WO2005059557A1 - Methods of predicting, diagnosing and monitoring term and pre-term labour - Google Patents

Abstract

The present invention provides a method of predicting term or pre-term labour in a subject, the method including the steps of determining a level of a cathelicidin or fragment thereof, or a polynucleotide encoding a cathelicidin or fragment thereof, in a biological sample derived from the subject, and comparing the level of the cathelicidin or fragment thereof to a predetermined value.

Description

METHODS OF PREDICTING. DIAGNOSING AND MONITORING TERM AND PRE-TERM LABOUR

This invention relates generally to methods, agents and kits for predicting, diagnosing or monitoring term or pre-term labour. The invention also provides a method of treating a subject at risk of pre-term labour.

BACKGROUND

Pre-term labour, characterized by cervical effacement and/or dilatation, and increased uterine irritability occurring before 37 weeks gestation, is one of the most important and costly problems in obstetric medicine and remains a leading cause of infant mortality and long-term neurological handicap. More importantly, the younger the fetus at birth, the more serious the problems it is likely to experience in surviving the neonatal period, and the greater the probability of ongoing physical and intellectual defects. Recent advances in the care of premature newboms include developments in respiratory technology. However, despite these advances, pre-term birth has been found to present serious fiscal and social implications for the families affected and consequently imposes an increased burden on both the health and education systems.

To address this issue, there has been some advancement in existing treatment strategies towards delaying pre-term gestation (e.g. tocolysis, corticosteroid therapy, bed rest). However, their effectiveness and/or benefit is largely dependent upon the practitioner's ability to predict the onset of a pre-term labour. In most cases, the mother presents with established labour and there is insufficient time to implement any of the existing treatments. At present, the rate of premature births is steadily increasing, where approximately one in every seven pregnancies in developed countries is a pre-term labour. Accordingly, there is a critical need to predict and diagnose the onset of pre-term labour.

A similar situation arises at the veterinary level, where any offspring delivered prematurely may have a reduced chance of survival, especially if exposed to inclement conditions. This can be extremely costly to breeders of elite animals. Other problems occur during breeding seasons when there is a large number of expectant animals. The general practice is that a veterinarian needs to be on call during this period, which could be quite costly for the breeders. If the birth at term of pregnancy could be predicted, then the veterinarian could be on call only during the predicted times for the birth of the animal.

Prematurely born infant animals are also disadvantaged because their mother's colostrum will be of poorer quality, and arguably, their specialised colostrum absorption cells will be too immature to function properly. Accordingly, if pre- term birth can be predicted, then animals may be treated for the purposes of delaying the onset of labour, allowing the mother's colostrum to reach its desired quality and for the specialised colostrum absorption cells of the unborn animal to fully develop.

Attempts to predict the onset of term or pre-term labour have proven ineffective. Previous attempts include routine weekly cervical assessment, transvaginal ultrasound examination of cervical length and home monitoring of uterine activity. All of these require constant monitoring and in the case of farm animals, the animal would need to be under constant supervision. This may be inconvenient, particularly when it is difficult to house animals for their constant surveillance.

The use of biological markers, such as fetal fibronectin, matrix metalloproteinase- 8, vaginal human defensins and interleukin-6, have also proven ineffective in predicting the onset of term or pre-term labour.

Thus, the limitations of presently available strategies as they relate to the assessment of risk and the prediction of term and pre-term labour highlight the need for a novel and reliable diagnostic and prognostic indicator of this condition.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. SUMMARY OF THE INVENTION

In an aspect of the present invention, there is provided a method of predicting term or pre-term labour in a subject, the method comprising the steps of determining a level of a cathelicidin or fragment thereof in a biological sample derived from the subject, and comparing the level of the cathelicidin or fragment thereof to a predetermined value.

In another aspect of the present invention, there is provided a method of predicting term or pre-term labour in a subject, the method including the steps of determining the level of a polynucleotide encoding a cathelicidin or a fragment thereof in a biological sample derived from the subject, and comparing the level of the polynucleotide encoding the cathelicidin or fragment thereof to a predetermined value.

In yet another aspect of the present invention, there is provided a method of monitoring a subject for term or pre-term labour, the method comprising the steps of determining a level of a cathelicidin or fragment thereof in a first biological sample derived from the subject, and comparing the level of the cathelicidin or fragment thereof to a level of the cathelicidin or fragment thereof identified in a second biological sample obtained from the same subject.

In yet another aspect of the present invention, there is provided a method of monitoring a subject for term or pre-term labour, the mej iod comprising the steps of determining a level of a polynucleotide encoding a cathelicidin or a fragment thereof in a first biological sample derived from the subject, and comparing the level of the polynucleotide to a level of a polynucleotide encoding the cathelicidin or fragment thereof identified in a second biological sample obtained from the same subject.

Any one of the cathelicidins or equivalents may be used to predict or monitor for term or pre-term labour including hCAP18, LL-37, bactenicin-1 , -5 and -7, eCATH-1 , -2 and -3, SMAP29, BMAP-27, BMAP-28, protegrin-1 to -5, PMAP- 23, PMAP-36, PMAP-37 and mCRAMP. In yet another aspect, the present invention provides a kit when used to predict or monitor term or pre-term labour in a subject, the kit comprising an agent which detects a cathelicidin or fragment thereof and a detection system for detecting the agent.

The agent may be an antibody to cathelicidin or fragment thereof.

In a further aspect, the present invention provides a kit when used to monitor or predict a subject in term or pre-term labour, the kit comprising an agent which detects a polynucleotide which encodes a cathelicidin or fragment thereof and a detection system for detecting the agent.

The agent may be a primer which hybridises to a polynucleotide which encodes a cathelicidin or fragment thereof.

In yet another aspect of the present invention, there is provided a method of treating a subject identified as being in, or at risk of, pre-term labour as herein described, the method comprising administering to^" the subject a treatment designed to delay or prevent the onset or progression of pre-term labour.

FIGURES

The invention is now more clearly described with reference to, but not limited to, the accompanying figures, wherein:

Figure 1 shows the statistical comparisons of a study assessing changes in protein expression between non-labour and labour samples of a spontaneous labour group and a dexamethasone-induced labour group. Comparisons were made within individual treatment groups, and between the two treatment groups.

Figure 2 shows a display of the rehydration tray and the application of contamination absorbers (wicks) beneath the immobilized protein gradient (IPG) strips for the purposes of first dimension protein separation. The first dimension separates proteins based on their isoelectric points (the specific point at which a protein will not migrate electrophoretically in a pH gradient, and carries zero charge).

Figure 3 shows a collection of different sized proteins which have been focussed on a pH 3-10 IPG strip according to each protein's isoelectric point (pi), and independently of size.

Figure 4 shows a schematic diagram of the separation of proteins during second dimension separation following isoelectric focusing (IEF).

Figure 5 shows a raw image of the protein expression from cervicovaginal fluid (CVF) suspended in a buffer containing 150mM NaCI from a spontaneous labouring ewe which is not in labour (GA137). 15μg of protein was loaded into the gel. Molecular weight markers are shown on the left.

Figure 6 shows a raw image of the protein expression from CVF suspended in a buffer containing 15mM NaCI from a spontaneous labouring ewe during labour onset at term (GA143). 15μg of protein was loaded into the gel. Molecular weight markers are shown on the left.

Figure 7 shows a raw image of a protein expression from CVF from a spontaneous labouring ewe which is not in labour (GA 137). 15μg of protein was loaded into the gel. Molecular weight markers are shown on the left.

Figure 8 shows a raw image of a protein expression from CVF following a TCA precipitation prior to first dimension separation from a spontaneous labouring ewe which is not in labour (GA 137). 15μg of protein was loaded into the gel. Molecular weight markers are shown on the left.

Figure 9 shows filtered images CVF protein expression of non-labour and labour states in spontaneous and induced labour ewes: (A) a CVF sample taken during non-labour (GA 137) from a spontaneous labour sheep; (B) the corresponding labour onset sample taken from sheep TV at term (GA144); (C) a CVF sample taken during non-labour (GA 135) prior to dexamethasone treatment; and (D) the corresponding labour onset sample from sheep 'C following labour induction with dexamethasone (GA 137).

Figure 10 shows a computer-generated distribution of all proteins expressed in this analysis series of cervicovaginal fluid known as the reference standard.

Original reference image was that of a spontaneous labour onset at term sheep (GA 145, spot number 534). Following series matching, spot number equals 2003, with 85 unmatched spots. Any protein spots without a letter, circle or box subjected to them are classed as unmatched. Spot no. 6003 is indicated by arrow for reference purposes later described.

Figure 11 shows a computer-generated profile of protein expression in CVF of a labour induced animal. Twenty eight proteins have been identified as being up- regulated by a factor of 2 in labour onset after dexamethasone treatment Vs non- labour samples prior to dexamethasone treatment.

Figure 12 shows a graphical display of the number of protein spots relative to the intensity by which they are up-regulated by in a comparison with spontaneous onset labour (L) at term and dexamethasone induced labour onset (L). Dexamethasone treated ewes show a higher number of proteins that are upregulated at each intensity factor in induced labour samples compared spontaneous labour samples at term. Untreated ewes during spontaneous labour onset possess approximately half as many proteins as dexamethasone ewes during labour onset, which have up-regulated intensities when compared with identical proteins in labour induced samples.

Figure 13 shows a graphical display of the number of protein spots relative to the intensity by which they are up-regulated by in a comparison with non-labour samples (NL) Vs labour onset (L) within both treatment groups. Dexamethasone treated ewes show a higher number of proteins that are up-regulated at each intensity factor in labour induced samples compared spontaneous labour samples. Untreated ewes during spontaneous labour onset possess approximately half as many proteins as dexamethasone ewes during labour onset, which have up-regulated intensities when compared with identical proteins in labour induced samples.

Figure 14 shows a graphical display of the number of protein spots relative to the intensity by which they are up-regulated by in a comparison between non-labour samples (NL) from both animal groups. Untreated ewes during their gestational period (non-labour, GA 137) possess several proteins which have increased intensities than in gestationally age-matched ewes prior to Dexamethasone administration (non-labour, GA 135). Dexamethasone ewes did not show any proteins which have higher non-labour intensities compared to spontaneously gestating ewes.

Figure 15 shows the intensity changes of five proteins from non-labour (NL) to labour onset (L) in spontaneous labour sheep. Protein identification numbers are indicated on the right. Two proteins (nos. 3408 and 6003) showed a significant increase during labour onset at term compared to during non labour.

Figure 16 shows the intensity changes of five proteins from non-labour (NL) to labour onset (L) in dexamethasone induced labour sheep. Protein identification numbers are indicated on the right. Two proteins (nos. 4713 and 6003) showed a significant increase during induced labour onset compared to non-labour samples taken prior to dexamethasone administration.

