WO2006110940A1

WO2006110940A1 - Biomarker for growth hormone

Info

Publication number: WO2006110940A1
Application number: PCT/AU2006/000484
Authority: WO
Inventors: Robert C Baxter; Liping Chung
Original assignee: Northern Sydney And Central Coast Area Health Service
Priority date: 2005-04-18
Filing date: 2006-04-12
Publication date: 2006-10-26

Abstract

The present invention relates to a biomarker for growth hormone, to uses of the biomarker in determining levels of growth hormone action and to detection kits for determining growth hormone action. Disclosed are methods for determining the level of growth hormone action in a subject, methods for diagnosing in a subject the over-expression or under-expression of endogenous growth hormone, methods for diagnosing a growth-related condition in a subject, methods for detecting in a subject the illicit administration of exogenous growth hormone and associated kits.

Description

Biomarker for Growth Hormone

Technical Field

The present invention relates to a biomarker for growth hormone, to uses of the biomarker in determining levels of growth hormone action and to detection kits for determining growth hormone action.

Background of the Invention

Growth hormone is a pituitary-derived protein that stimulates the growth and metabolism of many of the tissues in the body, either directly or by the generation of insulin-like growth factor-l

(IGF-I). IGF-I is produced by many tissues in response to the action of growth hormone, and may act locally in these tissues to stimulate growth, but it is growth hormone action on the liver that is mainly responsible for generating the IGF-I that is secreted into the circulation. Levels of circulating or "endocrine" IGF-I are closely related to growth status in humans and animals. For example, a low serum IGF-I level is associated with growth retardation in children, while a higher than normal serum IGF-I level is associated with tissue overgrowth, such as in subjects who have a growth hormone-secreting pituitary tumour (acromegaly).

The reliable detection of growth hormone in subjects has a number of important applications, including the diagnosis of disorders involving under- or over-expression of growth hormone, the monitoring of growth hormone status in subjects being treated with exogenous growth hormone as a drug to reverse a natural growth hormone deficiency, and testing to detect illicit use of growth hormone, for example in elite athletes, racehorses or other animals.

Growth hormone itself can be measured in serum. However it is secreted in a pulsatile manner by the pituitary gland, and has a very short half-life in circulation. Thus a simple measurement of growth hormone in a serum sample is not a reliable means of diagnosing a subject's growth hormone secretory status.

One approach to determining growth hormone levels is dynamic testing, wherein a subject is given a treatment to stimulate growth hormone secretion, and the increase in serum growth hormone in response to that stimulus is measured in a series of blood samples. While this test can utilize a variety of stimuli to increase serum growth hormone, some stimuli nevertheless carry a risk of adverse side-effects (e.g. insulin-induced hypoglycemia). In addition, as several blood samples are required over different periods of time, dynamic testing is more time-consuming and more intrusive to the subject than a single blood test. For this reason, other less direct methods requiring only a single blood test have been devised to determine growth hormone status, involving the measurement of marker proteins which increase in the bloodstream in response to growth hormone action. One marker of growth hormone action is IGF-I. A relatively non-invasive test for growth hormone levels based on IGF-I levels in serum has been established and is widely used, for example by sports drug testing agencies. Other markers include the liver-derived proteins IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS), and markers of collagen turnover such as procollagen type III aminoterminal peptide (PIIINP), However, diagnostic tests based on these markers have varying utility in different clinical situations. There is a need for the development of an accurate, reliable, simple and relatively noninvasive test for the determination of growth hormone levels in a subject as an adjunct or alternative to the already established IGF-I based test.

The present invention is predicated on the surprising and unexpected finding by the inventors that serum levels of the abundantly expressed and easily measured hemoglobin α-chain, are positively associated with growth hormone levels in human subjects.

Summary of the Invention

According to a first aspect of the present invention there is provided a method for determining the level of growth hormone action in a subject, the method comprising the steps of: (a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of hemoglobin α-chain present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of growth hormone action. The sample may be serum, plasma, urine, saliva or other body fluid.

The hemoglobin α-chain may be free hemoglobin α-chain including any of its natural variant forms. The hemoglobin α-chain may be encoded by the HBA1 or HBA2 gene.

