WO2004019041A1

WO2004019041A1 - Novel prognostic and diagnostic markers of an acute pulmonary exacerbation and recovery therefrom

Info

Publication number: WO2004019041A1
Application number: PCT/AU2003/001064
Authority: WO
Inventors: Nicolle Hannah Packer; Niclas Karlsson; Benjamin Luke Schulz
Original assignee: Proteome Systems Intellectual Property Pty Ltd
Priority date: 2002-08-20
Filing date: 2003-08-20
Publication date: 2004-03-04

Abstract

The present invention provides methods for the diagnosis and/or prognosis of respiratory infection and inflammation of the respiratory tract, in particular for the diagnosis/prognosis of an acute pulmonary exacerbation in a cystic fibrosis patient. In one embodiment, the methods of the present invention are based upon determining specific post-translational modifications to the MUC5B apoprotein in the sputum or saliva of a patient. In a further embodiment, the methods of the present invention are based upon determining specific modifications to the glycosylation of a mucin in the MUC5B-containing fraction of sputum or saliva of a patient and, in particular, upon modifications to the glycosylation profile of the MUC5B protein.

Description

Novel Prognostic and diagnostic markers of an acute pulmonary exacerbation and recovery therefrom

Field of the invention The present invention relates to a method for the diagnosis or prognosis of an inflammatory condition of the lung, a bacterial infection of the lung, a viral infection of the lung, a respiratory infection, a respiratory disease, or a lung disease in a subject, and, more particularly to a method for determining whether or not a subject suffering from cystic fibrosis has an exacerbated condition eg., as a consequence of lung infection and/or inflammation, or alternatively, has responded to treatment for an exacerbated condition.

Background of the invention General Information As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Unless otherwise stated or an appropriate construction would require otherwise, the integers, steps and features described herein for each embodiment shall be taken to apply mutatis mutandis to every other embodiment. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific examples described herein. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

All the references cited in this application are specifically incorporated by reference herein.

The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference: Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;

Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp1-22; Atkinson etal., pp35-81 ; Sproat etal., pp 83-115; and Wu et al., pp 135-151; Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text;

Perbal, B., A Practical Guide to Molecular Cloning (1984);

Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series;

J.F. Ramalho Ortigao, "The Chemistry of Peptide Synthesis" In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany);

Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976). Biochem.

Biophys. Res. Commun. 73 336-342 Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154.

Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and

Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.

Wϋnsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der

Organischen Chemie (Mϋler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.

Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag,

Heidelberg.

Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis,

Springer-Verlag, Heidelberg. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.

Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.

Blackwell, eds., 1986, Blackwell Scientific Publications).

2. Description of the related art Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disease affecting Caucasian populations. CF has an incidence in neonatals of about 0.05%, indicating a carrier frequency of about 5% of the population. Biological parents of subjects with CF are, by definition, obligatory carriers. Carriers are clinically normal and their detection prior to the birth of an affected child has been precluded by the absence of detectable effects of the gene in single dose. Methods for detecting CF include DNA sequencing, enzyme immunoassay (Sanguiolo et al., Int. J. Clin. Lab. Res., 25, 142-145, 1995), multiplex DGGE analysis (Costes et al, Hum. Mut. 2, 185-191, 1993), and the use of the polymerase chain reaction (PCR) in conjunction with allele-specific oligonucleotide probes (PCR-ASO). US Patent Application No. 20030008281 (Weston et al.) describes a two tube multiplex ARMS assay for simultaneously detecting 12 of the most prevalent CF mutations in humans, specifically for detecting CFTR gene mutations such as, for example, G542X, W1282X, N1303K, ΔF508, R553X, G551D, R117H, R1162X and R334W (Kazazian etal., Hum Mut. 4, 167-177, 1994), as well as the test distinguishing between CF ΔF508 heterozygotes and homozygotes. The principle of the ARMS test is that the 3'- end of an ARMS amplification primer confers allele-specificity, and an ARMS product is only generated if the primer is complementary to its target at the 3'-end under the appropriate conditions.

CF is a disease of the exocrine glands, affecting most characteristically the pancreas, respiratory system, and sweat glands. The disease usually begins during infancy and the prognosis for an affected child with CF is a median life expectancy currently estimated to be 30 years.