Figure 17 shows a 2D gel of human CVF.

DETAILED DESCRIPTION OF THE INVENTION

In an aspect of the present invention, there is provided a method of predicting term or pre-term labour in a subject, the method comprising the steps of determining a level of a cathelicidin or fragment thereof in a biological sample derived from the subject, and comparing the level of the cathelicidin or fragment thereof to a predetermined value

Cathelicidins are a family of antimicrobial and endotoxin-binding proteins found predominantly in peroxidase-negative granules of vertebrate neutrophils, whose name derives from the highly conserved N-terminal domain called cathelin. Synthesized as preproproteins, cathelicidins are subject to proteolytic digestion by serine proteases released from the azurophil granules of neutrophils, which in turn liberates the highly variable C-terminal domain in which the antimicrobial activity resides. Whilst highly cationic and hydrophobic, the C-termini of cathelicidins vary greatly in amino acid sequence and structure, ranging from proline- and arginine-rich sequences to sequences forming amphipathic α- helices.

In humans, there exists an 18 kDa cationic antimicrobial peptide (hCAP-18), present in the secondary (specific) granules of neutrophils. hCAP-18 is found in plasma bound to lipoproteins and is expressed in various non-myeloid tissues such as the epididymis, keratinocytes, epithelial cells and lymphocytes.

The biologically active C-terminal antimicrobial domain of hCAP-18 is a 37 amino acid peptide called LL-37, liberated by primary (azurophil) granule-derived proteinase 3 during degranulation and secretion. LL-37 shows broad antimicrobial activity toward both gram-negative and gram-positive bacteria, has synergistic antibacterial effects with the defensins and is a chemotactic agent for neutrophils, monocytes, and T lymphocytes via the formyl peptide receptor-like 1 receptor.

In spite of the aforementioned breadth of scientific development in the art, cathelicidins have not been previously implicated in reproductive physiology. Surprisingly, it has now been found that the level of cathelicidin in biological samples obtained from a pregnant subject is increased both prior to and during the onset of term or pre-term labour. The pattern of cathelicidin expression or the cathelicidin expressed in the subject can be used to predict the onset of term or pre-term labour.

As used herein, the terms "predicting", "diagnosing" and the like are used interchangeably with reference to identifying a "predisposition" or "propensity" and the like of a subject entering term or pre-term labour. The terms are also used interchangeably with reference to identifying a "risk" or "increased risk" of a subject entering pre-term labour. The terms specifically encompass identifying the propensity for a subject to enter into term or pre-term labour, whether or not there is prior knowledge of pregnancy in that subject by any other means available to the skilled addressee. Thus, the present invention may be used to predict the onset of term or pre-term labour at any time during pregnancy, whether labour appears be it days, weeks or months from the time of application of the methods described.

The term "pre-term labour", as used herein, denotes the expulsion from the uterus of a neonate before the normal end of gestation, as is well known to those skilled in the art. The term "neonate" denotes a newly born subject, whether it be a human or of another animal species. More particularly, pre-term labour is defined as the onset of labour, with effacement and dilation of the cervix. It may or may not be associated with vaginal bleeding or rupture of the membranes. By contrast, the term "labour", as used herein, denotes the expulsion from the uterus of an infant.

Analogous members of the cathelicidin family of antimicrobial proteins have been found in other species, including bovine (Bac5), equine (eCATH-1 , -2 and -3), ovine (SMAP29), leporine and murine (mCRAMP) cathelicidins. Accordingly, the methods of the present invention may be applicable to a subject of any number of species such as, but are not limited to, humans, sheep, pigs, cows, horses and mice. Accordingly, as used herein, the term "subject" is a reference to a human being, as well as a farm animal such as a cow, horse, goat, pig or sheep. Preferably, the subject is an elite animal such as a thoroughbred of horse or cattle. More preferably, the subject is a female of the species, whether or not the female subject presents with symptoms of pregnancy known to those in the art.

As used herein, the terms "control sample" or "standard sample" are used interchangeably and denote a sample comprising a pre-determined level of a cathelicidin or fragment thereof. Preferably, the control sample comprises a level of a cathelicidin or fragment thereof normally found in a population of subjects that are not pregnant, or those who are pregnant at the time of detection or measurement, but will not enter pre-term labour (negative control sample). This provides a "predetermined value" for a subject who is unlikely to enter into term or pre-term labour. Alternatively, the control sample may comprise a level of a cathelicidin or fragment thereof normally found in a population of subjects that are pregnant and are about to enter, or have entered into, term or pre-term labour at the time of detection or measurement (positive control sample). This provides a "predetermined value" for a subject who is likely to enter into term or pre-term labour. One of ordinary skill in the art can readily determine the normal range for a given population. The control sample may be prepared by diluting a known amount of a cathelicidin or fragment thereof into an appropriate assay diluent known to those skilled in the art, such as phosphate buffered saline. Preferably, the control sample is a biological sample obtained from a subject, or a pool of biological samples obtained from a number of subjects, whether or not they are of the same species as the biological sample that is subject to the measurement of a cathelicidin or fragment thereof. The biological sample may be modified prior to use, such as by dilution, purification of various fractions, centrifugation and the like. A control may also include a normalised standard curve representing varying levels of a cathelicidin or fragment thereof against which one can monitor and compare changes in the concentration of a cathelicidin or fragment thereof in a biological sample derived from a subject that is progressing towards term or pre-term labour.

In a preferred embodiment, the term "biological sample" includes, but is not limited to, bodily secretions, bodily fluids and tissue specimens. Examples of bodily secretions include cervicovaginal secretions, trachial-bronchial secretions and pharyngeal secretions. Examples of bodily fluids include amniotic fluid, maternal or fetal blood, serum or plasma, sweat, tears, cerebral spinal fluid, serum, sputum, urine, synovial fluid and saliva. Animals, cells and tissue specimens such as a biopsy, are also embraced by this term. Biological samples may be obtained from a subject by any number of means known to those skilled in the art, essentially consisting of collecting a few microlitres of fluid from the upper vagina at speculum examination (see, for example, Lucas et al., British Medical Journal 325:301-311 ). Preferably, the biological sample is obtained from human subjects during the period of 24-36 weeks gestation. In the case of non- human subjects such as farm or companion animals, sampling is preferably commenced at 2 standard deviations before the mean gestation length. In cattle, for example, the standard deviation of gestation length is about 10 days. Accordingly, in a preferred embodiment, sample collection would begin at 20 days before the expected day of term delivery. A "biological sample" may also be modified prior to use, such as by dilution, purification of various fractions, centrifugation and the like. Accordingly, a "biological sample" may refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. In a preferred embodiment, the biological sample is derived from female gestational fluid, tissue or secretions including, but not limited to, cervicovaginal fluid, amniotic fluid and uterine biopsy tissue.

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 exclude other additives, components, integers or steps.

In a preferred embodiment, the term "cathelicidin" encompasses cathelicidin polypeptides and fragments thereof in various forms, including naturally occurring variants. Examples of cathelicidins encompassed by the present invention include hCAP18, LL-37, bactenicin including bactenicin-1 , -5 and -7, eCATH-1 , - 2 and -3; SMAP29; BMAP-27, BMAP-28, protegrin-1 to -5, PMAP-23, PMAP-36, PMAP-37 and mCRAMP. These cathelicidins are preferably used for predicting term and pre-term labour in a subject, more preferably in human (hCAP18, LL- 37, bactenecin), cattle (bactenecin-5, bactenecin-7, BMAP-27 and BMAP-28), horses (eCATH-1 , -2 and -3), sheep (bactenicin-1 , bactenecin-5, SMAP-29), pigs (protegrins 1-5, PR-39, prophenins 1-2, PMAP-23, PMAP-36 and PMAP-37) or mice (mCRAMP). Preferably, in sheep the cathelicidin is bactenicin-1. The term "cathelicidin" also encompasses a naturally occurring nucleic acid molecule, or a fragment thereof, whose nucleotide sequence encodes a cathelicidin polypeptide or a naturally occurring variant or a fragment thereof. The nucleic acid molecule may be a deoxyribonucleic acid (DNA)^" molecule or a ribonucleic acid (RNA) molecule. Naturally occurring variants may exhibit amino acid sequences that are at least 80% identical to a native cathelicidin polypeptide or fragment thereof. Also contemplated are embodiments in which a variant comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the native cathelicidin polypeptide or fragment thereof. Percent identity may be determined by visual inspection and mathematical calculation. Among the naturally occurring variants and fragments thereof provided are variants of native cathelicidin that retain native biological activity or a substantial equivalent thereof. Also provided herein are naturally occurring variants that have no substantial biological activity.

Variants include polypeptides that are substantially homologous to the native form, but which have an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues when compared to a native sequence. A given sequence may be replaced, for example, by a residue having similar physiochemical characteristics. Examples of such conservative substitution of one aliphatic residue for another, such as lie, Val, Leu or Ala for one another; substitution of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp or Tyr for one another. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known in the art.

Preferably, the term "fragment" refers to a portion of a cathelicidin polypeptide, or a variant thereof, that comprises an immunogenic or antigenic region. A fragment therefore includes a portion of a cathelicidin polypeptide, or a variant thereof, that is recognized (i.e., specifically bound) by an immunoglobulin. Such fragments generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of a cathelicidin or a variant thereof. In a preferred embodiment, a cathelicidin fragment consists of the biologically active C-terminal domain of a cathelicidin polypeptide. Such fragments may generally be identified using well known techniques, such as those summarized in the prior art (see, for example, Paul, Fundamental Immunology (3rd ed., 243-247; Raven Press, 1993, and references cited therein). Such techniques include screening polypeptides for the ability to react with cathelicidin-specific antibodies and/or antisera.

As used herein, antisera and antibodies are "cathelicidin-specific" if they specifically bind to a cathelicidin polypeptide or a variant or fragment thereof (i.e., they react with a cathelicidin in an enzyme-linked immunosorbent assay [ELISA] or other immunoassay, and do not react detectably with unrelated polypeptides). Such antisera and antibodies may be prepared as described herein, and using well-known techniques (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

It is contemplated that procedures useful for measuring a cathelicidin or fragment thereof in a biological sample include, but are not limited to, immunological assays, such as immunoblotting, immocytochemistry, immunohistochemistry or antibody-affinity chromatography, electrophoretic analysis, such as one- or two- dimensional SDS-PAGE, Northern or Southern analysis, in vivo or in vitro enzymatic activity assay, the polymerase chain reaction (PCR), reverse- transcription PCR (RT-PCR), in situ nucleic acid hybridization, electrophoretic mobility shift analysis (EMSA), transcription assay, or variations or combinations of these or other techniques such as are known in the art. In general, a cathelicidin, or fragment thereof, may be detected in a biological sample obtained from a subject by any means available to the skilled addressee. In a preferred embodiment, the method of detection employs a binding agent. The binding agent provided herein generally permits detection of a level of a cathelicidin or fragment thereof that binds to the agent in the biological sample.

