with a detectable marker, typically an.enzyme marker such as, for example, peroxidase or alkaline phosphatase, 25 allowing quantitative detection by the addition of a substrate for the enzyme which generates a detectable product, for example a coloUred, chemiluminescent or fluorescent product. Other types of detectable labels known in the art may be used with equivalent effect. 30 The invention relates to a method of detecting antibodies that are biological markers of a disease state or disease susceptibility. This particular aspect of the invention preferably excludes assays designed to test for 35 antibodies produced as a result of a vaccine challenge or immunisation protocol, other than vaccination with cancer markers. Therefore, assays according to this aspect of the invention preferably do not include assays designed to test WO 2006/126008 PCT/GB20061001944 - 19 for the presence of anti-viral or anti-bacterial antibodies following vaccination/immunisation. In certain embodiments of the invention the antibody 5 may be an- autoantibody. As indicated above, the term "autoantibody" refers to a. naturally occurring antibody directed to an antigen which an' individual's immune system -.;. recognises as foreign even though that antigen actually originated in the individual. Atoantibodies include 10 antibodies directed against altered forms of naturally .. occurring proteins produced by a diseased .cell or during a disease process. The a-ltered-form of the protein originates in the individual but may be viewed by the individual's immune system as "non-self" and thus elicit an 15 immune response .in that individual in the form of autoantibodies immunologically specific to the altered protein. Such altered forms of a. protein can include, for example, mutants having altered amino acid sequence, optionally accompanied by changes in seconday, tertiary or 20 quaternary structure, truncated forms, splice variants, altered glycoforms etc. In other embodiments the autoantibody may be directed to a protein which is overexpressed in a disease state, for as a result of gene amplification or abnormal transcriptional regulation. 25 Overexpression of a protein which is not normally encountered by cells of the immune system in significant amounts can trigger an immune response leading to autoantibody production. In still further embodiments the autoantibody may be directed to a fetal form of a protein 30 which becomes expressed in a disease state. If a fetal protein which is normally expressed only in early stages of development before the immune system is functional becomes expressed in a disease state, the fetal form may be recognised by the immune system as "foreign", triggering an 35 immune response leading to autoantibody production. In one embodiment the antibody may be an autoantibody specific for a tumour marker protein, and more particularly a "cancer-asBsociated' anti-tumour autoantibody.