CF is typified by chronic respiratory infection, pancreatic insufficiency, and susceptibility to heat prostration. It is a major cause of death in children. It is estimated that there are between ten million and twelve million carriers for cystic fibrosis in the United States. Each year, between two thousand and three thousand children are born in the United States who are affected by cystic fibrosis. The cost of therapy for cystic fibrosis patients exceeds US$20,000 per year per patient. Of patients diagnosed in early childhood, fewer than fifty percent reach adulthood.

A serious consequence of CF is an exacerbated clinical condition or exacerbated state. As used herein the term "acute clinical exacerbation", "acute exacerbation", "clinical exacerbation", "exacerbation", or "exacerbated state" in the context of a CF patient shall be understood to mean an exaggeration of a pulmonary symptom of CF.

In most cases, such a clinical exacerbation will be a consequence of a respiratory infection, or increased inflammation. The term "respiratory infection" in this context includes invasion by and/or multiplication and/or colonisation of a pathogenic microorganism in one or more components of the respiratory tract, such as, for example, lung, epiglottis, trachea, bronchi, bronchioles, or alveoli. Commonly, such infections result in the inflammation of the respiratory tract.

CF patients are particularly susceptible to respiratory infections from organisms such as, for example, Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, Aspergillus fumigatus, Burkholderia cepacia complex, Stenotrophomonas maltophila, Alcaligenes (Achromobacter) xylosoxidans, B. gladioli, Ralstonia picketti Influenza A virus and Respiratory Syncytial Virus. The most common causes of respiratory infection are the bacteria S. aureus, P. aeruginosa, and H. influenzae.

For example a chronic respiratory infection, particularly an infection of the lung by P. aeruginosa, accounts for almost 90% of the morbidity and mortality in CF. By age 12, about 60-90% of CF patients are infected with P. aeruginosa.

Progressive loss of pulmonary function over many years due to chronic infection with mucoid P. aeruginosa is common in subjects suffering from CF. Smith et al., Cell. 85, 229-236, 1996, reported defective bacterial killing by fluid obtained from airway epithelial cell cultures of CF patients, and suggested that this phenomenon was due to the inhibition of an unidentified antimicrobial factor resulting from increased levels of sodium chloride in the airway epithelial fluid.

Severe chronic pulmonary disease is also associated with cases of CF wherein CFTR expression on the cell surface is reduced, such as, for example, in patients carrying the ΔF508 mutation. Pier et al. Science. 271, 64-67, 1996 proposed that ingestion and clearance of P. aeruginosa by epithelial cells may protect the lungs against infection, since the specific ingestion and clearance of P. aeruginosa was compromised in a cell line derived from a patient with the ΔF508 mutation.

US Patent No. 6, 245,735 to Brigham and Womens Hospital disclosed the binding of P. aeruginosa to CFTR via the core portion of the lipopolysaccharide of P. aeruginosa. Also disclosed was a method for up-regulating the CFTR in epithelial mucosa to thereby enhance clearance of P. aeruginosa. Such a method comprises contacting mucosal cells expressing the CFTR with the core portion of the lipopolysaccharide of P. aeruginosa.

Patients suffering from CF are extremely susceptible to acute clinical exacerbations, often resulting in an increase in inflammation and mucus production, thus increasing the risk of bronchiectasis and eventually respiratory failure.

An acute clinical exacerbation is generally assessed using the protocols described in Williams et al Australian Journal of Physiotherapy, 41 227 - 236, 2001 ; Dakin et al, Pediatr Pulmonol 34, 436-442, 2001 ; or Rosenfeld et al, J.Pediatr 139 359-365, 2001. In particular, several criteria are assessed, and a patient satisfying four or more of these criteria is considered to have an acute clinical exacerbation. These criteria are as follows: i. Change in sputum production (volume, colour, consistency); ii. New or increased haemoptysis; iii. Increased cough; iv. Increased dyspnoea (shortness of breath); v. Malaise, fatigue or lethargy; vi. Decreased exercise tolerance; vii. Fever; viii. Anorexia or weight loss; ix. Sinus pain/tenderness or change in sinus discharge; x. FVC or FEVi decreased 10% from previous recorded value; xi. Radiographic changes indicative of a pulmonary infection; and xii. Changes in chest sounds.

Alternatively, an acute clinical exacerbation is also diagnosed using by detecting the concentration of C-reactive protein, determining erythrocyte sedimentation rate, peripheral neutrophil counts and determining serum levels of haptoglobin, as reviewed in Hϋner ef a/, Med Bull Istanbul, 32(1), 1999.