There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptides in a sample. In general, the level of a cathelicidin in a biological sample obtained from a subject may be determined by contacting a biological sample obtained from a patient with a binding agent, detecting in the sample a level of a cathelicidin that binds to the binding agent and comparing the level of a cathelicidin with a predetermined value normally found in a control sample, whether it be a negative control sample or a positive control sample, as herein described.

In a preferred embodiment, the methods as herein described involve the use of binding agent immobilized on a solid support to bind to and remove a cathelicidin from the remainder of the sample. The bound cathelicidin may then be detected using a detection reagent that contains a reporter group and specifically binds to the cathelicidin/binding agent complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to a cathelicidin, or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a cathelicidin is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the biological sample. The extent to which components of the sample inhibit the binding of the labeled cathelicidin to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. A suitable cathelicidin for use within such assays include full-length cathelicidin polypeptides or variants or fragments thereof, to which the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skill in the art to which a binding agent may be attached. For example, the solid support may be a test well in a microtitre plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both non-covalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen or nucleotide and functional groups on the support, or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtitre plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable ^'buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtitre plate (such as polystyrene or polyvinylchloride) with an amount of a binding agent ranging from about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of binding agent.

Covalent attachment of a binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, for example, Pierce Immunotechnologv Catalog and Handbook. 1991 , at A12-A13).

Any agent that satisfies the above requirements may be a binding agent. For example, a binding agent may be a ribosome, with or without a peptide component or a polypeptide. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the cathelicidin polypeptide, or fragment thereof, is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the cathelicidin may serve as the immunogen without modification. Alternatively, particularly for relatively short cathelicidin polypeptide fragments, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for cathelicidin may then be purified from such antisera by, for example, affinity chromatography using the cathelicidin polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for a cathelicidin may also be prepared, for example, using the technique of Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976), and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with a cathelicidin). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against a cathelicidin. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Cathelicidin polypeptides, or fragments thereof, may be used in the purification process in, for example, an affinity chromatography step. Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.

In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtitre plate, with the sample, such that the cathelicidin within the biological sample is allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized cathelicidin- antibody complex and a detection reagent (preferably a second antibody capable of binding to a different site on the cathelicidin, containing a reporter group) is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support as described above, the remaining binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20 ™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample and the cathelicidin allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of a cathelicidin within a biological sample obtained from a subject. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between the bound and unbound cathelicidin. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient. Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20™. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include those groups recited below. The detection reagent is then incubated with the immobilized antibody-cathelicidin complex for an amount of time sufficient to detect the bound cathelicidin. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups, chromogenic enzymes and fluorescent groups. Chromogenic enzymes include, but are not limited to, peroxidase and alkaline phosphatase. Fluorescent groups include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), rhodamine, Texas Red and phycoerythrin. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products. The substrate can be selected from a group of agents consisting of 4-chloro-l- naphtol (4CN), diaminobenzidine (DAB), aminoethyl carbazole (AEC), 2,2'azino- bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ophenylenediamine (OPD) and tetramethyl benzidine (TMB).

It may also be desirable to couple more than one reporter group to a binding agent. In one embodiment, multiple reporter groups are coupled to one binding agent molecule. In another embodiment, more than one type of reporter group may be coupled to one binding agent. Regardless of the particular embodiment, immunoconjugates with more than one reporter group may be prepared in a variety of ways. For example, more than one reporter group may be coupled directly to a binding agent, or linkers that provide multiple sites for attachment can be used.

In a related embodiment, the method as herein described may be performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, a cathelicidin polypeptide within a biological sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-cathelicidin complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which binding agent is bound is immersed in a solution containing the biological sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. The concentration of second binding agent at the area of immobilized antibody indicates the^" presence of a cathelicidin in a biological sample. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of a cathelicidin that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.

In predicting term or pre-term labour, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined value normally found in a control sample. In one preferred embodiment, the predetermined value is a population average obtained when the immobilized antibody is incubated with samples from a normal subject, including, but not limited, to non-pregnant subjects (negative control sample). It is preferred that a sample generating a signal that is three standard deviations above this predetermined value is considered positive in identifying a subject in, or at risk of, term or pre-term labour. In an alternative embodiment, the predetermined value is a population average obtained when the immobilized antibody is incubated with a sample from a subject who has entered, or is about to enter into, term or pre-term labour (positive control sample). In this case, a sample generating a signal that is three standard deviations above or below this predetermined value may be considered positive in predicting term or pre-term labour in a subject. In a further preferred embodiment, the predetermined cut-off value is determined using a Receiver Operator Curve (ROC), according to the method of Sackett et al. (Clinical Epidemiology: A Basic Science for Clinical Medicine. Little Brown and Co., 1985, p106-7). Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the predetermined value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for predicting term or pre-term labour.

As used herein, "binding" refers to a non-covalent association between two separate molecules such that a complex is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In general, two compounds are said to "bind," in the context of the present invention, when the binding constant for complex formation exceeds about 10³ L/mol. The binding constant may be determined using methods well known in the art. Each binding agent should satisfy the above criteria; however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity.

Alternatively, the diagnostic methods of the present invention may adopt an automated analytic method using a biological microchip. For instance, a diagnostic kit can be structured to perform immunoblotting using an anti- cathelicidin antibody-coated slide glass. This diagnostic kit may comprise a biological microchip onto the surface of which an anti-cathelicidin antibody is immobilized, an appropriate buffer, a standardised sample comprising a known concentration of a cathelicidin, and a secondary anti-cathelicidin antibody.

Polynucleotide primers and probes may also be used to detect the level of messenger ribonucleic acid (mRNA) encoding a cathelicidin, or a variant or fragment thereof, whose levels are also expected to be of predictive value of term or pre-term labour. It would be expected that the level of expression of a cathelicidin polynucleotide would be greater in a biological sample from a subject with a propensity to enter term or pre-term labour, than in an analogous biological sample obtained from a non-pregnant subject, or a pregnant subject who is not about to enter term or pre-term labour.

It will therefore be apparent to those of ordinary skill in the art that a predisposition to term or pre-term labour may be identified by analysis of the level of a cathelicidin polynucleotide in a biological sample. In a preferred embodiment, a nucleotide probe that specifically hybridizes to a polynucleotide encoding a cathelicidin or a variant or fragment thereof may be used in a hybridization assay to detect the presence of polynucleotide encoding a cathelicidin in a biological sample, such as by Northern analysis, as is well known in the art (see Sambrook et al, 1989, Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). To permit hybridization under assay conditions, a nucleotide probe should comprise a nucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a cathelicidin that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, a nucleotide probe hybridizes to a polynucleotide encoding a cathelicidin under moderately stringent conditions, as can be readily determined by one skilled in the art. A nucleotide probe, which may be usefully employed in the diagnostic methods as herein described, preferably are at least 10-40 nucleotides in length. In a preferred embodiment, the oligonucleotide primer or probe comprises at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence encoding a cathelicidin.

As an alternative to Northern analysis, reverse transcription/polymerase chain reaction (RT-PCR) may be performed. Typically, RNA is extracted from a biological sample and reverse transcribed to produce complimentary DNA molecules (cDNA). Alternatively, a biological sample may be subjected to a reverse transcription reaction in the absence of any prior RNA extraction step. In the reverse transcription (RT) step of RT-PCR, mRNA is converted to first strand cDNA, which is relatively stable and is a suitable template for a PCR reaction. In the second step, the cDNA template of interest is amplified using PCR. This is accomplished by repeated rounds of annealing sequence-specific primers to either strand of the template and synthesizing new strands of complementary DNA from them using a thermostable DNA polymerase as previously described (Mullis and Faloona, 1987, Methods Enzymol., 155: 335-350, herein incorporated by reference). PCR, which uses multiple cycles of DNA replication catalyzed by a thermostable DNA-dependent DNA polymerase to amplify the target sequence of interest, is well known to those skilled in the art.

Oligonucleotide primers useful to this application are single-stranded DNA or RNA molecules that are hybridizable to a nucleic acid template to prime enzymatic synthesis of a second nucleic acid strand. ^" The primer is preferably complementary to a portion of a cathelicidin polynucleotide present in a pool of nucleic acid molecules of a given biological sample or a purified extract thereof. It is contemplated that such a molecule is prepared by synthetic methods, either chemical or enzymatic. Alternatively, such a molecule or a fragment thereof is naturally-occurring, and is isolated from its natural source or purchased from a commercial supplier. Oligonucleotide primers are 15 to 100 nucleotides in length, ideally from 20 to 40 nucleotides, although oligonucleotides of different length are of use. Typically, selective hybridization occurs when two nucleic acid sequences are substantially complementary (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary; see Kanehisa, M., 1984, Nucleic Acids Res. 12: 203, incorporated herein by reference). As a result, it is expected that a certain degree of mismatch at the priming site is tolerated. Such mismatch may be small, such as a mono-, di- or tri-nucleotide. Alternatively, it may encompass loops, which are defined as regions in which mismatch encompasses an uninterrupted series of four or more nucleotides. Overall, five factors influence the efficiency and selectivity of hybridization of the primer to a second nucleic acid molecule. These factors, which are (i) primer length, (ii) the nucleotide sequence and/or composition, (iii) hybridization temperature, (iv) buffer chemistry and (v) the potential for steric hindrance in the region to which the primer is required to hybridize, are important considerations when non- random priming sequences are designed. There is a positive correlation between primer length and both the efficiency and accuracy with which a primer will anneal to a target sequence; longer sequences have a higher melting temperature (TM) than do shorter ones, and are less likely to be repeated within a given target sequence, thereby minimizing promiscuous hybridization. Primer sequences with a high G-C content or that comprise palindromic sequences tend to self-hybridize, as do their intended target sites, since unimolecular, rather than bimolecular, hybridization kinetics are genererally favoured in solution; at the same time, it is important to design a primer containing sufficient numbers of G-C nucleotide pairings to bind the target sequence tightly, since each such pair is bound by three hydrogen bonds, rather than the two that are found when A and T bases pair. Hybridization temperature varies inversely with primer annealing efficiency, as does the concentration of organic solvents, e.g. formamide, that might be included in a priming reaction or hybridization mixture, while increases in salt concentration facilitate binding. Under stringent annealing conditions, longer hybridization probes (of use, for example, in Northern analysis) or synthesis primers hybridize more efficiently than do shorter ones, which are sufficient under more permissive conditions. Stringent hybridization conditions typically include salt concentrations of less than about 1M, more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures range from as low as 0°C to greater than 22°C, greater than about 30°C, and (most often) in excess of about 37°C. Longer fragments may require higher hybridization temperatures for specific hybridization. As several factors affect the stringency of hybridization, the combination of parameters is more important than the absolute measure of a single factor. Oligonucleotide primers are designed with these considerations in mind. While estimates of the relative merits of numerous sequences may be made mentally by one of skill in the art, computer programs have been designed to assist in the evaluation of these several parameters and the optimization of primer sequences. Once designed, suitable oligonucleotides are prepared by a suitable method, e.g. the phosphoramidite method described by Beaucage _. and Carruthers (1981 , Tetrahedron Lett., 22: 1859-1862) or the triester method according to Matteucci et al. (1981 , J. Am. Chem. Soc, 103: 3185), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer.