The hemoglobin α-chain may be measured using an immunoassay. The immunoassay may be an enzyme-linked immunoassay or radioimmunoassay. The level of growth hormone action in the subject may indicate the administration to the subject of exogenous growth hormone. Alternatively, the level of growth hormone action in the subject may indicate the level of expression of endogenous growth hormone in the subject.

The method may include comparing the level of hemoglobin α-chain in the sample obtained from the subject with the level of hemoglobin α-chain from one or more control samples. Typically a control sample may be a sample from a subject with normal levels of growth hormone and/or known not to use exogenous growth hormone.

According to a second aspect of the present invention there is provided a method for determining the level of growth hormone action in a subject, the method comprising the steps of: (a) obtaining a biological sample from the subject; and

(b) assaying the expression of hemoglobin α-chain in the sample, wherein the level of expression of hemoglobin α-chain is indicative of the level of growth hormone action.

The sample may be serum, plasma, urine, saliva or other body fluid. The method may include comparing the level of hemoglobin α-chain expression in the sample obtained from the subject with the level of hemoglobin α-chain expression from one or more control samples. Typically a control sample may be a sample from a subject with normal levels of growth hormone and/or known not to use exogenous growth hormone.

According to a third aspect of the present invention there is provided a method for diagnosing in a subject the over-expression or under-expression of endogenous growth hormone, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of hemoglobin α-chain present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of expression of growth hormone.

Over-expression of growth hormone in the subject may be associated with acromegaly, gigantism, or a growth hormone-secreting tumour.

Under-expression of growth hormone in the subject may be associated with growth retardation, dwarfism, Turner's syndrome, injury to the pituitary gland, or other forms of pituitary insufficiency.

The step of analysing the sample to determine the amount of hemoglobin α-chain may comprise measuring hemoglobin α-chain polypeptide levels, for example using an anti- hemoglobin α-chain antibody. According to a fourth aspect of the present invention there is provided a method for diagnosing a growth-related condition in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of hemoglobin α-chain present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of growth hormone action.

The growth-related condition may be selected from the group consisting of acromegaly, gigantism, hyperpituitarism, growth retardation, fetal growth retardation, dwarfism, a growth 5 hormone receptor defect, a growth hormone-secreting tumour, injury to the pituitary gland, or any other condition associated with growth hormone deficiency or excess.

The step of analysing the sample to determine the amount of hemoglobin α-chain may comprise measuring hemoglobin α-chain polypeptide levels, for example using an anti- hemoglobin α-chain antibody, o According to a fifth aspect of the present invention there is provided a method for detecting in a subject the illicit administration of exogenous growth hormone, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of hemoglobin α-chain s present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of growth hormone action.

In one embodiment the subject may be a sportsperson such as an elite athlete. In an alternative embodiment the subject may be an animal, such as a racehorse or animal raised for 0 breeding or consumption.

According to a sixth aspect of the present invention there is provided a diagnostic kit for use in determining the level of growth hormone in a subject, the kit comprising at least one agent for measuring hemoglobin α-chain in a biological sample.

The agent may be an antibody that recognises and binds hemoglobin α-chain. s Accordingly, in one embodiment a kit may comprise a first container containing an antibody raised against hemoglobin α-chain and a second container containing a conjugate comprising a binding partner of the antibody, together with a detectable label.

For the purposes of the above aspects and embodiments, the subject may be a human or any other animal. In particular embodiments the subject is selected from the group consisting of o human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline, canine and murine. Definitions

In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

5 The term "growth hormone" as used herein refers to the protein hormone also known as somatotropin.

The term "growth-related condition" as used herein refers to any condition, disorder or disease state associated with, resulting from or causing abnormal levels of growth hormone and/or abnormal activity or action of growth hormone. In such conditions, growth hormone levels may be io elevated or reduced in comparison to normal basal levels of growth hormone in the population.