WO 2006/126008 PCT/GB20061001944 - 20 The term "cancer-associated" anti-tumour autoantibody refers to an autoantibody which is directed against an epitope present on forms of tumour marker proteins which'are 5 preferentially expressed in the cancer disease state. The presence. of such autoantibodies is characteristic of the cancer disease state, or of pre-disposition to cancer in asymptomatic patients. 10 In preferred applications the method of the invention .. wll be used to detect the presence of cancer-associated anti-tumour autoantibodies in- human subjects or patients., and will most preferably take the form of an in vitro immunoassay, performed on a test sample comprising a sample 15 of bodily .fluid..taken from the subject/patient. The.sample of bodily fluid may be diluted in a suitable buffer or may be treated. for long .term storage or .otherwise. prior to testing. 20 In vitro immunoassays .are non-.invasive and can be repeated as often as is thought necessary to build up a profile of autoantibody production.in a patient, either prior to the onset of disease, as in the screening of "at risk' individuals, or throughout the course of disease 25 (further discussed below in relation to preferred applications of the method). In particular, but non-limiting, embodiments the methods of the invention may comprise immunoassays to 30 (simultaneously) detect two or more types of autoantibodies, each having specificity for different epitopes on the same or related tumour marker proteins (e.g. different isoforms or variants encoded by a single gene) or for epitopes on different tumour marker proteins (meaning proteins encoded 35 by different genes). These methods will typically involve use of a panel of two or more sets of antigens, each set of antigens usually being derived from a different tumour marker protein (different in this context meaning proteins that are the products of different genes) although as noted WO 2006/126008 PCT/GB2006/001944 - 21 above a set of antigens could also involve different epitopes on the same tumour marker protein. A set of antigens refers to single antigen to be tested at different amounts/concentrations in the method of the invention. 5 These methods,-which may be hereinafter referred to as "panel .assays", utillse A panel of two. or more sets of antigens to monitor the overall'immune response of an individual to a tumour or other carcinogenic/neoplastic change. These methods thus detect a "profile" of the immune 10 response in a given individual, indicating which tumour markers elicit an -immune response resulting in autoantibody production. The-use of a panel of two or more antigens to monitor production of autoantibodies against two or more different tumour markers is generally more sensitive than 15 the detection of autoantibodies to single markers..and gives a much lower frequency of false negative results (see WO 99/58978 and WO.2004/044590, the contents of which are incorporated herein in their entirety by reference).. 20 Therefore, in a non-limiting embodiment. the invention provides a method of detecting two or more antibodies in a test sample comprising a bodily fluid from a mammalian subject wherein at least one of said antibodies is a biological marker of a disease state or disease 25 susceptibility, the method comprising: (a) contacting.the test sample with two or more sets of antigens, wherein each one of said sets of antigens is specific for one of said antibodies to be detected in the test sample and wherein each set of antigens comprises a 30 plurality of different amounts of said antigen, (b) detecting the amount of specific binding between said antibodies and said antigens, and (c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each set 35 of antigens used in step (a). In one embodiment each of said two or more antibodies will be a biological marker of a disease state or disease susceptibility, however it is within the scope of the WO 2006/126008 PCT/GB2006/001944 - 22 invention to combine a titration assay for a disease marker antigen with a titration assay for any other type of antibody, which may or may not be a disease marker, in the same test sample. Either way the.judgement.as to whether the relevant antibodies are or are not present in the test sample is based upon the amount of specific binding observed at each of the different antigen c6ndentrations in respect of each 10 different antigen in the test, in other words the collective .values for each antigen rather than a reading at a single concentration for each- antigerr.-- Thus, the determination of presence or absence of disease state or disease susceptibility or antibodies to a foreign substance in a 15 patient.sample. based.upon presence of two or more types of antibody can be based on these collective values for each antigen. Preferably, the judgement is made on..the basis of the showing of a generally S-shaped or sigmoidal curve in respect of any or all of the antigens present in the test. 20 For the avoidance of doubt, assays based on the use. of a single type of antigen to detect antibodies may be. referred to herein as "single marker assays",- whereas assays based on the use of a panel of two or more antigens are 25 referred to as "panel assays". The method of the invention may be adapted for use in the detection of autoantibodies to essentially any tumour marker protein for which a suitable antigen may be prepared, 30 as a single marker assay or as a component of a panel assay. In particular, the method may be adapted to detect/measure autoantibodies to the epidermal growth factor receptor protein EGFR (Downward et al (1984) Nature. 307: 521-527; Robertson et al. (2001Archives of Pathology and Laboratory 35 Medicine 126;177-81), the glycoprotein MUC1 (Batra, S. K. et al. (1992) Int. J. Pancreatology. 12: 271-283) and the signal transduction/cell cycle regulatory proteins Myc (Blackwood, E. M. et al. (1994) Molecular Biology of the Cell 5: 597-609), p53 (Matlashewski, G. et al. (1984) EMBO WO 2006/126008 PCT/GB2006/001944 - 23 T. 3: 3257-3262; Wolf, D. et al. (1985) Mol. Cell. Biol. 5: 1887-1893) and ras (or Rae) (Capella, G. et al. (1991) Environ Health Perspectives. 93: 125-131), and also BRCA1 (Scully, R. et al. (1997) PNAS 94:~5605-10), BRCA2 (Sharan, 5 S. K. et al. (1997) Nature. 386: 804-810), APC (Su, L. K.. et al..(19.93) Cancer Res. 53: 2728-2731; Munemitsu, S. et al. (1995) PRAS 92: 3046-50), CA125- (Nouwen, B. J. et al. (1990). Differentiation. 45: 192-8), PSA (Rosenberg, R. S. et al. (1998) Blochem Biophys Res Commun. 248: 935-939), 10 carcinoembryonic antigen CEA (Duffy, M.J. (2001) ClIn Chem,' Apr 47(4):624-30), CA19.9 (Haga, Y. et al (1989) Clin. Blochem (1989) Oct 22(5): 363-8), NY-ESO-1 (cancer/testis_ antigen; Chen, Y.-T. et al., Proc. Nat. Acad. Sci. 94: 1914 1918, 1997), PSMA (prostate specific membrane antigen; 15 Israeli, R..S. et al., Cancer Res. 53: 227-230, 1993), PSCA (prostate stem cell antigen; Reiter, R. E. et al., Proc. Nat. Acad. Sci. 95: 1735-1740, 1998) and EpCam (epithelial cellular adhesion molecule; Szala, S. et al., Proc. Nat. Acad. Sci. 87: 3542-3546, 1990), HER2 (also known as c-erbB2 20 Coussens, L. et al., Science-230: 1132-1139, 1985), CAGE (Jager D, et al., Cancer Res. 1999 Dec 15;59(24):6197-204; Mashino K, et al., Br J Cancer. 2001 Sep 1;85(5):713-20), cytokeratins (Moll R, et al., Cell. 1982 Nov;31(1):11- 2 4; Braun S, et al., N Engl J Med. 2000; 342: 525-533), 25 recoverin (Maeda A, et al., Cancer Res. 2000 Apr 1;60(7):1914-20, kallikreins (Kim H, et al., Br J Cancer 2001;84:643-650; Yousef GM, et al., Tumor Biol 2002;23:185 . 192); annexins (Hudelist G, et al., Breast Cancer Res Treat. ' 2004 Aug;86(3):281-91), a-fetoprotein (Stiller D, et al., 30 Acta Histochem Suppl. 1986;33:225-31), GRP78 (Block TM, et al., Proc Natl Acad Sci USA. 2005 Jan 18;102(3):779-84; Hsu WM, et al., Int J Cancer. 2005 Mar 1;113(6):920-7), CA125 (Norum LF, et al., Tumour Biol. 2001 Jul-Aug;22(4):223-8; Perey L, et al., Br J Cancer. 1990 Oct;62(4):668-70; Devine 35 PL, et al., Anticancer Res. 1992 May-Jun;12(3):709-1 7 ); mammoglobin-(Zehentner BK, et al., Clin Chem. 2004 Nov;50(11):2069-76; Zehentner BK, Carter D. Clin Biochem. 2004 Apr;37(4):249-57), raf (Callans LS. et al.,.Ann Surg Oncol. 1995 Jan;2(1):38-42; Pratt MA, et al., Mol Cell WO 2006/126008 PCT/GB2006/O01944 -24 Biochem. 1998 Dec;189(1-2):119-25), beta-human chorionic gonadotropin b-HCG (Ayala AR, et al., Ani J Reprod Immunol. 1983 Apr-May;3(3):149-51; Gregory JJ Jr, et al., Drugs. 1999 Apr;57(4):463-7), or 4-5 antigen (Krause P, et al., J 5 Immunol Methods. 2003 Dec;283(1-2):261-7). However, the invention is not intended to be limited to the detection of autoantibodies to these particular tumour markers. Assay methods according to the invention based on 10 detection of anti tumour-marker autoantibodies (in single .marker or panel assay form) may be -employed in a variety of diffMent clinical situations. In particular, the method may be used in the detection or diagnosis of cancer, in assessing the prognosis of a patient diagnosed with cancer, 15 in predicting.response to therapy, in.monitoring.the. progress of cancer or other neoplastic disease in a patient, in detecting early.neoplastic or.early carcinogenic.change. in an asymptomatic human. subject, in screening a population. of asymptomatic human.subjects in order either to identify 20 those subjects who.are at increased risk.of developing cancer or to diagnose the presence of cancer, in predicting the response of a cancer patient to anti-cancer.treatment (e.g. vaccination, anti-growth factor or signal transduction therapies, radiotherapy, endocrine therapy, human antibody 25 therapy, chemotherapy), in monitoring the response of a cancer patient to anti-cancer treatment (e.g. vaccination, anti-growth .factor or signal transduction therapies, radiotherapy, endocrine therapy, human antibody therapy chemotherapy), in the detection of recurrent disease in a 30 patient previously diagnosed as having cancer who has undergone anti-cancer treatment to reduce the amount of cancer present, or in the selection of an anti-cancer therapy (e.g. vaccine, anti-growth factor or signal transduction therapies, radiotherapy, endocrine therapy, 35 human antibody treatment chemotherapy), for use in a particular patient. The inventors have generally observed that levels of cancer-associated autoantibodies show a positive correlation WO 2006/126008 PCT/GB2006001944 - 25 with disease state (see also Wo 99/58979, the contents of which are incorporated herein by reference). Hence, when the method of the invention is used in clinical applications increased levels of anti-tumour marker autoantibodies, as 5 compared to suitable controls, .are generally taken as an indication of the cancer disease state. For example, when the immunoassays are used in the diagnosis of cancer, the presence of an elevated level of 10 autoantibodies, as compared to "normal' control individuals, is taken as an indication that the individual has cancer. The -"normal" control individuals will -preferably be age matched controls not having any diagnosis of cancer based on clinical, imaging and/or biochemical criteria. 15 When the immunoassays are used in predicting the response of a cancer patient to.anti-cancer treatment. (e.g. vaccination, anti-growth factor or signal.transduction., therapies, radiotherapy, endocrine therapy, human antibody 20 therapy, chemotherapy), the presence of an elevated level..of autoantibodies, as compared to "normal" control individuals, may be taken as an indication of. whether or not the individual is likely to respond to the anti-cancer treatment. The "normal" control individuals will preferably 25 be age-matched controls not having any diagnosis of cancer based on clinical, imaging and/or biochemical criteria. For each of the treatments listed above, a relationship between the level of autoantibodies compared to controls and likely success of treatment can be established by observation of 30 the outcome of such treatment in patients whose autoantibody status is monitored throughout treatment. The previously established relationship may then be used to predict the likelihood success for each treatment in a given patient based on assessment of autoantibody status. 35 When the immunoassays are used in monitoring the progress of cancer or other neoplastic disease in a patient., the presence of an elevated level of autoantibodies, as compared to a normal control", is taken as an indication of WO 2006/126008 PCT/GB2006/001944 - 26 the presence of cancer in the patient. The *normal control" may be levels of autoantibodies present in control individuals, preferably age-matched, not having any diagnosis of cancer based on clinical, imaging and/or 5 biochemical criteria. Alternatively, the "normal control" may-be-a.."base-line". level established for the particular patient under-test. The "base-line" level may be, for example, the level of autoantibodies present when either a first diagnosis of cancer or a diagnosis of recurrent cancer 10 was made. Any increase above the base-line level would be .. taken as an indication that the amount of cancer present in the patient has increased, whereas any decrease- below-the base-line would be taken as an indication that the amount of cancer present in the patient has decreased. The 'base 15 line" value may also be, for.example,. the level.before a new treatment is commenced. A change in the level of autoantibodies would..be taken as, an indication .of .the effectiveness of the therapy. The direction of the 'change" (i.e. increase vs decrease) indicating a positive response 20 to treatment.will be dependent upon the precise nature of the treatment. For any given treatment the direction.of the %change" in autoantibody levels indicating a positive result may be readily determined, for example by monitoring autoantibody levels in comparison to other clinical or 25 biochemical indicators of response to the treatment. When the immunoassays are used in screening a population of asymptomatic human subjects this may be to identify those subjects who are at increased risk of 30 developing cancer, individuals having an elevated level of autoantibodies, as compared to "normal" control individuals, are identified as being 'at risk" of developing cancer. The "normal" control individuals will preferably be age-matched controls not identified as having any predisposition to 35 developing cancer or any significant elevated risk of developing cancer. An exception to this may be where age itself is a major risk factor.