Furthermore, several methods have been suggested for the detection of complications of CF, in particular bacterial infection, more specifically P. aeruginosa infection. Such methods include the monitoring of levels of IgG specific to core lipopolysaccharide (US Patent No. 5,179,001), IgG specific to P. aeruginosa (Brett etal, J. Clin. Pathol. 39(10) 1124-1129, 1986), and IgA specific to P. aeruginosa (Brett et al, J. Clin. Pathol. 41(10) 1130-1134, 1988). However, these assays require a significant response by the patient's immune system to a P. aeruginosa infection in order to detect these immunoglobulin molecules. Accordingly, such methods are only useful in the monitoring, or prognosis of an established P. aeruginosa infection, rather than being suitable for early detection of infection.

Alternative methods have been suggested for the diagnosis of P. aeruginosa infection in patients suffering from CF, including the detection of P. aeruginosa type-Ill secretory proteins (Roy-Burman et al, J. Infect. Dis. 183(12), 1767-1774, 2001 ), the detection of antibodies specific to P. aeruginosa flagellar types a or b in CF patients (Anderson et al, J. Clin. Microbiol. 27(12), 2789-2793, 1989), and the detection of antibodies to sodium alginate exo-polysaccharide of P. aeruginosa in CF patients (Bryan et al, J. Clin. Microbiol. 18(2), 276-282, 1983). Again these assays do not detect early P. aeruginosa infection.

Other pathogens, such as Staphylococcus aureus and non-typable Haemophilus influenzae, are also commonly isolated from the respiratory tract of CF patients. Whilst there has been significant progress in diagnosing CF, the need still exists for further diagnostic and prognostic assays for complications arising in patients suffering from the disease, in particular rapid and reliable methods for determining whether or not a subject suffering from CF is about to enter an exacerbated condition or state, eg., respiratory infection. Sensitive assays for accurately predicting whether or not a CF patient is entering an exacerbated state, whether this is caused by a microbial infection or not, are highly desirable, as are reliable prognostic indicator for determining whether or not such a subject is responding to treatment for the exacerbated condition.

Mucins constitute a large part of the total protein content of the lung mucosa and whole sputum. They are very high molecular weight glycoproteins, with post- translational oligosaccharide modifications accounting for up to 80% of their total molecular weight. These oligosaccharides comprise an enormous diversity of structures, which are involved in protein-protein interactions, including mediating leukocyte-pathogen interactions (Prakobphol et al. βiochemistry 38, 6817-6825, 1999).

Mucin oligosaccharides are synthesised through the action of a variety of glycosyltransferases, and changes in the activity of these enzymes alter the oligosaccharide structures present (Lamblin, et al. Glycoconjugate J. 18, 661- 684, 2001). Fucose, sulfate and sialic acid are typical terminal residues in oligosaccharides, and are therefore important in forming the structures of terminal epitopes which regulate many protein-protein interactions.

There are at least 14 mucin apoproteins encoded by the human genome, designated MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5B, MUC5AC, MUC6, MUC7, MUC8, MUC11, MUC12, MUC13 and MUC16 (Dekker et al., Trends Biochem Sci. 27, 126-131, 2002). Of these mucins, MUC1 , MUC2, MUC4, MUC5B, MUC5AC, MUC7 and MUC8 have been previously detected in human airways, however MUC2, MUC5B and MUC5AC are considered to be major gel- forming mucins in normal and pathological secretions of the airways (Kirkham et al., Biochem. J., 361 , 537-546, 2002).

Global alterations in glycosylation of sputum have been associated with CF, but the precise nature of the modified glycosylations, such as, for example, the apoproteins that are modified, has not been elucidated. Nor is known whether or not these global changes are a cause or an effect of CF pathology. In summary, Rhim et al., Glycoconjugate J. 18, 649-659, 2001 ) suggest that there is enhanced increased fucosylation and decreased sialylation of glycoconjugates generally on epithelial cells, in the absence of a functional CFTR. However, this pattern does not appear in the glycoconjugates of secreted airway mucins (Lamblin et al., Glycoconjugate J. 18, 661-684, 2001)..

Davril et al., Glycobiology 9, 311-321, 1999, described increased sulfation in respiratory and salivary mucins from CF subjects, while increased levels of sialylation and sulfation have also been associated with the severity of infection in CF, as well as in other pulmonary infections. Again, these were global alterations in mucin proteins. Accordingly, the changes reported by Davril et al (1999) may mask specific changes to particular mucin proteins.