The term "expression" as used herein refers interchangeably to expression of a gene or gene product, including the encoded protein. Expression of a gene product may be determined, for example, by immunoassay using an antibody(ies) that bind with the polypeptide. Accordingly, in the context of the present invention, expression may refer to the expression of a hemoglobin α- i5 chain polypeptide. As used herein the term "polypeptide" means a polymer made up of amino acids linked together by peptide bonds.

Brief Description of the Drawings

Embodiments of the present invention will now be described, by way of example only, with 20 reference to the accompanying drawings:

Figure 1. Mean peak intensities, determined by SELDI-TOF MS, of the 15.1 kDa peak identified as hemoglobin α-chain in three treatment groups of 20 athletes before treatment (dark; day 0) and at day 21 (light). Treatment groups are placebo, low-dose growth hormone (Low-GH;

0.1 IU/kg/day) and high-dose growth hormone (High-GH; 0.2 IU/kg/day). All values are presented

25 as mean ± SE.

Figure 2. Mean peak intensities, determined by SELDI-TOF MS, of the 15.1 kDa peak identified as hemoglobin α-chain in three treatment groups of 20 athletes at 7 day intervals before (day 0), during (days 7-28) and after (days 35-84) treatment. Treatment groups are placebo (PL- triangle), low-dose growth hormone (LO- square; 0.1 IU/kg/day) and high-dose growth hormone 30 (HI- diamond; 0.2 IU/kg/day). All values are presented as mean ± SE.

Best Mode of Performing the Invention

The present invention provides a novel biomarker that is shown herein to be useful in determining levels of growth hormone in subjects. The biomarker is the α chain of hemoglobin

35 (HBA1). HBA1 levels are shown to be positively correlated with growth hormone action. As exemplified herein the present inventors have found that subjects treated with growth hormone display higher serum levels of HBA1 than subjects treated with placebo, and higher than all subject groups before treatment. Further, the levels of HBA1 were observed to remain high throughout the treatment period before returning to baseline values within 2 weeks of cessation of treatment. Accordingly, the present invention can be used to identify individuals with over- or under-production of endogenous growth hormone or to identify individuals using, or having been administered, exogenous growth hormone.

Accordingly, one aspect of the present invention relates to a method for determining the level of growth hormone action in a subject, the method comprising the steps of obtaining a biological sample from the subject and analysing the biological sample to determine the amount of hemoglobin α-chain present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of growth hormone action.

It will be understood that the hemoglobin α-chain (HBA) to be measured may exist free from association with other molecules, or may be bound to other molecules, such as other hemoglobin chains.

For the purposes of the present invention HBA includes the product of the HBA1 and HBA2 genes and variant forms thereof. For example, a variant of HBA1 or 2 may include one or more amino acid substitutions such that although the primary sequence of the polypeptide is altered, the activity of the polypeptide is fully or partially retained. Many examples of HBA1 variants are known to those skilled the art, such as, for example, hemoglobin O-lndonesia and hemoglobin J-Oxford. Other known variants are listed in various publicly available databases such as HbVar at http://qlobin.cse.psu.edu/qlobin/hbvar/menu.html.

Further, those skilled in the art will appreciate that the level of HBA may be measured in a variety of body fluids or tissues, including but not limited to serum, plasma, urine and saliva. As exemplified herein, the method of surface-enhanced laser desorption-ionisation time-of- flight mass spectrometry (SELDI-TOF MS) may be used to measure HBA , but the invention is not limited to the use of such a method. Those skilled in the art will readily appreciate that any method that is able to measure HBA will be useful in determining the level of growth hormone and is contemplated as forming part of the present invention. Typically, levels of HBA polypeptide may be assayed.

Particular embodiments of the invention provide the use of one or more antibodies raised against HBA, either free or in association with other molecules, for the detection of HBA and the determination of HBA levels. The antibodies may be polyclonal or monoclonal and may be raised by the use of HBA or an antigenic fragment or portion thereof as an antigen. Antibody binding may be detected by virtue of a detectable label on the primary anti- HBA antibody. Alternatively, the anti- HBA antibody may be detected by virtue of its binding with a secondary antibody or reagent that is appropriately labeled to enable detection. A variety of methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example determinations of HBA levels can be accomplished by any one of a number of techniques known in the art including, for example enzyme-linked immunosorbent assays (ELISA); sandwich immunoassays, immunoradiometric assays (IRMA), radioimmunoassays (RIA), immunoelectrophoresis assays, In situ immunoassays, immunodiffusion assays, immunofluorescence assays, Western blots, ligand-binding assays, and the like.