WO 2006/126008 PCT/GB2006/001944 - 27 When the immunoassays are used in screening a population of asymptomatic human subjects this may be to diagnose cancer in those subjects who have already developed a cancer, individuals having an elevated level of 5 autoantibodies as compared to "normal" control individuals being scored as having cancer or some.form of neoplastic change. The "normal" control individuals will preferably_ be age-matched controls not identified as having any predisposition to developing cancer or any significant 10 elevated risk of developing cancer. An exception to this .may be where age itself is a major risk factor. A.tdrhatively; the "normal control" may be a "base-line" level established for the particular patient under test. The "base-line" level may be, for example, the level of 15 autoantibodies...present..when the..patient. was first tested and found to have levels not elevated above a "normal control" population. Any increase thereaf.ter against.this.baseline measurement would be taken as an indication of the presence of cancer in that individual. Thus the individual could 20 through such a baseline test.become their own.control for future autoantibody measurement. When the immunoassays are used in monitoring the response of a cancer patient to anti-cancer treatment (e.g. 25 vaccination, anti-growth factor or.signal transduction therapies, radiotherapy, endocrine therapy, human antibody therapy, chemotherapy), the presence of an altered level of autoantibodies after treatment is taken as an indication that the patient has responded positively to the treatment. 30 A base-line level of autoantibodies taken before treatment is commenced may be used for comparison purposes in order to determine whether treatment results in an "increase or decrease, in autoantibody levels. A change in the level of autoantibodies would be taken as an indication of the 35 effectiveness of the therapy. The direction of the 'change" (i.e. increase vs decrease) indicating a positive response to treatment will be dependent upon the precise nature of the treatment. For any given treatment the direction of the "change" in autoantibody levels indicating a positive result WO 2006/126008 PCT/GB2006/001944 - 28 may be readily determined, for example by monitoring autoantibody levels in comparison to other clinical or biochemical indicators of response to the treatment. 5 The method of the invention may used in predicting' and/or-monitoring response of an individual to essentially any known anti-cancer treatment.- This includes, for example human antibody therapy wherein monoclonal or polyclonal antibodies are infused into the patient, a non-limiting 10 specific example being treatment with the anti-growth factor antibody Herceptin" (Baselga,. J., D. Tripathy et al;,.J Clin Oncol., 14(3), 737-744, 1996). The presence of a natural autoantibody response may enhance or inhibit the effectiveness of treatment with artificially infused 15 therapeutic antibodies... Using the method.of the..invention. it is possible to correlate response to any anti-cancer treatment, including antibody therapy, with natural levels of autoantibodies prior- to and over the .course. of the -treatment in any patient or group of patients. This 20 knowledge may then in turn be used to predict.:how. other. patients (or the same patient in the case of repeated treatment) will respond to the same treatment. When the immunoassays are used in detection of 25 recurrent disease, the presence of an increased level of autoantibodies in the patient, as compared to a "normal control", is taken as an indication that disease has recurred. The "normal control" may be levels of autoantibodies present in control individuals, preferably 30 age-matched not having any diagnosis of cancer based on clinical, imaging and/or biochemical criteria. Alternatively, the "normal control may be a "base-line" level established for the particular patient under test. The "base-line" level may be, for example, the level of 35 autoantibodies present during a period of remission from disease based on clinical, imaging and/or biochemical criteria.

WO 2006/126008 PCT/GB2006/001944 - 29 The assay method of the invention may be applied in the detection of many different types of cancer, of which examples are breast, bladder, colorectal, prostate, lung, pancreatic and ovarian cancers. The assays may complement 5 existing methods of screening and surveillance. For - example, -in the case of primary breast cancer immunoassays for autoantibodies could be used to alert clinicians to , biopsy small lesions on mammograms which radiographically do not appear suspicious or to carry out breast imaging or to 10 repeat imaging earlier than planned. In the clinic, the apsay methods of the invention are expected to be m6re. objective and reproducible compared to current imaging techniques (i.e. mammography and ultrasound), the success of which can be operator-dependent. 15 "Panel assays" may be tailored having regard to the particular clinical application. A panel of antigens for. detection of autoantibodies to at least p53 and c-erbB2 is particularly useful for many types of cancer and can 20 optionally be supplemented with. other .markers. having a .known association with the particular cancer, or a stage of the particular cancer, to be detected. For example. for breast cancer the panel might include MUC 1 and /or c-myc and/or BRCA1 and/or BRCA2 and/or PSA whereas bladder cancer the 25 panel might optionally include MUC 1 and/or c-myc, for colorectal cancer ras and/or APC, for prostate cancer PSA and/or BRCA 1 and/or BRCA2 or for ovarian cancer BRCA1 and/or BRCA2 and/or CA125. There are other preferred embodiments in which p53 or.c-erbB2 are not necessarily 30 essential. In the case of breast cancer suitable panels could be selected from the following: 35 p53 and MUC 1 with optional c-erbB2 and/or c-myc, and/or BRCA1 and/or BRCA2 and/or PSA and/or NY-ESO-1 and/or BRC1; p53 and c-myc with optional c-erbB2 and/or MUC and/or BRCA1 and/or BRCA2 and/or PSA and/or NY-ESO-1 and/or BRC1; WO 2006/126008 PCT/GB2006/001944 - 30 p53 and BRCA1 with optional c-erB2 and/or MUC 1 and/or c-myc and/or BRCA2 and/or PSA and/or NY-ESO-1 and/or BRCl; p53 and BRCA2 with optional c-erbB2 and/or MUC 1 and/or C myc and/or BRCAl and/or PSA and/or NY-ESO-1 and/or BRC1; 5 c-erbB2 and MUC 1 with optional p53 and/or c-myc, and/or BRCA1 and/or BRCA2 and/or PSA and/or NY-ESO-1 and/or BRC1; c-erbB2 and c-myc with optional p53 and/or MUC1 and/or BRCA1 and/or BRCA2 and/or PSA and/or NY-ESO-1 and/or BRC1; c-erbB2 and BRCA1 ~iith optional p53 and/or MUC 1 and/or c 10 myc and/or BRCA2 and/or PSA and/or NY-ESO-1 and/or BRC1; .. c.-erbB2 and BRCA2 with optional p53 and/or MUC 1 and/or c . iyc -and/or BRCA1 and/or PSA; p53, c-myc, NY-ESO-1 and BRCA2. 15 In the case.of colorectal. cancer suitable panels could be selected for example from the following: p53 and ras with optional c-erbB2 and/or APC; p53 and APC with optional c-erbB2 and/or Ras; Rao and APC with optional p53 and/or c-erbB2 20 Such panels might.also include CEA or.CA19-9. In the case. of prostate. cancer suitable panels..could be selected for example from the following: p53 and PSA with optional BRCA1 and/or BRCA2 and/or c-erbB2; 25 c-erbB2 and PSA with optional p53 and/or BRCA1 and/or BRCA2; PSMA, PSCA and kallikreins. In the case of ovarian cancer suitable panels could be selected for example from the following: 30 p53 and CA125 with optional c-erbB2 and/or BRCA1 and/or BRCA2; c-erbB2 and CA125 with optional p53 and/or BRCA1 and/or BRCA2; HER2, annexins, CAGE and 4-5. 35 In the case of lung cancer suitable panels may be selected from: p53 and NY-ESO-1, optionally with further markers; HER2, annexins, CAGE and 4-5.