The length and temperature of each step of a PCR cycle, as well as the number of cycles, is adjusted in accordance to the stringency requirements in effect. Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated; obviously, when nucleic acid molecules are simultaneously amplified and mutagenized, mismatch is required, at ^"least in the first round of synthesis. In attempting to amplify a population of molecules using a mixed pool of mutagenic primers, the potential for loss, under stringent (high-temperature) annealing conditions, of products that would only result from low melting temperatures is weighed against the promiscuous annealing of primers to sequences other than the target site. The ability to optimize the stringency of primer annealing conditions is well within the knowledge of one of moderate skill in the art. An annealing temperature of between 30°C and 72°C is used. Initial denaturation of the template molecules normally occurs at between 92°C and 99°C for 4 minutes, followed by 20-40 cycles consisting of denaturation (94-99°C. for 15 seconds to 1 minute), annealing (temperature determined as discussed above; 1-2 minutes), and extension (72°C for 1 minute). Final extension is generally for 4 minutes at 72°C, and may be followed by an indefinite (0-24 hour) step at 4°C.

The amplified cDNA product (also referred to as an "amplicon") may then be separated from other components of the PCR reaction mixture using any number of methods known to those skilled in the art, such as gel electrophoresis. To facilitate visualization and quantification of the amplicon, a detectable reporter may be incorporated into the amplicon during separation by gel electrophoresis, such as ethidium bromide and other such nucleotide incorporating dyes. A detectable reporter label, such as fluoroscene isothiocyanate (FITC) or digoxigenin (DIG), may be incorporated into the oligonucleotide primer that is specific for a cathelicidin polynucleotide, such that each amplicon generated by PCR will comprise a detectable reporter label. Such labels can then be detected by standard methodologies known to those skilled in the art, such as via the use of a binding agent that is selective for the detectable reporter, including, but not limited to, a reporter-specific antibody conjugated with a colorimetric enzyme. In general, the amount of detectable reporter label is proportional to the amount of amplicon in a PCR reaction mixture, which is in turn proportional to the amount of a cathelicidin polynucleotide in the starting material.

In another preferred embodiment, detection of a cathelicidin polynucleotide may advantageously be performed in a single tube reaction -for reverse transcription of RNA and specific amplification of the transcript of interest. This system utilizes two enzymes, a reverse transcriptase to prepare first strand cDNA, and the thermostable Tfl DNA polymerase for second strand cDNA synthesis and subsequent DNA amplification, with an optimized single buffer system that permits RT-PCR to be performed in one step, simplifying the assay and minimizing the chance for contamination during preparation of a separate PCR reaction. Commercial kits are available which have conveniently assembled all materials (except primers) necessary to carry out the method in this way. Alternatively, it is possible to use an enzyme such as rTth polymerase that has reverse transcriptase activity in the presence of Mn²⁺ and has DNA polymerase function at higher temperatures (Juhasz et al., 1996, BioTechniques, 20: 592- 600). Such an enzyme system allows for single tube and single enzyme RT- PCR. PCR product detection has been performed both by polyacrylamide gel electrophoresis and ethidium bromide staining, as well as by performing the PCR reaction in combination with a detection system such as the one described above. Utilization of such a detection system in the one-tube system allows for the simple addition of RNA to a well containing the buffer, enzymes, dNTPs, primers and the detection probe followed by RT-PCR and a luminescent reading.

Quantitative RT-PCR assays may also be employed, such as by real-time RT- PCR. The term "real-time RT-PCR" is intended to mean any amplification technique that makes it possible to monitor the evolution of an ongoing amplification reaction, as is known to those skilled in the art. During polymerization, a reporter fluorescence dye and a quencher dye are attached to a nucleotide probe that is specific to a nucleic acid sequence that encodes a cathelicidin. Negligible fluorescence from the reporter dye's emission is observed once both dyes are attached to the probe. Once PCR amplification begins, DNA polymerase cleaves the probe, and the reporter dye is released from the probe. The reporter dye, which is separated from the quencher dye during every amplification cycle, generates a sequence-specific fluorescent signal. The signal increases in real time as the PCR cycles continue; and the fluorescence intensity increases proportionally with amplification of the target gene of interest. Thus, in predicting term or pre-term labour by the methodology as herein described, the level of cathelicidin gene-specific fluorescence detected in a biological sample during real-time PCR is generally compared to the level of cathelicidin polynucleotide-specific fluorescence detected in a control sample, whether it be a negative or positive control sample as herein described. In general, a biological sample generating a signal that is three standard deviations above the predetermined value derived from a negative control sample is considered positive in predicting term or pre-term labour. Alternatively, a biological sample generating a signal that is three standard deviations above or below a predetermined value derived from a positive control sample is considered positive in predicting term or pre-term labour in a subject.

In a further preferred embodiment, a cathelicidin polynucleotide may be detected in a biological sample by in situ hybridization using either ^'squashed' cellular material or sectioned tissue samples affixed to glass surfaces, prepared as described below. Either paraffin-, plastic- or frozen (Serrano et al., 1989, Dev. Biol. 132: 410-418) sections are used in the latter case. Following preparation of either squashed or sectioned tissue, mRNA transcripts present in the biological sample are reverse-transcribed in situ to complementary cDNA. Following reverse transcription, the sample are subjected to localized in situ amplification (LISA) by methods well known to those skilled in the art (Tsongalis et al., 1994, Clinical Chemistry, 40: 381-384), in the presence of both the forward and reverse primers complementary to a cathelicidin polynucleotide or a variant or fragment thereof and a Taq polymerase. LISA is accomplished over a number of amplification cycles as previously described for the PCR, each consisting of a primer annealing step, an extension step and a denaturation step. These amplification cycle profiles may differ slightly from those used in tube amplification in order to preserve optimal tissue morphology, hence allowing the distribution of reverse transcripts and the products of their amplification to be identified on the slide. Amplified products containing an incorporated detectable reporter, such as fluoroscene isothiocyanate (FITC)^' or digoxigenin (DIG), are detected with a binding agent that is able to bind to the detectable reporter (e.g. alkaline phosphatase-conjugated anti-digoxigenin) and the addition of an appropriate chromogen (e.g. nitroblue tetrazolium chloride and 5-bromo-4-chloro- 3-indolyl phosphate). The detection reaction is then monitored for optimal staining and stopped by rinsing in an appropriate buffer comprising, for example, ethylene diamine tetra acetate (EDTA). Samples can then be examined by light microscopy, whereby areas of colour stain are indicative of cathelicidin polynucleotide expression. Accordingly, it is expected that one is able to predict term or pre-term labour in a subject by comparing the spatial distribution of cathelicidin polynucleotide expression in a biological sample in situ with that identified in a control sample, whether it be a negative control sample or a positive control sample. In general, it is expected that one is able to predict term or pre-term labour in a subject if the in situ distribution of cathelicidin polynucleotide expression is substantially comparable to a predetermined degree of distribution identified in a negative control sample. To improve sensitivity of the methods herein described, multiple markers of term or pre-term labour may be assayed within a given sample. It will be apparent to those skilled in the art that binding agents specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of additional markers of term or pre-term labour may be based on routine experiments to determine whether their combination with the measurement of a cathelicidin or fragment thereof can result in optimal sensitivity.

In a further aspect of the present invention, there is provided a method of monitoring a subject for term or pre-term labour, the method comprising the steps of determining a level of a cathelicidin or fragment thereof in a first biological sample derived from the subject, and comparing the level of the cathelicidin or fragment thereof to a level of the cathelicidin or fragment thereof identified in a second biological sample obtained from the same subject.

In yet another aspect of the invention there is provided a method of monitoring a subject for term or pre-term labour, the method including the steps of determining the presence of a polynucleotide encoding a cathelicidin or a fragment thereof in a first biological sample derived from the subject, and comparing the level of the polynucleotide to a level of a polynucleotide encoding the cathelicidin or fragment thereof identified in a second biological sample obtained from the same subject

It is expected that the predictive value of the present invention may be improved by measuring of the level of a cathelicidin or fragment thereof in a biological sample over a period of time, and evaluating the rate of change in the level of said cathelicidin or fragment thereof over that period of time. For example, the methods as herein described may be performed every twenty-four to seventy-two hours for any period of time during gestation (typically the time elapsed from the first day of the last menstrual period of the mother until birth). In general, term or pre-term labour is imminent in a subject when the level of a cathelicidin or fragment thereof begins to increase towards a predetermined value normally found in a sample derived from a subject who has entered into labour, whether it be term or pre-term labour (positive control sample). In contrast, labour is not imminent if the level of a cathelicidin either remains constant at a level normally found in a non-pregnant subject or a pregnant subject who is not about to enter term or pre-term labour (negative control sample), or if the level of a cathelicidin decreases with time towards a level normally found in a negative control sample. In a preferred embodiment, the level of a cathelicidin or fragment thereof is measured in each of two or more biological samples obtained from a subject, whether or not they have entered term or pre-term labour, and analysing the difference in the level of the cathelicidin or fragment thereof between each biological sample. Preferably, the biological samples are derived from the same source. The difference in the level of a cathelicidin or fragment thereof can be calculated either by manual means, or by incorporation into the analysis software applicable to the aforementioned methodologies, such that the final value for a given biological sample is automatically adjusted by subtracting a measurement obtained from a biological sample obtained at an earlier or later time point. In doing so, it is expected that an increase in the level of a cathelicidin or fragment thereof in a biological sample, when compared to a level found in a preceding biological sample obtained from the same individual, would be indicative of an increased propensity of the subject to enter term or pre-term labour. Conversely, it is expected that a decrease in the level of a cathelicidin or fragment thereof in a biological sample, when compared to a level found in a preceding biological sample obtained from the same individual, would be indicative of a decreased propensity of the subject to enter term or pre-term labour. It would also be expected that a lack of change in the level of a cathelicidin or fragment thereof in a biological sample, when compared to a level found in a preceding biological sample obtained from the same individual, would be indicative that there is no change in the propensity of the subject to enter term or pre-term labour.