Kirkham et al., Biochem. J., 361 , 537-546, 2002, also reported an increase in the relative amount of MUC5B relative to MUC5AC in the sputum of CF and asthma patients, and a corresponding increase in a low charge isoform of MUC5B, which was attributed to enhanced submucosal gland secretion of mucins.

There have been no reports which directly compared sputum mucin glycosylation in a large sample size of clearly-defined CF subjects and non-CF healthy control subjects, let alone a clearly defined clinical group of CF subjects with acute pulmonary exacerbation, that analyzes the glycosylation profiles of the same subjects after a period of hospital treatment. In summary, there have been no detailed reports correlating modifications to the glycosylation status of specific respiratory and/or salivary mucin proteins with the aetiology of an acute pulmonary exacerbation in a CF subject, or the recovery from such an exacerbation.

Summary of invention In work leading up to the present invention, the inventors sought to characterize the glycoproteome of CF subjects during acute pulmonary exacerbations and following treatment, and compare these profiles to the glycoproteome of non-CF subjects, including an analysis of (i) the MUC5B apoproteins of non-CF and CF subjects during and following an acute pulmonary exacerbation; and (ii) the glycosylation status of the MUC5B-containing mucin fraction of sputa that characterizes the acute pulmonary exacerbation and recovery phases.

Glycoproteomic analysis was performed on the high molecular weight proteins in induced sputum from 19 CF subjects with acute pulmonary exacerbations, 13 of these subjects after discharge from hospitalisation, and 19 non-CF healthy controls. Significant differences were observed in both the MUC5B apoprotein and MUC5B-containing fraction glycosylation profiles of these groups. These modifications indicate novel diagnostic and prognostic approaches to monitoring lung function in CF and non-CF subjects.

In particular, the inventors have shown that the MUC5B apoprotein is modified in CF subjects suffering from an acute clinical exacerbation. When CF is exacerbated, such as for example by infection of the epithelial mucosa of the lung, the N-terminal and C-terminal portions of the MUC5B apoprotein are cleaved, as determined by analysis of tryptic peptide digests of the MUC5B apoproteins present in the sputa of non-CF and CF subjects suffering from a clinical exacerbation. The present inventors have further shown that CF subjects successfully treated with an antibacterial compound and/or an anti-inflammatory compound following acute clinical exacerbation produce sputa comprising native or uncleaved MUC5B apoprotein. Conversely, in CF patients that do not respond to anti-bacterial treatment and/or anti-inflammatory treatment, the cleaved MUC5B apoprotein remains detectable in patient samples. The glycosylation profiles of the most abundant mucin band in sputum from exacerbated CF subjects showed a 1.5-fold decrease in fucosylation, a 2.8-fold increase in sialylation, and a 4.3-fόld decrease in sulfation, compared to the most abundant sputum mucins in non-CF healthy control sputum. These differences were all significant at the 95% confidence interval.

Comparison of sputum from individual CF subjects at acute pulmonary exacerbation, and the same subjects upon discharge from hospital, showed a there was large variation in the individual response to hospital treatment, both in terms of clinical recovery, and differences in sputum mucin protein profile and glycosylation profile. However, CF subjects showing a clear improvement in clinical signs also exhibited a large change in mucin protein and glycosylation profiles post-discharge from hospital compared to their profiles during the acute exacerbation phase. In particular, the glycosylation profile of the MUC5B- containing mucin fraction from these CF subjects exhibited a 1.8-fold increase in fucosylation, a 2.0-fold decrease in sialylation and a 1.6-fold decrease in sulfation post-discharge from hospital compared to during the acute exacerbation phase. The fucose and sialic acid content at discharge more closely resemble the mucin glycosylation profiles of non-CF healthy control subjects. However, the sulfate content of the MUC5B-containing mucin fraction from the sputum of these recovered CF subjects was even lower than at exacerbation, and remained significantly less than that of non-CF healthy controls. These data suggest the following glycan-based diagnostic/prognostic assays: (i) a novel diagnostic assay of CF or a past or present acute pulmonary exacerbation in a CF subject based upon determination of a specific decrease in sulfation of sputum mucins relative to a non-CF subject;

(ii) a novel diagnostic/prognostic assay of an acute pulmonary exacerbation in a CF subject based upon determination of the relative amounts and/or changes to the degree of fucosylation and/or sialylation and/or sulfation of a MUC5B-containing mucin fraction of human sputum; and (iii) a novel diagnostic/prognostic assay of recovery from an acute pulmonary exacerbation in a CF subject based upon determination of the relative amounts and/or changes to the degree of fucosylation and/or sialylation and/or sulfation of a MUC5B-containing mucin fraction of human sputum.