WO 2006/126008 PCT/GB2006/001944 - 31 Where the method of the invention is used to perform a "panel assay" based on two or more tumour marker antigens derived from different proteins, at least one of the 5 antigens in the panel must be tested in an assay according to the-invention based on testing of multiple different amounts of the antigen to form 'a 'titration curve. Preferably each of the antigens forming the panel is tested according to the assay of the invention and a titration 10 curve plotted/calculated for each individual antigen in the p.anel. The. invention also contemplates that a titration assay for detection of at least one anti-tumour marker antibody. 15 may be used in.combination. with an assay designed.to..detect. at least one tumour marker protein (which may be related or unrelated.to. the antigen used in the titration.assay) in the same patient sample. Thus assays for anti.-tumour. marker autoantibodies and assays for tumour marker proteins .may be 20 performed in. parallel on a single patient. sample.. In a further. embodiment, the immunoassay .method of the invention may be used in the selection of an anti-cancer vaccine for use in a particular patient. In this embodiment 25 a sample of bodily fluid taken from the patient is tested using a panel of two or more antigens, each corresponding to a different tumour marker protein, in order to determine the relative strength of the patient's immune response to each of the different tumour marker proteins. The "strength of 30 immune response" to a given tumour -marker protein or proteins is indicated by the presence and/or the amount of cancer-associated autoantibodies specific to that tumour .marker protein detected using the immunoassay; where autoantibodies are quantified, the greater the level of 35 cancer-associated auto-antibodies, the stronger the immune response. The tumour marker protein or proteins identified as eliciting the strongest immune response or strong responses in the patient (i.e. the highest level of WO 2006/126008 PCT/GB2006/001944 - 32 autoantibodies) is or are then selected to form the basis of an anti-cancer vaccine for use in the patient. 'he utility of the method of the invention is not 5 limited to detection of anti-tumour autoantibodies, although the-assay is particularly useful for this purpose. Cancer is just one example of a disease- wherein detection of autoantibodies may be used as a biological marker for disease state/disease susceptibility. The inventors have 10 shown that substantial advantages are gained by the use of a titration approach to detect autoantibodies in patient. samples. It is therefore reasonable to conclude that similar advantages will be gained by the use of the titration approach to detect autoantibodies that are 15 biological markers for diseases.other. than .cancer. The method is therefore applicable to detection of any autoantibody which .serves. as a biological marker for. a disease state or disease susceptibility, in any disease which has been shown (or can be shown) to be associated with 20 autoantibody production. Other applications.. of .the method..of the invention include, but are not limited to, detection of autoantibodies that are biological markers of autoimmune disease, such as 25 for example rheumatoid arthritis, systemic lupus erythematous (SLE), primary biliary cirrhosis (PBC), autoimmune thyroiditis (e.g. Hashimoto's thyroiditis), autoimmune gastritis (e.g. pernicious anaemia), autoimmune adrenalitis (e.g. Addison's disease), autoimmune 30 hypoparathyriodism, autoimmune diabetes (e.g. Type 1 diabetes) or myasthenia gravis and screening of patient samples for kidney or hepatic disease leading to insufficiency or failure of either organ, and for screening of patient samples post-transplantation to detect the 35 presence of antibodies directed against either the diseased tissue (which has been left in-situ post-transplantation) or against the transplanted tissue.

WO 2006/126008 PCT/GB2006/001944 - 33 In a further aspect the invention relates to a method of detecting an antibody in a test sample comprising a bodily fluid from a mammalian subject, wherein said antibody is directed to a foreign substance'introduced into said 5 mammalian subject, the method comprising: (a).contacting the test.sample with a.plurality of different amounts of an antigen specific for said antibody, (b) detecting the amount of specific binding between said antibody and said antigen, and 10 (c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each amount of-antigen used in step (a). Preferably, in this embodiment of the invention the 15 method includes as.step (d) detecting. the presence..of said. antibody based upon the amount of specific.binding between said antibody.and said antigen at each different.antigen concentration used, in other words the collective.values... observed for a particular antigen. Preferably, the presence. 20 in the test sample of antibody reactive with the: antigen used in the assay is indicated by a generally.S-shaped or sigmoidal curve. In this aspect of the invention the titration 25 methodology may be used to evaluate the immune response of a mammalian subject, and preferably a human subject, to any foreign substance introduced into said subject. In one embodiment the foreign substance may be a therapeutic agent, such as for example a drug or prodrug, 30 human antibody therapy or vaccine. The method of the invention may be used to assess whether administration of a therapeutic agent to a patient triggers an immune response leading to the production of antibodies specific for an epitope on the therapeutic .agent, or a component of a 35 delivery vehicle, excipient, carrier etc. administered with the therapeutic agent. The precise nature of the therapeutic agent is not limiting to-the invention. In non-limiting embodiments the method of the invention may be used to assess immune WO 2006/126008 PCT/GB2006/001944 - 34 response to synthetic small molecules, naturally occurring substances, naturally occurring or synthetically produced biological agents, or any combination of two or more of the foregoing, optionally in combination with excipients, 5 carriers or delivery vehicles.. In one useful embodiment the method of the invention may be used to assess the immune- response to a non-target portion of a therapeutic agent or vaccine. By unon-target" portion is meant a component part of the administered 10 therapeutic agent or vaccine which, in the case of a therapeutic agent, does not contribute directly to therapeutic activity or, in-the case of a vaccine, is not intended to elicit production of antibodies in the host. .The non-target portion may be present, for example, to 15 facilitate pur.ification..of the therapeutic. agent or vaccine. or may be designed to assist with delivery, uptake or targeting .of the therapeutic. agent/vaccine.. Examples .of such Onon-target" portions .include, but are not limited. to, linkers or makers commonly attached to recombinantly 20 expressed polypeptides such as biotin labels, histidine tags etc. In another embodiment of this aspect of the invention, the foreign substance may be an infectious agent, such as 25 fungus, bacteria, virus or parasite. The invention will be further understood with reference to the following non-limiting experimental examples: 30 Example 1 - general protocol for titration of antigen in an autoantibody assay Samples of (biotinylated) tumour marker antigens may be 35 prepared by recombinant expression, following analogous methods to those described in WO 99/58978. Briefly, cDNAs encoding the marker antigens of interest were cloned into the pET21 vector (Invitrogen) which has been WO 2006/126008 PCT/GB2006/001944 - 35 modified to encode a biotin tag and a 6xhistidine tag to aid in purification of the expressed protein. The resulting clones are grown in a suitable bacterial host cell (in inclusion bodies), -the bacteria lysed and denatured and the 5 expressed- antigens recovered via Nickel chelate affinity columns .(Hi-trap, commercially available from Amersham, following manufacturer's protocbl-). The expressed antigens were renatured by dialysis in appropriate buffer and the yield of expressed protein assessed by SDS-PAGE, western 10 blot and ELISA and quantitated prior to storage. The negative control VOL is empty vector (i.e. no cloned cDNA) which still includes the His and biotin tag sequences) 15 GenBank accession numbers for a number of marker cDNAs are as follows: P53: B003596 20 c-myc: V00568 ECD6 (HER2) extracellular domain: M11730 NY-ESO: NM_001327 BRCA2: U43746 BRCA1 delta 9-10: NM_007302 25 1. Antigens and VOL (negative control) were diluted to appropriate concentrations in 0.1 M carbonate buffer then diluted serially to form a semi-log titration range (see 30 table 1). Antigen dilutions were dispensed at 50pl/well into the rows of a Falcon micotitre plate according to plate layout using an electronic multi-channel pipette. Plates were covered and stored at 4 0 C for 48 h. 35 2. Plates were washed once in PBS + 0.1W tween 20 using an automated plate washer then tapped dry on tissue paper.