Methods of the invention for determining growth hormone action may include the step of comparing the level of HBA in a sample obtained from the subject of interest, for example an individual suspected of the illicit use of exogenous growth hormone, with the level of HBA from one or more control samples. Typically the control sample may be a sample from an individual with normal levels of growth hormone and/or known not to use exogenous growth hormone.

Antibodies suitable for use in the methods of the present invention can be raised against HBA using techniques known to those in the art. Suitable antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanised, single chain, Fab fragments, and a Fab expression library.

Suitable antibodies may be prepared from discrete regions or fragments of the HBA polypeptide. An antigenic HBA polypeptide contains at least about 5, and typically at least about 10, amino acids.

Methods for the generation of suitable antibodies will be readily appreciated by those skilled in the art. For example, an anti-HBA1 monoclonal antibody, typically containing Fab portions, may be prepared using the hybridoma technology described in Antibodies-A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, N.Y. (1988). In essence, in the preparation of monoclonal antibodies directed toward HBA, fragment or analogue thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include the hybridoma technique originally developed by Kohler et aL, Nature, 256:495-497 (1975), as well as the trioma technique, the human B-cell hybridoma technique [Kozbor et a/., Immunology Today, 4:72 (1983)], and the EBV-hybridoma technique to produce human monoclonal antibodies [Cole et aL, in Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., (1985)]. Immortal, antibody-producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein- Barr virus. See, e.g., M. Schreier et aL, "Hybridoma Techniques" (1980); Hammerling et al., "Monoclonal Antibodies and T-cell Hybridomas" (1981); Kennett et aL, "Monoclonal Antibodies" (1980). A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity, The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques.

Similarly, there are various procedures known in the art which may be used for the production of polyclonal antibodies to HBA, or fragments or analogues thereof. For the production of polyclonal antibody, various host animals can be immunized by injection with the HBA polypeptide, or a fragment or analogue thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. Further, the HBA polypeptide or fragment or analogue thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Also, various adjuvants may be used to increase the immunological response, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Screening for the desired antibody can also be accomplished by a variety of techniques known in the art. Assays for immunospecific binding of antibodies may include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, and ^'Immunoelectrophoresis assays, and the like (see, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).

Methods of the present invention as disclosed herein may be used in conjunction with other diagnostic methods for the determination of the level of growth hormone action. For example, it may be beneficial to utilise a method of the present invention based on measurement of HBA levels, as an adjunct to one or more alternative methods based on measurement of growth hormone biomarkers such as IGF-I, lGFBP-3, ALS and PIIINP.

The present invention also provides kits for the determination of the level of growth hormone action, the diagnosis of altered growth hormone expression levels or growth-related conditions, wherein the kits facilitate the employment of methods of the invention. Typically, kits for carrying out a method of the invention contain all the necessary reagents to carry out the method. For example, in one embodiment the kit may comprise a first container containing an antibody raised against HBA and a second container containing a conjugate comprising a binding partner of the antibody, together with a detectable label.

Typically, the kits described above will also comprise one or more other containers, containing for example, wash reagents, and/or other reagents capable of quantitatively detecting the presence of bound antibodies, Preferably, the detection reagents include labelled (secondary) antibodies or, where the antibody raised against HBA is itself labelled, the compartments comprise antibody binding reagents capable of reacting with the labelled antibody,

In the context of the present invention, a compartmentalised kit includes any kit in which reagents are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of reagents from one compartment to another compartment whilst avoiding cross-contamination of the samples and reagents, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion. Such kits may also include a container which will accept the test sample, a container which contains the antibody(s) used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and like), and containers which contain the detection reagent,

Kits of the invention may incorporate a variety of methodologies for determining levels of growth hormone action. For example it may be advantageous for a kit to comprise not only reagents for carrying out one or more of the methods of the present invention but also reagents for carrying out other methods, such as IGF-I, PIIINP, IGFBP-3 or ALS based immunoassays for example.