In a further aspect, the present invention provides a kit when used to predict term or pre-term labour in a subject as herein described, the kit comprising a binding agent which detects a cathelicidin or fragment thereof and a detection system for detecting the agent.

In yet a further aspect, the present invention provides a kit when used to monitor a subject in term or pre-term labour as herein described, the kit comprising an agent which binds to a cathelicidin or fragment thereof and a detection system for detecting the agent.

Based on the aforementioned methods encompassing the present invention, a diagnostic kit can be configured, allowing the prediction or monitoring of term or pre-term labour to be assessed quantitatively or qualitatively with relative convenience. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a cathelicidin. Such antibodies or fragments may be provided attached to a support material, as described above. Accordingly, it is preferred to include a solid support. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as herein described that contains a reporter group suitable for direct or indirect detection of antibody binding. Alternatively, a kit may be designed to detect the level of a polynucleotide encoding a cathelicidin or a variant or fragment thereof in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer that hybridizes to a polynucleotide encoding the cathelicidin or a variant or fragment thereof. Such an oligonucleotide may be used, for example, within a polymerase chain reaction or a hybridization assay as herein described. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a cathelicidin or a variant or fragment thereof. Certain in vivo diagnostic methods may be also performed directly on a subject. One such method involves contacting cells lining the uterus with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications.

In a further embodiment of the present invention, there is provided a method of evaluating the course of therapeutic intervention designed to delay the onset of pre-term labour in a subject, the method comprising obtaining a biological sample from a subject undergoing treatment for pre-term labour, measuring a cathelicidin in said biological sample, and comparing the level of the cathelicidin to a predetermined value normally found in a control sample.

It is expected that the change in the level of a cathelicidin or fragment thereof is indicative of the clinical efficacy of the treatment employed to delay or prevent pre-term labour in a subject. Examples of such clinical treatment strategies are well known to those skilled in the art, such as intravenous re-hydration, treatment of urinary tract infection if present, and treatment designed to stop the progression of labour, including the use of beta agonists such as terbutaline, magnesium, prostaglandin inhibitors, or calcium channel blockers. Additionally, such treatment may also comprise corticosteroid administration in an attempt to reduce the incidence of respiratory disorders in premature infants.

Examples of the procedures used in the present invention will now be more fully described. It should be understood, however, that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above. EXAMPLES

Example 1 : Identification of cathelicidin as a predictive marker of term and pre- term labour.

For the purpose of this study, statistical comparisons were made between non- labour and labour samples of a spontaneous labour group, and an induced labour group. Comparisons were made within individual treatment groups, and between the two treatment groups. Limited differences between the protein and MMP expressions in spontaneous labour sheep at gestational age 137 (GA137) versus dexamethasone-treated ewes at gestational age 135 (GA135) prior to dexamethasone administration were expected (see Figure 1 ).

(0 Animals

Border-Leicester-Merino cross-bred pregnant ewes of known gestational age contributed to this study. Prior to surgery, the sheep, were housed for at least 3 days in standard individual metabolism cages, with feeding (1 kg Lucerne chaff) occurring between 9am and 12 noon daily. The sheep were exposed to a constant 12 hour light/dark cycle (0700 hours - 1900 hours), and the temperature was maintained at 22°C. Free access to water was constantly provided. Surgery was performed between 120-125 days of gestational age. The ewes were starved for 24 hours prior to surgery.

(ii) Surgical Procedures

(a) Pre-operative preparation

General anesthesia was induced in both the mother and the fetus with an intravenous injection of 20mg/kg thiopentone sodium in water (Pentothal 50mg/ml, Bomac Laboratories Ltd, Asquith, New South Wales, Australia). Anesthesia was maintained by inhalation through an endotracheal tube (cuffed 7- 9mm tube, Portex Laboratoire, Berck Sur Mer, France) with 0.5-2.0% halothane (Fluothane, ICl, Villawood, Australia) and a nitrous oxide/oxygen mixture (50/50 v/v) contained in a closed circuit apparatus (CIG Midget 3 Medishield, Alexandria, New South Wales, Australia). The animal was ventilated during surgery with an automatic ventilator through positive pressure breathing (Cambell anaesthetic ventilator, Ulco Engineering, Marrickville, New South Wales, Australia). In preparation for surgery, shearing of the abdomen, right flank, groin and anterior neck was followed with washing in savlon antiseptic (ICl, Australia). The groin and udder region were then wiped with cotton wool, followed by a betadine surgical scrub (7.5% w/v polyvinylpyrrolidone, F.H.Fauldings Co.Ltd, S.A., Australia). This scrub was applied to the operative sites in an outward motion advancing from the proposed incision site. Following removal of the solution with cotton wool, the procedure was repeated. A final coating of Betadine antiseptic solution (10% w/v polyvinylpyrrolidone-iodine, F.H. Fauldings Co. Ltd, S.A., Australia) was applied to the surgical sites. The ewes were then transferred to the operating table and secured in a dorsal recumbent position. Sterile surgical drapes covered the ewe once placed on the operating table, and the surgery itself was performed under strict aseptic conditions, with incision sites coated in a 5% hibitane in 70% alcohol solution (ICl, Villawood, N.S.W., Australia). (b) Surgery

A 10-15cm rnidline incision was made in the skin from the umbilicus to the mammary glands, followed by an incision through the linea alba. A diathermy (SSE2-K electrosurgical generator, Valleylab Inc., U.S.A.) was utilized to control any subsequent bleeding, and a 6cm incision was then made through the uterine wall between cotyledons to exteriorize the fetal head and neck. Amniotic fluid loss was controlled with the use of babcock clamps at the incision site. In the case of multiple fetuses, only one fetus was instrumented and catheterized for this study.

(c) Uterine vein catheters

A saline filled medical grade polyvinyl catheter (i.d. 0.81 mm, o.d. 1.52 mm, 210cm in length, Dural plastics and Engineering, Silverwater B.C., N.S.W., Australia) was inserted into each uterine vein. A small incision overlying a tributary vessel was made, and blunt dissection was used in order to locate and separate the vessel from the loose connective tissue on the external surface of the uterus. The vessel was then occluded with a silk ligature at the distal end, and a bulldog clip was used to control blood flow at the cardiac end of the exteriorized vessel. A transverse section was made with iris scissors, and the catheter was then inserted. A ligature tied to the catheter was used to mark the catheter for determining its insertion depth. Depending on the structure of the vessel network, the catheter was inserted in a central direction to a depth of 8- 20cm so that the tip lay in the utero-ovarian vein. Catheters were then tied in place, tested and flushed prior to closing the incision. Incisions were closed with 3/0 silk (Cyanamid) using a continuous mattress stitch.

(d) Fetal catheters

Fetal carotid artery (FCA) and fetal jugular vein (FJV) saline filled medical grade polyvinyl catheters (i.d. 0.81 mm, o.d. 1.52 mm, 155cm in length, Dural plastics and Engineering, Silverwater B.C., N.S.W., Australia) were surgically implanted and inserted 8-10cm toward the heart into each vessel following blunt dissection, location and separation of the carotid artery and vagus nerve. Prior to insertion of the catheter, the vessel was occluded with a silk ligature at the cranial end, and a bulldog clip at the cardiac end. This enabled a transverse section to be made in the vessel with iris scissors without excess blood flow and leakage. All catheters were tied in place, tested and flushed prior to wound closure. The fetal skin incision was closed with 3/0 silk (Cyanamid) using a continuous mattress stitch. Catheterization was performed in all animals that underwent surgery to enable infusions and routine blood sampling.

An amniotic catheter with a perforated end was sutured to the nape of the fetal neck in order to allow collection of amniotic fluid. The three fetal catheters were then tied to the fetal ear prior to the fetus being returned to the uterus. A simple continuous stitch was used to close the membranes, while an inverting stitch was used to close the muscular layer (chromic 2/0 catgut, Ethicon, Ethnor Pty. Ltd, Sydney, N.S.W., Australia). The fetal catheters were ^"passed between individual stitches one at a time.

(e) Uterine electroymyograph leads

Electromyography (EMG) electrodes made from 3 teflon coated, braided stainless steel wires (Cooner Wire Company, California, USA) surrounded by polyvinyl catheter (i.d. 1.50 mm, o.d. 2.70, Dural Plastics Engineering) and sealed with silicone (RTV sealant, Sow Corning Corporation) were sutured to the myometrium to monitor uterine electrical activity, and to determine the onset of labour; using a recording system (Grass instruments Co, Quincy, Massachusetts, USA).

(f) Exteriorization of catheters. Fetal, amniotic, uterine catheters and EMG wires were exteriorized through a 2cm incision in the right flank of the ewe. The peritoneum and the muscle layers of the flank were closed with a continuous suture using chromic gut suture (2/0 cat gut, Ethicon). The skin was closed with a continuous mattress stitch using Vetafil Bengen synthetic suture (Clements Stansen, Nth Ryde, N.S.W., Australia).

The linea alba and subcutaneous fatty layers of the abdominal incision were closed with a continuous locking stitch, and a simple continuous stitch using chromic 2 cat gut suture. The skin was then closed separately with Vetafil Bengen synthetic suture using an everting mattress stitch.

(g) Maternal catheters Maternal carotid artery (MCA) and jugular vein (MJV) catheters were implanted via an incision in the jugular groove of the neck in a similar fashion described for catheterization of the fetal neck. The polyvinyl catheters (i.d. 1.50 mm, o.d. 2.70 mm, Dural Plastics and Engineering) were inserted 10-15cm into the vessels. A continuous mattress stitch was used to close the maternal neck incision with vetafil synthetic suture. All catheters were fitted with three way stopcocks and stored in plastic bags.

(h) Post operative care and maintenance

Following the completion of surgery, the administration of halothane was withdrawn. An elasticized tubular netting (Surgifix, size 7, Australian Home Healthcare Group, North Melbourne, Australia.) was placed around the torso of the ewe, and plastic bags containing catheters were secured underneath the netting. Mechanically assisted breathing was maintained until the ewes displayed spontaneous breathing and a swallowing reflex. The endotracheal tube was then removed, and ewes were then transferred on a trolley to their metabolic cage for recovery. Ewes were then provided with food, and monitored until standing upon which water was provided.