WO 2006/126008 PCT/GB2006/001944 - 36 3. Plates were blocked with high salt incubation buffer (HSB, PBS + 0.5M NaCl + 0.l% casein) at 200 pl/well for one hour or until required for use (store covered at 4 0 C). 5 4. Serum samples were defrosted, vortexed and diluted' 1/100 in.HSB at room tentp. 5. Plates were emptied and tapped dry on tissue paper. Each diluted serum sample was dispensed at 50pl/wdll into 10 all wells of the microtitre plate using an electronic multi .channel pipette. Control antibodies were diluted 1/1000 in HSB-and dispensed into appropriate wells of plate final plate. Plates covered and incubated for 1.5 hour at room temp with shaking. 15 6. Wash step: Plates were washed three times in PBS + 0.1% tween 20 using an automated plate washer then tapped dry on tissue paper. 20 7. Horseradish peroxidase conjugated rabbit anti-human.Ig (Jackson, 1/10,000 in HSB) was dispensed .at 50 pl/well into all wells of the microtitre.plate. HRP-conjugated rabbit anti-mouse Ig (1/1000 in HSB) was dispensed into control wells containing anti-antigen antibody. Plates were then 25 incubated at room temp for 1 hour with shaking. 8. Plates were washed as in .step 6. 9. Pre-prepared TMB substrate was added at 50pl/well and 30 plate incubated on bench for 10 min. Plates were gently tapped to mix. 10. Optical density of wells was determined at 650 nm using a standard plate reader protocol. 35 Table 1; standard plate layouts P53 plates.