Typically, a kit of the present invention will also include instructions for using the kit components to conduct the appropriate methods.

Methods and kits of the present invention are equally applicable to any animal, including humans, for example including non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline, canine and murine species. Accordingly, for application to different species, a single kit of the invention may be applicable, or alternatively different kits, for example containing reagents specific for each individual species, may be required.

Methods and kits of the present invention find application in any circumstance in which it is desirable to determine growth hormone levels or obtain an indication of growth hormone action. The invention can be applied to the determination of endogenous growth hormone production in a subject or to the identification of the presence of exogenous growth hormone in a subject. For example, the determination of levels of endogenous growth hormone action using methods and kits of the invention finds application in the diagnosis of growth hormone deficiency or excess in individuals, such as in children or teenagers with growth retardation or in the diagnosis of children or teenagers with accelerated growth. The invention also finds application in the diagnosis of growth hormone deficiency in adults, the diagnosis of conditions of growth hormone excess, such as acromegaly, in adults or in the monitoring of patients treated with growth hormone for growth hormone deficiency or other conditions.

5 Exogenous growth hormone is used illicitly by some athletes and sportspeople to enhance performance. The detection of such illicit use is a constant concern for sporting authorities and drug testing agencies are typically employed to carry out screening programs for illicit use of substances including growth hormone. The methods and kits of the present invention provide a simple, rapid and reliable means of detecting the use of exogenous growth hormone. io Similarly, animals such as racehorses may be administered exogenous growth hormone to enhance performance. The methods and kits of the present invention may also be applied to the testing and screening of such animals. Further, some animals intended for human consumption or that produce products for human consumption, such as dairy cattle, may be administered growth hormone. It may be beneficial to be able to reliably detect such animals, for example in i₅ establishing and certifying that a product for human consumption is growth hormone-free. Tests may also be developed based on the methods of the present invention to measure growth hormone status in animals, for example to monitor animal growth studies.

The present invention will now be further described in greater detail by reference to the 20 following specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples

Example 1. Subjects

25 Serum samples were obtained from the former program established to devise new methods of detecting growth hormone abuse by competitive athletes (GH2000). The original study design was a double-blind, placebo-controlled trial of 49 women and 50 men, physically fit and aged 26±1 years, in which blood was collected weekly during a 4-week treatment period (days 1-28). The subjects were subsequently followed for 8 more weeks (days 29-84; the washout period). The

3o treatment arms included: placebo, (n=40), 0.1 IU/kg/day growth hormone, (low growth hormone, n=30), 0.2 IU/kg/day growth hormone, (high growth hormone, n=29). Most but not all serum samples from these three treatment groups at the various timepoints were available for the present study. All samples were stored at -80⁰C until use. Example 2. Cu2+ -IMAC ProteinChip Array Analysis

In preliminary experiments, it was established that immobilized metal affinity capture (IMAC) ProteinChip arrays (Ciphergen Biosystems, Inc.) loaded with Cu²⁺ provided the best discrimination between serum from subjects treated with growth hormone compared to untreated subjects, when using protein chip mass spectrometry as the detection method,

120 serum samples from 60 athletes prior to treatment at day 0, and after 21 days of treatment, were analyzed on Cu²⁺-IMAC ProteinChip arrays. The treatment arms included: Placebo, (n=20), 0.1 IU/kg/day growth hormone (low growth hormone, n=20), 0.2 IU/kg/day growth hormone (high growth hormone, n=20). The array spots were pre-activated with 100 mM CuSCU for 10 minutes at room temperature, followed by one wash with dH∑O. 20 μl serum were mixed with 30 μl denaturing buffer (8 M urea, 1% CHAPS, 1OmM Na Phosphate pH 7.2, 15OmM NaCI), and diluted 1:25 in binding buffer (2OmM Na Phosphate pH 7.2, 0.5M NaCI) and pipetted onto the spots, Following incubation for 30 min, the unbound proteins were removed by washing with binding buffer (3x5 μl), After two rinses with dhbO, an energy absorbing matrix solution (20 mg/mL sinapinic acid, 50% acetonitrile, 0.5% trifluoroacetic acid; 2 x 1 μl) was applied and air-dried for 10 min. Finally, the arrays were placed into the ProteinChip Reader (ProteinChip Biology System II, Ciphergen Biosystems, Inc.).