Daily flushing of all catheters was maintained in order to retain patency of the catheters. All catheters excluding the amniotic fluid catheter were flushed with heparinised saline (50iu heparin/1 mL saline) in order to prevent the formation of blood clots. Saline (Baxter Healthcare, Toongabbie, N.S.W, Australia) was used to flush the amniotic fluid catheter to prevent surrounding membranes from occluding the terminal ports.

Uterine EMG activity was continuously recorded from a gestational age of 130 days until elective post mortem. A grass 7P3 amplifier was used to amplify the signal (Grass instruments, Quincy, M.A, U.S.A.), with high pass filtering at 3Hz. (iii) Experimental Protocols

Pregnant ewes were allocated to one of the following treatment groups; intact fetuses receiving an infusion of dexamethasone at 135 days gestational age, and intact fetuses receiving no infusions and permitted to follow a spontaneous transition into labour. Swabs of cervico-vaginal fluid (CVF) occurred at days 123, 130, 135, 136, and 137 (in labour) for dexamethasone sheep, and at days 123, 130, 135, 137, 143 and in labour of spontaneous gestation sheep.

(a) Fetal arterial blood gas measurements Fetal well being was determined with measurements of fetal arterial blood gas tensions. Blood (0.4mL) was collected from fetal carotid artery catheter into 1 mL syringes rinsed with heparinised saline. Samples were collected daily following surgery. Fetal blood was measured for the partial pressure of oxygen (Pa0₂), carbon dioxide (PaC0₂), hemoglobin concentration (Hb), oxygen saturation (Sa0₂) and pH. An ABL30 acid-base analyzer and OSM2 hemoximeter (Radiometer, Copenhagen, Denmark) were used to determine the gas measurements. Measurements were corrected for estimated fetal body temperature (39°C) and barometric pressure.

(b) Administration of dexamethasone

Labour induction was achieved with a continuous infusion of dexamethasone (1 mg/day in sterile saline 1 mL/hr; David Bull Laboratories, Vic, Australia) to the fetus via the fetal jugular vein. The infusion ran continuously from 0900 hours on the 135^th day of gestation until the animal was advanced in the first stage of labour. A post mortem was carried out before the animal was permitted to deliver after the ewe and fetus were killed by an intravenous barbiturate overdose. Cervical-vaginal swabs and amniotic fluid samples were taken at time 0 of infusion (day 135), at 28 hours (day 136), at 56 hours (day 137), and immediately before euthanasia, when labour was established.

(c) Obtaining cervico-vaginal fluid samples

The pregnant ewe was restrained in the metabolic cage with a collar and lead, along with physical restraint, and rope barriers to prevent any backward movement while sampling took place. Following sufficient restraint, a small proctoscope was coated in an antibacterial lubricant, and inserted into the lumen of the vagina. The removable probe of the proctoscope was replaced with 2 pre- weighed sterile 15cm swab sticks, and allowed to sit in the high vaginal/cervical region for 30 seconds. Following removal of the swabs, the swabs were reweighed to detect a change in wet weight. If the change in wet weight was less than 20mg, the swabbing procedure was repeated. Swabs were then added to 1 ml of buffering solution (Table 1 ) in a 10mL plastic tube.

Table 1 : Swab suspension buffer (pH 7.5) Reagent Final Conc/δOml Amount SDS 0.1 % 50mg Hepes 50mM 0.596g NaCI 15mM 0.0438g EDTA 1 mM 18.6g PMSF 1 mM 8.71 mg Milli Q H₂0 Adjust to 50mL

The sample was then vortexed for 1 minute, followed by the inversion of the swabs before centrifuging at 3000rpm. Following this, the swabs were carefully discarded, and 3x 110μL aliquots of the sample were taken, and 1x 400μL aliquot to be stored at -70°C until analysis was required.

(d) Obtaining amniotic fluid

In conjunction with a cervico-vaginal fluid sample, amniotic fluid was obtained via the amniotic fluid catheter providing there were no blockages. A 5ml syringe containing 3mL sterile isotonic saline was inserted into the three-way tap after removal of any air bubbles, and the tap was rotated to the open position to the sheep. The syringe plunger was withdrawn until the syringe contained 5mL of fluid. This fluid was then discarded, the syringe was then reinserted, and a pure sample of 2mL of amniotic fluid was collected, transferred to a plastic tube, and centrifuged for 10 minutes at 3000rpm. The volume of fluid removed from the sheep was then slowly replaced with equal volume of sterile isotonic saline in a lOiTiL syringe. Two aliquots of 200μL were then taken and stored immediately at -70°C.

(iv) Two Dimensional Polvacrylamide Gel Electrophoresis Two dimensional polyacrylamide gel electrophoresis (2D PAGE) was used to display proteins. This method separates proteins in a two dimensional array. The first dimension separates proteins based on their isoelectric points (the specific point at which a protein will not migrate electrophoretically in a pH gradient, and carries zero charge). Following this, proteins are separated perpendicularly to their isoelectric point alignments based on their molecular weight. In this step, the SDS contained in the equilibration buffer coats the proteins based on a negative charge to mass ratio. When a current is applied, proteins migrate in proportion to their size to charge ratio.

(a) Determination of total protein content

Six serial, twofold dilutions were set up in 1.5mL microfuge tubes using Bovine Serum Albumin as the standard (2mg/ml in 0.9% aqueous solution containing sodium azide at room temperature). Standards were diluted using 1x PBS, in order to obtain a range of standards of 2mg/mL, 1 mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, and 0.0625mg/mL A BIORAD protein assay based on the modified Bradford dye-binding method (Bradford 1976) whereby the colorimetric assay is based on the colour change of Coomassie brilliant blue G-250 dye in response to various concentrations of protein. Briefly, a 5μL duplicate of 1x PBS, each standard dilution, and each cervical fluid sample was added in duplicate to each well of a 96-well microtitre plate (Nunc, Roskilde, Denmark), followed by the addition of 250μL of reagent at room temperature. Plates were vortexed gently, and absorbance was then determined at 595nm using a microplate reader (Model 3350, BIORAD Laboratories, California, USA). Standard curves and unknown protein concentrations were generated with the software program 'Microplate Manager 4.0 (BIORAD Laboratories, Inc). The results were used to load a standard mass of protein to each gel.

(b) TCA precipitation Following preliminary trials with cervico-vaginal fluid samples using the 2 Dimensional Polyacrylamide Gel Electrophoresis described in this section, it was established that these samples contained a variety of contaminants which resulted in poor resolution. To separate proteins from contaminating substances, a TCA (Trichloroacetic acid) precipitation was carried out on each sample prior to first dimension separation.

100μL of sample was added to 10OμL of 20% TCA. Samples were left on ice for 20 minutes, and microcentrifuged for 15 minutes at 4°C. Any supernatant was then removed, and the remaining pellet was washed in ice-cold ethanol to extract any remaining water. Samples were then re-suspended in various volumes of multiple chaotropic agent solution buffer (Table 2) sb that final concentrations were 15μg of protein per 200μL. This assumes there was zero or equal protein loss from each sample during the procedure.

(v) First Dimensional Separation

(a) Sample solubilization and preparation

A solubilizing solution (Multiple Chaotropic Agent Solution) was used as an extraction solution, and also for the immobilized pH gradient (IPG) strip rehydration. Following TCA precipitation and sample suspension in the buffering solution, samples were vortexed, microcentrifuged, and permitted to equilibrate for one hour.

Table 2: Multiple chaotropic agent solution Reagent Amount 7M Urea 4.2g 2M Thiourea 1.52 g 100mM DTT 0.154 g 4% Chaps 400 mg 40mM Tris 48.4mg 3/10 Pharmalytes 50 μL Bromophenol Blue 2 grains Milli Q H₂0 Adjust to 10 mL (b) Sample application

Using an 11cm rehydration tray, 200μL of the solubilized sample was evenly pipetted between electrodes, taking care not to produce bubbles or splash the solution into a neighboring well. Once all protein samples were loaded into the rehydration tray, the 11cm immobilized protein gradient strips (IPG) (pH 4-7) were removed from -20°C. Using two forceps, the plastic coversheet was peeled from the IPG strip. The strip was gently placed gel side down onto the sample. The rehydration tray was then left to sit in a safe position for Y∑ hour before the strips were overlaid with 2mL to 3mL of mineral oil to prevent evaporation during the rehydration process. The rehydration tray was then placed in the PROTEAN^® IEF cell for active rehydration with positive and negative electrodes directly connected to their respective electrode in the cell. This is shown in Figure 2.

(c) Active rehydration

The IPG strips were rehydrated under active conditions. The Bio-Rad PROTEAN^® IEF cell was programmed to apply a constant voltage of 50 volts for 12-16 hrs at 20°C. This process was carried out overnight, and a pause instruction was incorporated into the program following this time period to allow the insertion of a pair of electrode wicks (4mm x 20mm) between the IPG strip and electrode at each end before isoelectric focusing began. This allows for the collection of salts and other contaminants remaining in the sample (Figure 2).

(d) Isoelectric focusing During isoelectric focusing, charged contaminants move to electrodes and proteins move to the pH equal to their pi (Figure 3). The PROTEAN^® IEF cell was reprogrammed to maintain a current dependent on the number of strips contained in the rehydration tray (maximum of 12) and also the type of sample/rehydration buffer being used. The current applied was a quadratically increased voltage (0-250 V in fifteen minutes, 250-8000 V over 1 hour, 8000 V for 20-35,000 volt-hours) which is used for high-resistance samples/buffer systems in order to minimize high power input initially, while achieving a high voltage as quickly as possible. The maximum voltage reached is 8000 V. The wicks were discarded and replaced following the method previously described at -2,100/35,000 volt-hours. This aids in reducing the total time for the samples to reach their maximum voltage, as it is affected by the charged contaminants absorbed by the wicks. This process requires approximately 8 hours.

(e) Storage of IPG strips after IEF

IPG strips were individually removed from the rehydration tray, and held in vertical contact with filter paper to drain off excess mineral oil. They were then placed in 15 ml screw cap plastic tubes and stored at -80°C until required for second dimension separation. The rehydration tray was then scrubbed with dilute detergent, and soaked in a hot pyroneg solution overnight before being rinsed and dried for reuse.

(vi) Second Dimension Separation

This step required the use of ready made 8-16% gradient gels (BIORAD Laboratories, Inc.).

(a) IPG equilibration

Prior to running the second dimension, it is necessary to equilibrate the IPG strips in a buffer containing SDS. This results in each protein obtaining a uniform shape, single subunits, and a uniform negative charge/mass ratio (Figure 4).