WO 2006/126008 PCT/GB2006/001944 -37 1 1 2 3 4 | 5 | 6 7 8 9 10 11 12 A p53 10 pg/ml c-myc 10 pg/ml NY-ESO 10 pg/ml VOL 10 pg/ml B p53 3 pg/ml c-myc 3 pg/ml NY-ESO 3 pg/ml VOL 3 pg/ml C p53 1 pg/ml c-myc 1 pg/mi NY-ESO 1 pg/ml VOL 1 pg/ml D P53 0.3 pg/ml c-myC 0.3 pg/mi NY-ESO 0.3 VOL 0.3 pg/inl pg/mi I E p53 0.1 pg/ml c-myc 0.1 pg/ml' NY-BSO 0.1 VOL 0.1 pg/ml - .. .pg/ml.__ _ _ _ _ _ F p53 0.03 pg/mi c-myc 0.03 NY-ESO 0.03 VOL 0.03 pg/ml. pg/mi pg/ml G p53 0.01 pg/ml c-myc 0.01 NY-80 0.01 VOL 0.01 pg/mi pg/ml pg/ml H carbonate carbonate carbonate carbonate buffer buffer buffer buffer BRCA plates 1 2 3 4 5 6 7 8 9 10 11 12 A BRCAI 10 pg/mi BRCA2 10 pg/ml EcD-6 10 pg/ml. VOL.10 pg/ml B BRCA1 3 pg/ml BRCA2 3 pg/ml ECD-6 3 pg/ml VOL 3 pg/ml. C BRCA1 1 pg/ml BRCA2 1 pg/ml EcD-6 1 pg/ml VOL 1 pg/ml D BRCA1 0.3 pg/ml BRCA2.0.3 pg/ml BCD-6 0.3 pg/ml VOL 0.3 pg/ml E .BRCA1 0.1 pg/ml BRCA2 0.1 pg/ml EcD-6 0.1 pg/ml VOL 0.1 pg/ml F BRCA1 0.03 BRCA2 0.03 ECD-6 0.03 VOL 0.03 pg/ml pg/mi pg/ml pg/ml _ G BRCAi 0.01 BRCA2 0.01 SCD-6 0.01 VOL 0.01 pg/ml pg/mi pg/mi pg/mi H carbonate carbonate carbonate carbonate buffer buffer buffer buffer 5 Example 2 - detection of autoantibodies in primary breast cancer 10 The following data were obtained from a pilot study to assess the sensitivity and reproducibility of a panel of titration autoantibody assays in primary breast cancer (PBC). The study included serum from 17 women with no 15 evidence of cancer and pre-operative serum samples from 20 women with primary breast cancer. Normal and cancer samples were age matched. One normal and three cancer samples had to be removed from the study because they showed evidence of anti-biotin antibody responses and therefore could not be WO 2006/126008 PCT/GB2006/001944 - 38 assessed using the assay in its present format. Approximately 10%r of the population is thought to develop an immune response against biotin. 5 The assay.was.carried out according to the protocol givdn in example 1 using the .antigens p53, c-myc, NY-ESO-1 and BRCA2 Figure 1 gives examples of curves obtained when the antigen titration assay was used to measure p53 autoantibodies in 10 serum. It can be seen that the..cancer patient's serum 17766(C) binds strongly to the test antigen (p5 3 ) with a characteristic sigmoidal curve but does not-bind.to the negative control, VOL. In comparison, serum from a normal individual, 18052(N) does not give a titration curve for 15 binding to the test antigen or the negative control. Autoantibody levels were expressed as the optical density (65Onm) due to binding to the test antigen minus that due to binding to the negative control (VOL). The normal cut-off 20 was calculated as.the 95th percentile (mean + 2 standard deviations) of the normal group. This is .shown as the dotted line in Figure 2, in which anti-p53 autoantibody levels in normal individuals are compared with cancer patients. It can be seen that the cancer group show 25 generally higher autoantibody levels and also have a greater proportion of individuals with levels above cut-off. The panel consisted of four antigens; p53, c-myc, NY-ESO-1 and BRCA2. The sensitivity of the individual assays is 30 given in table 2 along with the combined sensitivity of the panel of four antigens in the detection of primary breast cancer (63%). p53 c-myc INY-ESO-1 BRCA2 Panel 6/17 (35%) 5/17 (29%) 4/17 (24%) 4/17 (24%) 63% 35 Table 2: Sensitivity of the antigen titration autoantibody assay. Autoantibodies against four different antigens were WO 20061126008 PCT/GB2006/001944 - 39 measured and the combined sensitivity of the panel calculated. Cut-off levels were calculated as mean + 2 standard deviations of the normal sample set. Individuals with anti-biotin antibody response's were excluded as not 5 capable of assessment. In order'to assess whether or not the measurements obtained. using the titration autaoantibody assay were reproducible', assays were performed on three separate days and the results 10 are shown in table 3. An assay. was deemed to be reproducible if all three results agreed. Reproducibility -- - -of measurements made on normal serum was 94V (15/16) and 88V (14/16) in breast cancer samples. 15 Run Run Normals A B C PBCO A B C 20 18017 - + + 17733 + - 18018 - - - 17734 AB + - 18019 - - - 17735 - - 18020 - - - 17742 + + + 18021 - - - 17743 + + + 25 18047 - - - 17744 + + + 18048 AB + - + 17755 - + 18049 - - - 17756. - - 18050 ND - - 17757 - - 18051 - - - 17758 ND - 30 18052 - - - 17759 + + + 18053 - - - 17766 + + + 18054 - - - 17774 - - 18055 - - - 17775 AB + + 18056 - - - 17776 - - 35 18057 - - - 17777 - - ND 18058 - - - 17796 - - 17797 AB + - 17832 + + + 17450 - - - WO 2006/126008 PCT/GB2006/001944 - 40 Table'3r Reproducibility of the antigen titration assay for p53 autoantibodies. Assays were perfoiined on three separate 5 days (Runs A, :B and C) on serum samples from patients with primary breast cancer (PBC) or normal.controls. Cut-off levels were calculated on a daily basis as mean + 2 standard deviations of the normal sample set. AB denotes individuals that showed evidence bf an anti-biotin antibody response and 10 whom can not be assessed by the present assay format. An ..assay was deemed to be reproducible if all three results agreed. Reproducibility of -the normal individuals was 94% (15/16)- and the PBC patients was 881 (14/16). 15 Example 3 - detection of autoantibodies in lung cancer Analysis of autoantibody responses against 2 antigens (p53 and NY-ESO) in a pilot lung cancer study (10 normal and 9 20 lung cancer plasma) revealed a detection rate of 78% (Figure 3). The assay was carried out according to the general protocol of example 1, except that plasma samples were used instead 25 of serum. Positive patient samples exhibited sigmoidal titration curves similar to that shown in Figure 1. Figure 3 shows a comparison of p53 and NY-ESO autoantibody levels in normal 30 individuals and patients with lung cancer as measured using the antigen titration assay. Normal cut-offs were calculated as the mean plus 2 standard deviations of the normal population. 35 Example 4 - Additional titration curves The following additional titration curves were all generated in assays based on the general methodology described in WO 2006/126008 PCT/GB2006/001944 - 41 Example 1. The results indicate that the titration curve approach may be applied to detection of a variety of different antigens, in different types of bodily fluids and in different disease (exemplified by different types of 5 cancer) and also illustrate the advantage of the method of the -invention in distinguishing between "true" and "false" positive results. Figure 4 shows- a titration curve for detection of 10 autoantibodies against p53-and NY-ESO in a sample of ascites .. fluid taken from a patient with breast cancer. .This patient was tested but found not to produce autoantibodies-against c-myc. 15 Figure 5 shows a titration curve for detection.of autoantibodies against BRCA1, BRCA2 and HER2 in a sample of serum from a patient with breast.cancer (ductal.carcinoma in situ). 20 Figure 6 .shows a titration curve for detection. of. autoantibodies against NY-ESO in a sample of serum from a patient with lung cancer. This patient was tested .but found not to produce autoantibodies against p53 or c-myc. 25 Figure 7 shows a titration curve for detection of autoantibodies against NY-ESO and p53 in a sample of serum from a patient with lung cancer. This patient was tested but found not to produce autoantibodies against c-myc. 30 Figures 8(a) and 8(b) illustrate the results on two independent titration assays for autoantibodies against p53, c-myc and NY-ESO-1 in samples of serum from a "normal" subject (i.e. an individual with no evidence of cancer). In the assay shown in Figure 8(a) a flat line is observed with 35 increasing amounts of antigen, indicating that the serum sample does not contain autoantibodies to any of the tested antigens. When a second aliquot of the same patient serum sample was tested again using the same assay methodology the assay failed producing the anomalous results shown in Figure WO 2006/126008 PCT/GB2006/001944 - 42 8 (b). The absence of the characteristic titration curve with increasing amounts of antigen indicates that this is an anomalous result, rather than a true positive. Had this sample been tested in a single point assay using a single, 5 fixed amount of antigen then it may have appeared as a "false-positive" result,. Thus, these.results illustrate the advantage of the titration curve approach in distinguishing between true and false positive results. 10 Figures 9(a) and 9(b) shows the results of two independent titration assays carried out on samples of serum from.a single patient with invasive breast cancer using a range of. different antigens. This particular patient shows autoantibodies to NY-ESO-1, HER2 and BRCA2. For each 15 positive antigen a positive .assay result is indicated.by increasing signal strength as the concentration of antigen increases, i.e. a titrating signal. Figures 10(a) and 10(b) illustrate the utility of the 20 invention in distinguishing between..anti-biotin responses and "true" autoantibodies to a specific antigen (i.e. a tumour marker). In these assays samples. of serum.from a clinically normal human subject were tested for the presence of autoantibodies using biotinylated antigens BRCA2, HER2, 25 c-myc and NY-BSO-1, non-biotinylated BRCA1 and control expression products of the "empty" vector VOL, which encodes the biotin tag but no additional antigen. The tested individual exhibits a titrating response to both biotinylated antigens and to the empty vector VOL, which is 30 effectively biotin alone, but no response to the non biotinylated antigen BRCA1, indicating that the "positive" results with the biotinylated markers are in fact due to the presence of anti-biotin antibodies in this individual. 35 WO 2006/126008 PCT/GB2006/001944 -43 Example 5 - Analysis of the sensitivity and specificity of antigen titration assays compared with single point measurement 5 Autoantibody (AAb) measurements were. performed on 100 women with primary breast .cancer (PBC) and 80 women with no evidence of malignant disease using both the titration method and by measuring at -a single concentration of antigen (10pg/ml). Tables showing a direct comparison of the two 10 methods are shown below: Table 4. Comparison of the-sensitivity of the titration AAb assay with a measurement at a single antigen concentration in PBC. 15 Antigen Single point Titration assay p53 17.5% 18.9% c-myc 6.2t 22.9% NY-ESO-1 24.7% 25.0% 20 BRCA2 20.6% 31.3% HER2 23.7% 25.0% MUC1 18. 5% 19.8% Panel (of 6 Ags) 54.6% 62.2% 25 Table 5. Comparison of the specificity of the titration AAb assay with a measurement at a single antigen concentration in normal women. Antigen Single point Titration assay 30 p53 93.8% 97.3% c-myc 93.8%. 94.6% NY-ESO-1 90.0 93.3% BRCA2 90.0% 94.6% HER2 91.3% 95.9% 35 MUC1 92.5% 95.9% Panel (of 6 Ags) 71.3% 78.4% WO 2006/126008 PCT/GB20061001944 - 44 It can be seen that by using several points on the antigen titration curve, both a higher sensitivity.-and specificity were obtained compared with a single point measurement. 5 Example 6 - Potential Reasons for higher sensitivity and specificity with antigen titration assay Without being bound by theory, the applicant considers there are a number of reasons for the observed higher specificity 10 and sensitivity in an assay where the antigen is titrated. (i) Figure 11 shows the results of AAb analysis of. serum from a patient with primary breast cancer (PCB) using antigens p53, c-myc and NY-ESO-1 at varying 15 concentration. These results demonstrate.that. in some cases the titration curve dips at high antigen levels (NY.-ESO-1 curve).. This is .a commonly observed phenomenon in immunochemistry. If a. single point measurement at 10 pg/ml were used, this patient 20 would have.been classified as negative with.respect to NY-ESO-l autoantibodies when in fact there is clearly a positive response.. (ii) Figure 12 shows the analysis of serum from a normal 25 individual, also using titrated antigen p53, c-myc and NY-ESO-1. This figure demonstrates an affect which the applicants have observed in approximately 10% of assays in which the baseline of the antigen measurement (in this case p53) is shifted to a level 30 above that of the negative control (VOL). This produces a falsely high reading. This type of result is easily identifiable with the titration assay, but would be invisible in a set of single point assays. This would result in reduced specificity due to these 35 false positives (see Table 5). (iii) As discussed in Example 4, the antigens that are used in the Titration AAb Assay have a biotin tag that can be used in purification of the protein. However, it WO 2006/126008 PCT/GB2006/001944 - 45 is known that approximately 10% of the population produce an antibody response to biotin, which is a vitamin. Figure 13 demonstrates an anti-biotin response in a patient with PBC. .This response can be .5 clearly-identified using. the titration curve as a strong antibody response against the negative control, VOL (which is also biotinylated)*. These individuals must be regarded as unassessible with the assay in this format. However, if a single point 10 assay were used, the- anti-biotin responders could not be distinguished from true responses. Example 7 - Demonstration of increased sensitivity of antigen titration compared with serum titration 15 AAb measurements were performed in two ways in two quite separate experiments. The firat was using the .standard format in which .the.plate was coated with antigen in.a semi log titration from 10 pg/ml down to 0.01 pg/ml. After 20 blocking, serum.was added at a dilution.of 1 in 100. In the second, the plates were coated with antigen at a concentration.of 3 pg/ml and after blocking.,. serum was added in a semi-log titration range from a dilution of 1 in 10 down to 1 in 10,000. The methods for the remainder of the 25 assays were identical. Two pools of serum known to be positive for p53 and c-myc were assayed along with 8 sera from women with PBC and 10 sera from normal individuals. The results are shown in table 6 below. 30 Table 6. Sensitivity of AAb assays involving titration of antigen compared with those involving titration of serum.