Time-of-flight spectra of proteins were generated by using an average of 80 laser shots at a laser intensity of 210 to 220 arbitrary units. For data acquisition of low-molecular weight proteins, the detection size range was between 5000 and 25,000 Da, Detector sensitivity was set at 8 and laser intensity was set at 210. For the high-molecular weight proteins, the detection size range was between 25,000 and 70,000 Da. The detector sensitivity was set at 8 and the laser intensity was set at 220, The surface-enhanced laser desorption-ionisation time-of-flight (SELDI-TOF) mass spectrometer was externally calibrated using the [M+H]⁺ ion peaks of insulin (bovine) at 5734.51 m/z, cytochrome c (equine) at 12,361.96 m/z, apomyoglobin (equine) at 16,952.27 m/z, Carbonic anhydrase (bovine) at 29, 023.70 m/z, aldolase (rabbit muscle) 39,212.28 m/z, glucose-6- phosphate dehydrogenase at 57,432.72 m/z and albumin (bovine serum) at 66,430.09 m/z, respectively (Sigma).

Intra-chip reproducibility was checked by spotting eight different aliquots of one sample on the same array, and inter-chip reproducibility was checked by including one given sample on every different array. The intra- and inter-chip coefficients of variation of peak intensity were assessed for all protein peaks of more than background according to the setting of detection. The means of intra- and inter-chip coefficient of variations of peak intensity were 16.2% and 24.4%, respectively. According to the manufacturer, the mass accuracy of the spectrometer is 0.1%. The detection of proteins in serum samples at various sampling times other than day O and day 21 was also performed for ten subjects in each of the three treatment groups. Samples taken at days 0, 21 and 28 after the administration of placebo, low growth hormone or high growth hormone, and days 30, 33, 42 and 84 of the washout period, were subjected to SELDI-TOF analysis using the Cu²⁺-IMAC chip method as described above, The statistical analyses were done using the Statview software package (SAS institute, Inc.Cary, NC). Repeated measures ANOVA was followed by Fisher's PLSD test, Results with P values below 0.05 were regarded as significant.

All spectra were compiled, and qualified mass peaks (signal-to-noise ratio >5) with mass-to- charge ratios [mil) between 3000 and 70,000 were autodetected. Peak clusters were completed using second-pass peak selection (signal-to-noise ratio >2, within 0.3% mass window), and estimated peaks were added. The data were analyzed with ProteinChip Biomarker Wizard Software version 3.0.2 (Ciphergen Biosystems, Inc.). The peak intensities were normalized to the total ion current of the mass spectrometry signal between 5,000 and 25,000 Da for the low- molecular weight range and between 25,000 and 70,000 for the high-molecular weight range. To characterize protein peaks of potential interest, serum proteomic profiles of athletes after 21 days treatment (n = 60) or before treatment (n = 60) were compared.

Example 3. Purification and identification of HBA1 as a protein biomarker

A protein of apparent molecular mass approximately 15.1 kDa consistently appeared to discriminate between serum samples from subjects after growth hormone treatment, and those before growth hormone treatment. Since this protein appeared to have potential utility as a biomarker of growth hormone action, it was purified and the N-terminal amino acid sequence determined.