Accordingly, the data presented herein support the development and production of novel diagnostics for the detection of an acute clinical exacerbation in a CF subject, and novel prognostic indicators for the progression of the exacerbated state, preferably for the early diagnosis of acute clinical exacerbation. It will also be apparent to the skilled person that such prognostic indicators as described herein may be used in conjunction with therapeutic treatments for CF or an acute clinical exacerbation associated therewith.

As used herein, the term "progression" is not to be taken as necessarily indicating a worsening of a clinical condition or disease state. In the present context, the term "progression" is to be given its widest meaning, to include any prediction of the course of a clinical condition or disease state over time, such as a deterioration or improvement in clinical symptoms, or alternatively, no change in clinical symptoms over time

As used herein, the term "MUC5B-containing mucin fraction" or similar term such as "MUC5B-containing" shall be taken to refer to a fraction of saliva or sputum that comprises respiratory mucins wherein the predominant mucin is a glycosylated form of MUC5B having an estimated molecular weight of between about 1 MDa and about 4 MDa as determined by one-dimensional SDS- AgPAGE. However, the invention is not to be limited to a requirement for a partial or complete purification of MUC5B because, having provided the diagnostic markers described herein, the skilled artisan would readily and without undue experimentation be capable of performing the diagnostic assays described herein using crude sputum or saliva. Without limiting the various embodiments described herein to the actual step of partially or completely purifying MUC5B, it is preferred for said MUC5B-containing mucin fraction to be derived from the 1-4 MDa region of an SDS- agarose/polyacrylamide gel following by one-dimensional SDS-AgPAGE, or by other size exclusion method known to the skilled artisan. Alternatively or in addition, a MUC5B-containing mucin fraction provides MUC5B in a sequencably pure form sufficient for amino acid sequence determination of tryptic peptides derived therefrom.

One aspect of the present invention provides methods of diagnosing an inflammatory condition or infection of the respiratory tract in a subject.

As used herein, the term "inflammatory condition" shall be understood to mean a state of the respiratory tract that is characterised by one or more changes in the physical appearance of functions of a portion of the respiratory tract, such as, for example, dilation of arterioles, capillaries and venules with increased permeability and blood flow, exudation of fluids (e.g. plasma proteins), leukocytic infiltration, swelling and/or loss of function. In most cases, such an inflammatory response is caused by injury to the respiratory tract or through infection.

As used herein, the term "infection" shall be understood to mean invasion and/or colonisation by a microorganism and/or multiplication of a micro-organism, in particular, a bacterium or a virus, in the respiratory tract of a subject. Such an infection may be unapparent or result in local cellular injury. The infection may be localised, subclinical and temporary or alternatively may spread by extension to become an acute or chronic clinical infection.

Preferably, the infection is a respiratory infection and more preferably a respiratory infection in a CF subject, such as, for example, occurs during an acute pulmonary exacerbation. Respiratory infections characterised by the same changes in MUC5B apoprotein, or alternatively, characterised by the same modifications to glycosylation in the MUC5B-containing fractions of sputum, as occur in acute pulmonary exacerbation of CF, such as, for example, conditions such as chronic bronchitis, bronchiectasis, allergic pulmonary aspergillosis, or ciliary dyskinesia, or infections associated therewith, can also be assayed using the diagnostics/prognostics referred to herein.

As used herein the term "respiratory tract" shall be taken to mean a system of cells and organs functioning in respiration, in particular the organs, tissues and cells of the respiratory tract include, lungs, nose, nasal passage, paranasal sinuses, nasopharynx, larynx, trachea, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, pneumocytes (type 1 and type 2), ciliated mucosal epithelium, mucosal epithelium, squamous epithelial cells, mast cells, goblet cells, and intraepithelial dendritic cells.

In accordance with this aspect of the invention, one embodiment provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising detecting in a biological sample from said subject a modified MUC5B apoprotein that does not comprise an amino acid sequence selected from the group consisting of:

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

NO: 1.