WO 2006/126008 PCT/GB2006/001944 - 46 Antigen Serum Antigen Serum Antigen Serum PBC p53 p53 c-myc c-myC BRCA2 BRCA2 p53 +ve ++ +++ ++ ++ c-myc+ve - + + 17179. +++ +++ .. .. ++ + 19451 + ++ - - 18237. ++ -++- ++. + + 18489 . . - + 19510 - - - 19190 + . - - + ++ 18610 + - + + 18458 + - - + - - postivity 70% 40% 60% 50% 40% 20%*~ It can be seen that the assay format involving titration.of 5 antigen is more sensitive than.the format involving. titration of serum. Samples that were strongly positive were detected by both formats, however weak positives. in the antigen titration assay were generally not detected in the serum titration .assay. 10 The lower sensitivity demonstrated by the serum titration format is due to the inherent.non-specific binding of serum at high concentration. This causes a level of binding to the proteins even in normal samples (see Figure 14), which 15 elevates the normal cut-off value with subsequent. reduction in sensitivity. The specificity was also reduced in the serum titration format (BRCA2 = 90%) compared with the antigen titration format (BRCA2 = 100%). 20 Figure 15 reflects the experiments shown in Figure 4 but with serum from a patient with primary breast cancer. The patient was found to be positive for auto-antibodies against p53 and c-myc but negative for auto-antibodies against BRCA2 when the antigen titration method of the invention was used. 25 However, when auto-antibodies were measured within a serum titration range no auto-antibodies were detected (see Table 6). This is due to the non-specific binding exhibited by serum at high concentrations (demonstrated by the high level WO 2006/126008 PCT/GB2006/001944 - 47 of binding to the negative control protein (VOL)) which masks signals due to specific auto-antibody binding. The consequence is that with serum dilution the assay could designate a patient with primary breast cancer, negative. 5 Since sensitivity = True positives/True positives + falge negatives this has the effect of increasing the denominator and therefore -decreasing the sehsitivity. In Summary, the applicants have shown that Antigen Titration 10 Autoantibody assays are more sensitive and specific than measuring autoantibody reactivity at a single antigen.. concentration. This is because the antigen titration' provides the scope to detect both low abundance, low affinity antibodies at high antigen concentrations as.well 15 as higli abundance..antibodies. which.,would otherwise.hook. at high antigen concentration but bind maximally further.down the titration curve. It also allows.discrimination.of non assessable assays from true results, which is not possible with single point measurement. It is believed an antigen 20 titration AAb assays are.more sensitive than mere titration of serum because the high level of non-specific binding. observed with serum at high concentration raises.normal cut off levels thereby decreasing sensitivity. 25 30 35

Claims (33)

1. A method of determining an antibody profile in a test sample comprising a bodily fluid from a mammalian subject 5 wherein said antibody is a biological marker of a disease state or disease susceptibility, the method comprising: a) contacting the test sample with a plurality of different amounts of an antigen specific for said antibody, 0 b) detecting the amount of specific binding between said antibody and said antigen and c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each amount of antigen used in step (a), to build up a profile of antibody 5 production in said subject either prior to the onset of disease, or throughout the course of disease.

2. The method according to claim 1, wherein the method is repeated to build up a profile of antibody production. 0

3. The method of claim 1 or claim 2 wherein the antibody is an autoantibody.

4. The method of claim 3 wherein the antibody is an 5 autoantibody specific for a tumour marker protein.

5. The method of claim 4 wherein the antigen is a tumour marker protein or an antigenic fragment or epitope thereof. 30

6. The method of claim 5 wherein the tumour marker protein is MUC1, MUC16, c-myc, EGRF, p53, ras, BRCA1, BRCA2, APC, HER2, PSA, CEA, CA19.9, NY-ESO-1, 4-5, CAGE, PSMA, PSCA, EpCam, a cytokeratin, recoverin, a kallikrein, an annexin, AFP, GRP78, CA125, mammoglobin, or raf. 35

7. Use of the method of any one of claims 4 to 6 in the diagnosis, prognosis or monitoring of cancer.

8. Use of the method of any one of claims 4 to 6 for: I0 (a) screening a population of asymptomatic human subjects to identify those subjects who are at increased risk of developing 4Jdenm.e> 49 cancer, wherein the samples to be tested using the method are samples of bodily fluid taken from the subjects, and wherein subjects having an elevated level of autoantibodies, as compared to a normal control which represents either the level 5 of autoantibodies present in control individuals or a base-line level for that subject, are identified as being at risk of developing cancer; or (b) detecting early neoplastic or early carcinogenic change in an asymptomatic human subject, wherein the sample to be tested 0 using the method is a sample of bodily fluid taken from the subject, and wherein the presence of an elevated level of autoantibodies, as compared to a normal control which represents either the level of autoantibodies present in control individuals or a base-line level for that subject, is 5 taken as an indication of early neoplastic or early carcinogenic change in the subject; or (c) screening a population of asymptomatic human subjects to identify those subjects who have developed a cancer, wherein 0 the samples to be tested using the method are samples of bodily fluid taken from the subjects, and wherein subjects having an elevated level of autoantibodies, as compared to a normal control which represents either the level of autoantibodies present in control individuals or a base-line level for that 5 subject, are diagnosed as having a cancer; or (d) testing a population of symptomatic human subjects to identify those subjects who have developed a cancer, wherein the samples to be tested using the method are samples of bodily 30 fluid taken from the subjects, and wherein subjects having an elevated level of autoantibodies, as compared to a normal control which represents either the level of autoantibodies present in control individuals or a base-line level for that subject, are diagnosed as having a cancer; 35 (e) monitoring the progress of cancer or other neoplastic disease in a patient, wherein the sample to be tested using the method is a sample of bodily fluid taken from a human patient, and wherein the level of autoantibodies present in said patient 40 is compared to a normal control which represents either the <lename> 50 level of autoantibodies present in control individuals or a base-line level established for that patient; (f) assessing the prognosis of a patient diagnosed with cancer; 5 (g) predicting the response of a cancer patient to anti-cancer treatment, such as anti-growth factor or signal transduction therapy, radiotherapy, endocrine therapy, human antibody therapy, chemotherapy or vaccination; 0 (h) monitoring the response of a cancer patient to anti-cancer treatment, such as anti-growth factor or signal transduction therapy, radiotherapy, endocrine therapy, human antibody therapy, chemotherapy or vaccination; or 5 (i) selecting an anti-cancer vaccine for use in a cancer patient.