For purification, sera were diluted in 1:10 with binding buffer, 5OmM NhU acetate pH 6.0, and centrifuged at 12,000 rpm for 10 min at room temperature. The proteins were loaded onto an HiTrap CM Sepharose FF column (1 ml, Amersham Biosciences) using AKTA purifer (Pharmacia), and the material was eluted with a linear gradient 0-1 M NaCI in binding buffer at a flow rate of 1 ml/min. Eluted fractions were analyzed on a Cu²⁺-IMAC ProteinChip array to monitor the elution and recovery of the protein of interest. Proteins eluted between 0.15 M and 0,2 M NaCI were pooled and loaded onto a RP-HPLC C18-column (250x4.6 mm, 3OθA pore size, Phenomenex), eluted with a linear gradient of 15-60% acetonitrile in 0.1 % TFA over 30 min at 1.5 ml/min. All peaks were collected, lyophilized and analyzed on a hydrophilic NP20 ProteinChip array (Ciphergen Biosystems, Inc.). Subsequent N-terminal sequencing was carried out using an Applied Biosystems 494 Procise Protein Sequencing System at the Australian Proteome Analysis Facility. N-terminal sequencing of the 15.1kDa protein revealed the sequence VLSPADKT- VKA which corresponds to hemoglobin α-chain (Swiss-Prot accession no. P69905) a protein of 15126 molecular weight.

Example 4. Use of HBA1 as a biomarker for growth hormone status

The levels of HBA1 were determined as described in previous Examples in samples from athletes treated daily for 21 days with either placebo, (n=20), 0,1 IU/kg/day growth hormone (low growth hormone, n=20), 0.2 iU/kg/day growth hormone (high growth hormone, n=20). HBA1 levels were measured by the intensity of the peak at an approximate mass/charge ratio of 15,100, as determined by SELDI-TOF MS. (As described in Example 3, this peak was identified as being HBA1.) As shown in Figure 1 , athletes treated with exogenous growth hormone displayed higher serum levels of HBA1 than did those athletes treated with placebo. Further the levels of HBA1 were significantly higher in those athletes treated with growth hormone (at day 21) than in any of the treatment groups (placebo, high GH or low GH) at day 0. Figure 2 shows a time course of HBA levels in the three treatment groups as defined above.

HBA levels were determined as described above at 7 day intervals prior to treatment (day 0), during treatment (days 7-28) and following treatment (the washout period; days 35-84). As illustrated in Figure 2 levels of HBA1 remained high through the treatment period (up to day 28), but fell rapidly after the cessation of treatment, returning to baseline levels by approx. 2 weeks post treatment (day 42).

These results indicate that the measurement of HBA1 in serum provides a novel means of detecting the action of growth hormone, and is a biomarker of growth hormone action.

Claims

1. A method for determining the level of growth hormone action in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and 5 (b) analysing the biological sample to determine the amount of hemoglobin α-chain present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of growth hormone action.

2. The method of claim 1 wherein the sample is serum, plasma, urine, saliva or other body fluid. io

3. The method of claim 2 wherein the sample is serum.

4. The method of any one of claims 1 to 3 wherein the hemoglobin α-chain is free hemoglobin α-chain or a variant thereof.

5. The method of any one of the preceding claims wherein the hemoglobin α-chain is encoded by the HBA1 gene or variant thereof or HBA2 gene or variant thereof. i5

6. The method of claim 5 wherein the hemoglobin α-chain is encoded by the HBA1 gene or variant thereof.

7. The method of any one of the preceding claims wherein the amount of hemoglobin α-chain is measured using an immunoassay.

8. The method of claim 7 wherein the immunoassay is an enzyme-linked immunoassay or 20 radioimmunoassay.

9. The method of any one of the preceding claims wherein the level of growth hormone action in the subject is indicative of the administration to the subject of exogenous growth hormone.

10. The method of any one of claims 1 to 8 wherein the level of growth hormone action in the subject is indicative of the level of expression of endogenous growth hormone in the subject.

25 11. The method of any one of the preceding claims wherein the method comprises the further step of:

(c) comparing the level of hemoglobin α-chain in the sample obtained from the subject with the level of hemoglobin α-chain from one or more control samples.

12. The method of claim 11 wherein the one or more control samples are from one or more 30 subjects with normal levels of growth hormone and/or known not to use exogenous growth hormone.

13. The method of any one of the preceding claims wherein the subject is selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline, canine and murine.

35 14. The method of claim 13 wherein the subject is human.

15. The method of claim 13 wherein the subject is equine,

16. method of claim 13 wherein the subject is bovine or ovine.