The solution used is shown in Table 3, and is generally made in bulk, with 10 mL and 25 mL aliquots stored at -80°C. Strips were removed from storage, and 5mL of tributyl phosphine (TBP) equilibration buffer at room temperature was added to each plastic tube containing 1 strip. Tubes were then placed on a slow rocker and rocked for 20 minutes.

Table 3: TBP Equilibration Buffer Reagent Final Conc./200mL Amount 1.5M Tris HCI pH 8.8 50mM 6.7mL Urea 6M 72.07g Glycerol (87% v/v) 30% v/v 69mL SDS 2% w/v , 4.0g TBP 200mM 50mL Bromophenol Blue trace Few grains Milli Q H₂0 Adjust to 200mL

(b) Second Dimension Gel Cassette preparation

Gel cassettes were removed from storage at 4°C. Labels were removed, and control numbers/expiry dates recorded. Cassettes were rinsed thoroughly with milli Q water, and plastic backing tape was removed. A plastic plug was cut using the original plug to the strip well, and was placed at one end of the cassette to create a well for the application of a molecular weight standard following the embedding of the IPG strip, and the cementing with agarose solution.

(c) Placement and Agarose Embedding of IPG strips

The IPG strips were applied to the SDS-PAGE gels by placing in the space in the cassette above the separation gel after the strip was dipped in running buffer (Table 4). The strips were then sealed in place with ~1 mL 0.5-1% agarose prepared in SDS-PAGE running buffer with a small amount of bromophenol blue. The agarose was allowed to set, and this step was followed by the removal of the molecular weight standard plug. A small amount of running buffer (BIORAD Laboratories, Inc.) was then added to the well, and 8μL of Unstained Broad Range molecular weight standard (BIORAD Laboratories, Inc.) was added to the well with a gel loading tip. The cassette was then added to the electrophoresis tank, and running buffer was poured into the tank until air bubbles reached the bottom of the cartridge. The cartridge was also filled with running buffer.

Table 4: SDS-PAGE Running Buffer Reagent Amount 10 x TGS 200 mL Milli Q H₂0 Adjust to 2L

(d) Running the Second Dimension

During this step, SDS charged proteins are resolved according to their molecular weight/size in the SDS-PAGE gel (Figure 4). Gels were inserted into the appropriate electrophoresis cell (depending on gel number), and ran according the following conditions: • 10mA per gel for 1hr • 20mA per gel for 2hrs • 30mA per gel for 20 min

When running more than 2 gels at once, gels were placed in a PROTEAN Plus Dodeca cell which holds a maximum of 12 gels. In these instances, the cell was placed on a magnetic stirrer, with temperature held constant at 12°C in order to counteract any heat build up as a result of the high current application. At the end of this process, the bromophenol blue tracking dye should have reached the bottom of the gel, if not disappeared into the running buffer solution. If not, the cell was programmed to run for a longer period.

(vii) Gel Staining Three stains that interact differently with different proteins were used. Sypro Ruby and Colloidal Coomassie stains were used on all 2D gels which were presented in the results of this investigation. This was to allow for the identification of proteins via mass spectrometry if required.

Gel staining is carried out immediately following second dimension electrophoresis. The gel was separated from one side of the cartridge by snapping the cartridge and pulling the layers apart. The bottom of the gel was cut with a scalpel to remove any remnant tracking dye and to neaten curled and non-flattened ends. The IPG strip was also sliced off the gel and a diagonal incision was made across the corner of the gel which contained the protein standard. This aids in orientation of the gel for later scanning and analysis. The gel was then placed in solution (depending on the stain used), and detached from the remaining side of the cartridge.

To detect protein spots, all gels were stained at room temperature in plastic BIORAD gel containers. Gloves were worn in all three staining procedures, and good laboratory precautions were carried out with staining chemicals.

(a) Modified Rabilloud Stain The highly sensitive modified Rabilloud stain was used to obtain gels which were to be dried and not analyzed by mass spectrometry for any spots excised from the gel. This stain was used during preliminary practices of the 2D-PAGE technique to determine what type of gel gave optimal results with this type of sample, and to minimize the costs of other more expensive stains.

A volume of 100mL was used for each solution during the staining procedure to ensure the gel was completely immersed. Gels were agitated on a rotary shaker at low speed during all steps. The six solutions (for 12 gels) are shown in Tables 5-10.

Table 5: Fix Solution 1 Reagent Amount Ethanol 480 mL Acetic Acid 120 mL Milli Q H₂0 600 mL

Table 6: Fix Solution 2 Reagent Amount Ethanol 360 mL Glutaraldehyde (50%) 12 mL Potassium Tetrathionite 3.0 gms Sodium Acetate 81 .6 gms lilli Q H₂0 Adjust to 1200mL

Table 7: Silver Solution Reagent Amount Silver Nitrate 2.40 gms Formaldehyde 300μL Milli Q H₂0 Adjust to 1.2L Table 8: Developer Reagent Amount Potassium Carbonate 36 gms Sodium Thiosulphate 9.0mg Formaldeyhde 180μL i Q H₂0 Adjust to 1.2L

Table 9: Stop Solution Reagent Amount Tris Base 60 gm Acetic Acid 24 mL Milli Q H₂0 Adjust to 1 .2L

Table 10: Gel Soak (2L) Reagent Amount Ethanol 600 mL Glycerol 80mL Milli Q H₂0 Adjust to 2 L

Gels were fixed in fixative 1 solution for 1 hour. They were then placed in fixative 2 solution for 1 hour or up to overnight. Following this, the gels were washed in 4 x 15 minute Milli Q (100ml) washes. Gels were then placed in silver solution and were able to remain in the solution from 30 minutes to 48 hours if necessary. Gels were then washed again for 1 minute in Milli Q water. Gels were then placed in developer solution until spots appeared (from 5 mins to 20 mins). Once faint spots were developed, gels were placed in stop solution for 10 minutes to prevent development of background staining and loss of clear spots. The gel was then scanned using Molecular Imager FX (BIORAD laboratories, Hercules, California).

For each gel, two layers of cellophane were placed in a pyrex container until the gel soak solution covered the cellophane. Cellophane was soaked for 20-30 minutes, and gels were soaked in 100mL of the gel soak for 10 minutes. Gels were then enclosed in cellophane, and dried using the BIORAD GelAir™ drying system (BIORAD Laboratories, Inc.).

(b) Sypro Ruby Staining Sypro Ruby staining was one of the two primary staining techniques used as it has little background staining, and is sensitive. Unlike silver staining, it does not detect nucleic acids and is compatible with downstream analysis, including mass spectrometry and Edman sequencing.

Gels were removed from the gel cassette and placed in the plastic BIORAD container from which the gel originated. The gel was then washed for 30 minutes in 40% methanol, 10% acetic acid prior to staining. The gels were then removed from the solution and added to 70mL of Sypro Ruby gel stain which had been recycled after use on one other occasion (BIORAD Laboratories, Inc.). An inverted plastic BIORAD container was then clipped in place as a lid, and the containers were entirely covered in aluminum foil to prevent light exposure. Gels were then stained for 3 hours, and rinsed in 10% methanol, 7% acetic acid for 30-60 minutes while still enclosed in foil. Gels were then placed into 100mL of Milli Q water and scanned using the BIORAD molecular imager FX at medium stain intensity.

(c) Colloidal Coomassie Staining

Colloidal Coomassie stain (Table 11 ) was used following some (but not all) gels stained with Sypro Ruby, as it appears to stain the broadest spectrum of proteins. It enables proteins to be visualized, and is also compatible with mass spectrometry.

Gels were then placed in 100 ml of Colloidal Coomassie (G250) and rocked overnight. Following this period, gels were rinsed several times in Milli Q water, and stored in the plastic BIORAD containers with Milli Q water.

Table 11 : Colloidal Coomassie (G250) or Modified Neuhoff Reagent Final Conc./1L Amount Ammonium sulphate 17% w/v 170gm 85% Phosphoric acid 3% 36mL Coomassie G-250 0.1% ig Methanol 34% 340mL Milli Q H₂0 Adjust to 1L

(d) Image analysis

Once images were digitized with the molecular imager FX, images were integrated with PDQuest™ software. Raw images were corrected, and the gel background was subtracted. A model of the protein spots from each gel was devised (3-D gaussian distribution), and spot intensities were obtained. The software program was also used to add spots that were not detected initially with the imaging wizard, to the spot list, or to delete spots that were artifacts.

From the set of gels for analysis, the gel containing the most number of spots was selected and set as a reference or standard gel. All other gels were then compared to this gel. Proteins from the series of gels ^"were then compared using PDQuest, and minor shifts in individual spot position were detected after marking landmark spots and comparing the neighbouring spots to the fixed landmark points. Protein spots which were not present in the reference gel were manually added in order to include all of the proteins from the gel series. In this way, an image was developed containing all the proteins found in all the gels.

Non-labour and labour onset gels from spontaneous labour sheep, and dexamethasone treated sheep were then viewed as a unit, and compared to the reference gel. Spots were assigned numbers, with any identical spots between units receiving identical numbers.

(viiO Analysis of Amniotic Fluid A protein assay was performed on the amniotic fluid samples following the protocol as herein described. Sixteen amniotic fluid samples of varying gestational age and treatment were chosen for analysis for the expression of matrix metalloproteinases.

(ix) Statistical Analysis For analysis of 2D gels, protein intensity (Log10 (density +1)) and number were analyzed using a paired student's t-test. Individual animals and animal groups were compared for protein differences. Significant differences are reported at the p≤O.05 level, except where noted, and all data are presented as means with standard deviation bars.

RESULTS

(\) Cervico-vaginal Fluid Protein Expression

(a) Development of CVF suspension buffer Figure 5 displays a raw two dimensional image of a CVF sample which has been suspended in a buffer containing 150mM NaCI. In the presence of this high salt concentration, CVF samples were not compatible with the first dimensional focusing as demonstrated by the gel streaking and blurry protein spots on the raw image. Following a reduction in the concentration of NaCI to 15mM, an improvement in the resolution of a gel can be noted (Figure 6).

(b) Development of CVF samples with TCA precipitation

While reducing salt concentration enhances first dimension focusing, CVF samples underwent a TCA precipitation prior to first dimension focusing to further minimize the affects of salt and other contaminants on first dimension focusing. As shown in Figure 5, two dimensional gels show further increased resolution of proteins; however there is a marked reduction in the number of proteins expressed. This is due to protein loss during a TCA precipitation. Consequently TCA precipitations were carried out consistently on each experimental sample assuming equal loss of proteins from each sample.