9. A method according to claim 3 wherein the antibody is an .0 autoantibody characteristic of or associated with an autoimmune disease.

10. The method according to claim 3 wherein the autoantibody is an autoantibody characteristic of or associated with a !5 disease leading to insufficiency or failure of an organ.

11. A method according to claim 10 wherein the disease is kidney or hepatic disease. 30

12. The method of claim 1 wherein the antibody is directed towards an epitope present or tissue transplanted into a mammalian subject.

13. The method according to any one of claims 1 to 12 wherein 35 the antigen is a naturally occurring protein or polypeptide, a recombinant protein or polynucleotide, a synthetic protein or polypeptide, synthetic peptide, peptide mimetic, polysaccharide or nucleic acid. 40

14. A method of profiling two or more antibodies in a test sample comprising a bodily fluid from a mammalian subject Oflename> 51 wherein at least one of said antibodies is a biological marker of a disease state or disease susceptibility, the method comprising: (a) contacting the test sample with two or more sets of 5 antigens, wherein each one of said sets of antigens is specific for one of said antibodies to be detected in the test sample and wherein each set of antigens comprises a plurality of different amounts of said antigen, (b) detecting the amount of specific binding between said 0 antibodies and said antigens, and (c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each set of antigens used in step (a), to build up a profile of antibody production in a patient either prior to the onset of disease or 5 throughout the course of disease.

15. The method of claim 14 wherein the profiling of each antibody is determined based upon the collective values of the amount of specific binding for all the antigen concentrations 0 tested for that antibody.

16. The method according to claim 14 or 15 wherein at least one or each of said antibodies is an autoantibody specific for a tumour marker protein. 5

17. The method according to claim 16 wherein each antigen is a tumour marker protein or an antigenic fragment or epitope thereof. 30

18. The method according to claim 17 wherein each of said tumour marker proteins is selected from the group consisting of MUC1, MUC16, c-myc, EGRF, p53, ras, BRCA1, BRCA2, APC, HER2, PSA, CEA, CA19.9, NY-ESO-1, 4-5, CAGE, PSMA, PSCA, EpCam, a cytokeratin, recoverin, a kallikrein, an annexin, AFP, GRP78, 35 CA125, mammoglobin, and raf.

19. Use of the method of any one of claims 16 to 18 in the diagnosis, prognosis or monitoring of cancer. 40

20. Use of the method of any one of claims 16 to 18 for: <llonam> 52 (a) screening a population of asymptomatic human subjects to identify those subjects who are at increased risk of developing cancer, wherein the samples to be tested using the method are samples of bodily fluid taken from the subjects, and wherein 5 subjects having an elevated level of autoantibodies, as compared to a normal control which represents either the level of autoantibodies present in control individuals or a base-line level for that subject, are identified as being at risk of developing cancer; or 0 (b) detecting early neoplastic or early carcinogenic change in an asymptomatic human subject, wherein the sample to be tested using the method is a sample of bodily fluid taken from the subject, and wherein the presence of an elevated level of 5 autoantibodies, as compared to a normal control which represents either the level of autoantibodies present in control individuals or a base-line level for that subject, is taken as an indication of early neoplastic or early carcinogenic change in the subject; or 0 (c) screening a population of asymptomatic human subjects to identify those subjects who have developed a cancer, wherein the samples to be tested using the method are samples of bodily fluid taken from the subjects, and wherein subjects having an 5 elevated level of autoantibodies, as compared to a normal control which represents either the level of autoantibodies present in control individuals or a base-line level for that subject, are diagnosed as having a cancer; or 30 (d) testing a population of symptomatic human subjects to identify those subjects who have developed a cancer, wherein the samples to be tested using the method are samples of bodily fluid taken from the subjects, and wherein subjects having an elevated level of autoantibodies, as compared to a normal 35 control which represents either the level of autoantibodies present in control individuals or a base-line level for that subject, are diagnosed as having a cancer; (e) monitoring the progress of cancer or other neoplastic 40 disease in a patient, wherein the sample to be tested using the method is a sample of bodily fluid taken from a human patient, filename> 53 and wherein the profile of autoantibodies present in said patient is compared to a normal control which represents either the level of autoantibodies present in control individuals or a base-line level established for that patient; or 5 (f) assessing the prognosis of a patient diagnosed with cancer; (g) predicting the response of a cancer patient to anti-cancer treatment, such as anti-growth factor or signal transduction 0 therapy, radiotherapy, endocrine therapy, human antibody therapy, chemotherapy or vaccination; (h) monitoring the response of a cancer patient to anti-cancer treatment, such as anti-growth factor or signal transduction 5 therapy, radiotherapy, endocrine therapy, human antibody therapy, chemotherapy or vaccination; or (i) selecting an anti-cancer vaccine for use in a cancer patient. 0

21. A method of determining an antibody profile in a test sample comprising a bodily fluid from a mammalian subject wherein said antibody is directed to a foreign substance introduced into said mammalian subject, the method comprising: 5 a) contacting the test sample with a plurality of different amounts of an antigen specific for said antibody, b) detecting the amount of specific binding between said antibody and said antigen and 30 c) plotting or calculating a curve of the amount of said specific binding versus the amount of antigen for each amount of antigen used in step (a), to build up a profile of antibody production in said subject. 35

22. The method according to claim 21 wherein the foreign substance is: (i) a therapeutic agent, such as a drug, prodrug, human antibody therapy or vaccine; (ii) a component of a delivery vehicle, carrier or excipient 40 administered with a therapeutic agent; Oflns8me> 54 (iii) a non-target portion of a therapeutic agent or vaccine; or (iv) an infectious agent, such as a fungus, bacteria, virus or protein. 5

23. A method of detecting an antibody in a test sample comprising a bodily fluid from a mammalian subject, wherein said antibody is directed to a foreign substance introduced into said mammalian subject, the method comprising: 0 (a) contacting the test sample with a plurality of different amounts of an antigen specific for said antibody, (b) detecting the amount of specific binding between said antibody and said antigen, and (c) plotting or calculating a curve of the amount of said 5 specific binding versus the amount of antigen for each amount of antigen used in step (a).

24. A method as claimed in claim 23 which comprises as step (d) detecting the presence of said antibody based upon the 0 amount of specific binding between said antibody and said antigen at each different antigen concentration used.

25. The method of claim 23 or 24 wherein detecting the presence of said antibody is based upon the collective values 5 of the amount of specific binding for all the antigen concentrations tested.

26. The method as claimed in any one of claims 23 to 25 wherein the presence in the test sample of antibody reactive M with the antigen used in the assay in indicated by a generally S-shaped or sigmoidal curve.

27. A method according to any one of claims 23 to 26 wherein the mammalian subject is a human. 55

28. A method according any one of claims 23 to 27 wherein the foreign substance is a therapeutic agent.

29. A method according to claim 28 wherein the therapeutic O agent is a drug, prodrug, or antibody therapy. <fdename> 55

30. A method according to any one of claims 23 to 27 wherein the foreign substance is a vaccine.

31. A method according to claim 29 or 30 wherein the foreign 5 substance is a non-target portion of a therapeutic agent or vaccine.

32. A method according to claim 31 wherein the non-target portion is biotin. 0

33. A method according to claims 23 to 27 wherein the foreign substance is an infectious agent such as fungus, bacteria, virus or parasite. <illename>