17. A method for diagnosing in a subject the over-expression or under-expression of endogenous growth hormone, the method comprising the steps of: (a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of hemoglobin α-chain present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of expression of growth hormone,

18. The method of claim 17 wherein the sample is serum, plasma, urine, saliva or other body fluid.

19. The method of claim 17 or 18 wherein the hemoglobin α-chain is free hemoglobin α-chain or a variant thereof.

20. The method of any one of claims 17 to 19 wherein the method comprises the further step of: (c) comparing the level of hemoglobin α-chain in the sample obtained from the subject with the level of hemoglobin α-chain from one or more control samples,

21. The method of claim 20 wherein the one or more control samples are from one or more subjects with normal levels of growth hormone.

22. The method of any one of claims 17 to 21 wherein over-expression of growth hormone in the subject is associated with a condition selected from the group consisting of: acromegaly, gigantism, a growth hormone-secreting tumour or any other condition resulting from over-expression of growth hormone.

23. The method of claim 22 wherein under-expression of growth hormone in the subject is associated with a condition selected from the group consisting of: growth retardation, dwarfism, Turner's syndrome, injury to the pituitary gland, other forms of pituitary insufficiency, or any other condition resulting from under-expression of growth hormone..

24. A method for diagnosing a growth-related condition in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and (b) analysing the biological sample to determine the amount of hemoglobin α-chain present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of growth hormone action.

25. The method of claim 24 wherein the sample is serum, plasma, urine, saliva or other body fluid.

26. The method of claim 24 or 25 wherein the hemoglobin α-chain is free hemoglobin α-chain or a variant thereof.

27. The method of any one of claims 24 to 26 wherein the growth-related condition is selected from the group consisting of acromegaly, gigantism, hyperpituitarism, growth retardation, fetal

5 growth retardation, dwarfism, a growth hormone receptor defect, a growth hormone-secreting tumour, injury to the pituitary gland, or any other condition associated with growth hormone deficiency or excess.

28. A method for detecting in a subject the illicit administration of exogenous growth hormone, the method comprising the steps of: Q (a) obtaining a biological sample from the subject; and

(b) analysing the biological sample to determine the amount of hemoglobin α-chain present, wherein the amount of hemoglobin α-chain present in the sample is indicative of the level of growth hormone action. s 29. The method of claim 28 wherein the sample is serum, plasma, urine, saliva or other body fluid.

30. The method of claim 28 or 29 wherein the hemoglobin α-chain is free hemoglobin α-chain or a variant thereof.

31. The method of any one of claims 28 to 30 wherein the method comprises the further step of: Q (c) comparing the level of hemoglobin α-chain in the sample obtained from the subject with the level of hemoglobin α-chain from one or more control samples.

32. The method of claim 31 wherein the one or more control samples are from one or more subjects with normal levels of growth hormone and/or known not to use exogenous growth hormone.. ₅

33. The method of any one of claims 28 to 32 wherein the subject is a sportsperson.

34. The method of claim 33 wherein the sportsperson is an elite athlete.

35. The method of claim any one of claims 28 to 32 wherein the subject is a non-human animal.

36. The method of claim 35 wherein the non-human animal is a racehorse.

37. The method of claim 35 wherein the non-human animal is raised for breeding or human 0 consumption.

38. A diagnostic kit for use in determining the level of growth hormone in a subject, the kit comprising at least one agent for measuring hemoglobin α-chain in a biological sample.

39. The kit of claim 24 wherein the agent is an antibody that recognises and binds hemoglobin α-chain or a variant thereof.

40. The kit of claim 38 or 39 wherein the kit comprises a first container containing an antibody raised against hemoglobin α-chain and a second container containing a conjugate comprising a binding partner of the antibody, together with a detectable label.

41. The kit of any one of claims 38 to 40 wherein the kit further comprises at least one agent for measuring one or more other growth hormone biomarkers.

42. The kit of claim 41 wherein the one or more other growth hormone biomarkers are selected from the group consisting of: IGF-I, PIIINP, IGFBP-3 and ALS.