(ii) Analysis of 2D Gelsfa) Protein characterization 2D PAGE was used to determine the proteins expressed in cervico-vaginal fluid from pregnant ewes during late gestation and not in labour, induced labour onset, and spontaneous labour onset at term. Proteins identified in any sample were able to be compared with all other samples based on their orientation on the 2D gel (Molecular weight Vs isoelectric point). To eliminate the possibility of regional variation in protein expression, all cervical swabs were placed into the vagina at an equal depth in each animal and underwent the same sample optimization techniques prior to first dimension separation. Only samples from individual animals whose membranes were still intact were used for the experiment to prevent uncontrolled sample contamination from other intrauterine sources such as amniotic fluid. Figure 9 displays typical raw protein expressions from non- labour and labour onset samples in both treatment groups.

(b) Computer assisted 2D gel analysis

All 16 two-dimensional gels underwent 5 major computer assisted image analysis steps before any evaluations of protein expression of CVF could be drawn. These steps were; • Cropping the important sections of the 2D gels Raw images were corrected and background was subtracted Editing automatically marked and unmarked protein spots Identification of proteins that were present in all gels of a series A reference or standard gel was selected for comparison to all other gels • Proteins not in reference gel were manually added Protein landmarks were manually identified Gels were systematically investigated for the presence/absence of matching spots

As shown in Figure 10, the reference standard (the integration of all sample proteins onto one computer generated spot distribution) expressed 2003 proteins, with 85 of those being unmatched with any other gel image.

(c) Data analysis of 2D gels With PDQuest software, all 16 gels of the investigation were viewed as unit and compared to the reference standard. In the comparisons shown on page 16, each spot is automatically assigned a number so that identical spots corresponding to particular proteins have identical numbers (Figure 11 ). Gels were analyzed for spot number and unique number of spots (Table 16) and also for up-regulated or down-regulated spots between comparative groups (Figure 11 ). In this case, proteins were quantified and compared in the following ways (see also Table 17). • Spontaneous labour GA 137 (non-labour) Vs Spontaneous onset labour at term • Dexamethasone treated GA 135 (non-labour) Vs Induced labour onset GA 137 • Induced labour onset GA 137 Vs Spontaneous labour onset at term • Spontaneous labour GA 137 (non-labour) Vs Dexamethasone treated GA 135 (non-labour).

Quantitative analysis found no proteins that were identified as being downregulated during labour onset. As shown in Figures 12-14, the number of proteins up-regulated during labour onset within or between treatment groups varies. As expected, no up-regulation of proteins was found when comparing non-labour samples prior to dexamethasone treatment with non-labour samples of the spontaneous labouring group. Importantly, the analysis also found 5 proteins that displayed up-regulation in all labour onset samples. Of these five proteins, three showed significant up-regulation during labour, whilst only 1 protein (no. 6003) showed significant (p<0.05) up-regulation during labour in both spontaneous and induced labour treatment groups (Figures 14-16). Further analysis by mass spectrometry identified protein no. 6003 as bactenecin-1 , an ovine cathelicidin.

Table 17: Spot data from individual animals after 2D PAGE of CVF samples. There are no statistical differences between total numbers of spots in non-labour Vs labour onset from both treatment groups

Treatment Individual Gestational Age Total Unique No. Group Animal (days) Spot No. Spots SPL* 2001 137 229 6 SPL* 2055 137 ^" 176 4 SPL* 2068 137 375 20 SPL* 2075 137 275 21 AVERAGE 263.75 12.75 Std.Dev 84.48 9.00 SPL 2001 150 260 26 SPL 2055 144 316 12 SPL 2068 143 - 443 53 SPL 2075 145 534 45 AVERAGE 388.25 34.00 Std.Dev 123.70 18.53 DEX* 2006 135 194 18 DEX* 2026 135 290 13 DEX* 2067 135 252 9 DEX* 2109 135 267 27 AVERAGE 250.75 16.75 Std.Dev 40.93 7.76 DEX 2006 137 280 12 DEX 2026 137 194 13 DEX 2067 137 344 14 DEX 2109 137 395 48 AVERAGE 303.25 21.75 Std.Dev 86.71 17.52

• Indicates samples collected from spontaneous labour ewes during late gestation and from dexamethasone ewes prior to dexamethasone administration.

Example 2: Determination of hCAP-18 in human CVF samples CVF was collected from 12 women at term and assayed by Enzyme linked immunosorbent assay (ELISA). Mean sample concentration was 560 +/- 592 ng/ml (range 0 - 1835 ng/ml). Determination of hCAP-18 during gestation and at term (in-labour and not-in-labour) is in progress.

Example 3: Proteomic comparison of CVF protein profiles in pregnant women.

(i) Recruitment and sampling

Several experimental approaches have been adopted for the determination of differential expression of CVF proteins relevant to spontaneous labour. These include (a) identifying and characterising CVF protein changes during normal gestation 20-36 weeks); (b) comparison of CVF protein profiles in normal pregnant women at term (>37 weeks) and in-labour; (c) identifying and characterising CVF protein changes during gestation (20-36 weeks) in women at risk of premature labour; and (d) characterising CVF proteins present during "threatened" pre-labour between women who progress into labour and those that do not.

(ii) Methods Samples from each group were assessed by 2D gel electrophoresis and comparisons are made between groups to establish differential expression of proteins. Only proteins that show a statistical difference in their expression intensity were further characterised to determine their molecular weight and pi , and further characterised by mass spectrometry as outlined above. The same samples were also assayed further to determine which proteins are associated with spontaneous labour and to determine their temporal expression prior to labour.

(iii) Results

Pilot studies have verified that human CVF sampling is readily achievable and the sample provides sufficient protein (range 400μg - 1800μg) for the performance of these studies. For 2D gel electrophoresis, sample proteins were concentrated by TCA precipitation and resuspended in 2D electrophoresis rehydration buffer as outlined above. Figure 17 illustrates a 2D gel of human CVF. A number of "landmark" proteins characteristic of human CVF have been identified. Preliminary analysis indicates altered protein expression with advancing gestation and spontaneous labour.

Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims

Claims

1. A method of predicting term or pre-term labour in a subject, the method including the steps of determining a level of a cathelicidin or fragment thereof in a biological sample derived from the subject, and comparing the level of the cathelicidin or fragment thereof to a predetermined value.

2. A method of predicting term or pre-term labour in a subject, the method including the steps of determining a level of a polynucleotide encoding a cathelicidin or a fragment thereof in a biological sample derived from the subject, and comparing the level of the polynucleotide encoding the cathelicidin or fragment thereof to a predetermined value.

3. The method of claim 1 or claim 2, wherein the predetermined value is derived from a biological sample obtained from a non-pregnant subject or a population thereof.

4. The method of claim 1 or claim 2, wherein the predetermined value is derived from a biological sample obtained from a pregnant subject that has not entered labour, or a population thereof.

5. A method of any one of claims 1 to 4, wherein the predetermined value is a predetermined level of a cathelicidin or fragment thereof or a polynucleotide encoding a cathelicidin or a fragment thereof.

6. A method of monitoring a subject for term or pre-term labour, the method including the steps of determining a level of a cathelicidin or fragment thereof in a first biological sample derived from the subject, and comparing the level of the cathelicidin or fragment thereof to a level of the cathelicidin or fragment thereof identified in a second biological sample obtained from -the same subject.

7. A method of monitoring a subject for term or pre-term labour, the method including the steps of determining a level of a polynucleotide encoding a cathelicidin or a fragment thereof in a first biological sample derived from the subject, and comparing the level of the polynucleotide to a level of a polynucleotide encoding the cathelicidin or fragment thereof identified in a second biological sample obtained from the same subject.

8. The method of claim 6 or 7, wherein the second biological sample is obtained from the subject after obtaining the first biological sample.

9. The method of claim 6 or 7, wherein the second biological sample is obtained from the subject before obtaining the first biological sample.

10. The method of any one of claims 1 to 9, wherein the cathelicidin is selected from the group including hCAP18, LL-37, bactenicin-1 , -5 and -7, eCATH-1 , -2 and -3, SMAP29, BMAP-27, BMAP-28, protegrin-1 to -5, PMAP- 23, PMAP-36, PMAP-37 and mCRAMP.

11. The method of any one of claims 1 to 10, wherein the subject is selected from the group of species including human, bovine, ovine, equine and canine.

12. The method of claim 11 , wherein the subject is a human.

13. The method of claim 11 , wherein the subject is of the ovine species.

14. The method of claim 11 , wherein the subject is of the bovine species.

15. The method of claim 11 , wherein the subject is of the equine species.

16. The method of claim 12, wherein the cathelicidin is hCAP18.

17. The method of claim 13, wherein the cathelicidin is bactenicin-1, bactenicin-5, SMAP-29 or any combination thereof.

18. The method of claim 17, wherein the cathelicidin is bactenicin-1.

19. The method of claim 14, wherein the cathelicidin is bactenicin-5, bactenicin-7, BMAP-27, BMAP-28 or any combination thereof.

20. The method of claim 15, wherein the cathelicidin is eCATH-1 , eCATH -2, eCATH-3 or any combination thereof.

21. The method of any one of claims 1 to 20, wherein the biological sample is selected from the group including cervicovaginal secretion, trachial-bronchial secretion, pharyngeal secretion, amniotic fluid, maternal blood, fetal blood, serum, plasma, sweat, tears, cerebral spinal fluid, sputum, urine, synovial fluid, saliva, a cell and tissue.

22. The method of claim 21 , wherein the biological sample is a cervicovaginal secretion.

23. The method of claim 21 , wherein the biological sample is amniotic fluid.

24. The method of any one of claims 1 to 23, wherein the biological sample is derived from the subject during the period of about the first day of the last menstrual period until birth.

25. The method of claim 12, wherein the biological sample is derived from the subject between about 24 weeks and 36 weeks gestation.

26. The method of any one of claims 1 to 25, wherein the fragment includes an antigenic fragment.

27. A kit when used to predict term or pre-term labour in a subject, the kit including an agent which detects a cathelicidin or fragment thereof and a detection system for detecting the agent.

28. A kit when used to predict term or pre-term labour in a subject, the kit including an agent which detects a polynucleotide encoding a cathelicidin or fragment thereof and a detection system for detecting the agent.

29. A kit when used to monitor a subject in term or pre-term labour, the kit including an agent which detects a cathelicidin or fragment thereof and a detection system for detecting the agent.

30. A kit according to claim 27 or 29 wherein the agent is an antibody to the cathelicidin or fragment thereof.

31. A kit according to claim 28 wherein the agent is a primer which can hybridise to a polynucleotide that encodes the cathelicidin or fragment thereof.

32. A method of preventing preterm labour, the method including the steps of identifying a subject at risk of pre-term labour according to the method of any one of claims 1 to 26 and administering to that subject a treatment designed to delay or prevent the onset or progression of pre-term labour.