In a related embodiment, the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising detecting in a biological sample from said subject a modified MUC5B apoprotein having an amino acid sequence consisting of residues from about 2345 to about 4922 of SEQ ID NO: 1.

The term "about" in this context shall be taken to mean that the position of the stated amino acid residue may be varied by an amount that extends N-terminally along SEQ ID NO: 1 from position 2344 to the nearest lysine or arginine residue in said sequence, or alternatively, that extends C-terminally along SEQ ID NO: 1 from position 4923 to the nearest lysine or arginine residue in said sequence. Peptides that are not defined by the specific stated amino acid positions are said to "consist essentially of the amino acid residues at those stated positions.

Preferably, the modified MUC5B apoprotein is detected by a process comprising: (i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of

SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of SEQ ID NO: 1 ; (ii) contacting a reference sample from a healthy subject with the antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed; and

(iii) comparing the amount of complex formed at (i) and (ii) wherein a modifed amount of complex at (i) compared to (ii) indicates a modified level of the modified MUC5B apoprotein.

More preferably, the antigen-antibody complex formed at (i) is reduced or absent. As will be apparent from the exemplified subject matter, a reduction in the amount of native or uncleaved MUC5B apoprotein in the mucin fraction is correlated with enhanced levels of the cleaved form of the apoprotein during infection/inflammation, eg., during an acute pulmonary exacerbation of a CF patient. Accordingly, a reduced level of the antibody-antigen complex at (i) compared to (ii) is indicative of an enhanced level of the modified MUC5B apoprotein and wee versa.

In a further embodiment, the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction an elevated level of an oligosaccharide relative to the level of the oligosaccharide in a healthy control subject, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂Fuc₁NeuAcι; HexNac₃Hexι; HexNac₃Hex₃Fuc₂NeuAcι ; HexNac₃Hex₃Fucι NeuAc₂Sulf₁ ; HexNac₃Hex₃Fuc₂NeuAc₂; HexNac₃Hex₂Fuc₂; HexNac₃Hex₂Fuc₂Sulfι; HexNac₂Hex₂FucιNeuAcιSulfι; HexNac₂Hex₂FucιNeuAc₂; and HexNac₃Hex₃FucιNeuAc-|. Preferably, the oligosaccharide comprises a composition selected from the group consisting of:

HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂Fucι NeuAci ; HexNac₃Hex₃Fucι NeuAc₂Sulfι ; HexNac₃Hex₃Fuc₂NeuAc₂; HexNac₃Hex₂Fuc₂Sulfι; and

HexNac₂Hex₂FucιNeuAcιSulfι. Even more preferably, the oligosaccharide comprises a composition selected from the group consisting of: HexNac₃Hex₃Fuc₂NeuAc₂; HexNac₂Hex₂NeuAcι; and HexNac₂Hex₂FucιNeuAc₂

Alternatively, or in addition, an elevated level of the oligosaccharide is determined by reference to an internal control for the sample, in particular an oligosaccharide comprising a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; HexNac₂Hex₂Fuc-|NeuAci; and HexNac₂Hex₂NeuAc₂. In a particularly preferred embodiment, the ratio of the level of an oligosaccharide comprising the composition HexNac₂Hex₂FucιNeuAc₂ to the level of an oligosaccharide comprising the composition HexNac₂Hex₂NeuAc₂ is determined, wherein a higher level of this ratio in the subject compared to the healthy control indicates that the subject is suffering from an inflammatory condition or infection of the respiratory tract.

In an alternative embodiment, the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction a reduced level of an oligosaccharide relative to the level of the oligosaccharide in a healthy control subject, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNacsHexaFuciSulfi; HexNac₃Hex₂Fuc₂Sulf₂; HexNac₃Hex₃FucιSulf₂; HexNac₃Hex₂Fuc₂Sulf₂; HexNac₄Hex₃FucιSulf₂; HexNac₂HexιNeuAcι;

HexNac Hex₃Fuc₂Sulf₂; HexNac₄Hex₃Fuc₃Sulf₂; HexNac₄Hex₄Fuc₂Sulf₂;

HexNac₃Hex₂Sulfι; HexNac₄Hex4Fuc₃Sulf₂; HexNac₂Hex₂NeuAcιSulfι;

HexNac₃Hex₂FucιSulfι; and HexNac₂Hex₂FucιNeuAcιSulf|.

In a further embodiment, the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin in a healthy control subject, wherein the modified glycosylation is selected from the group consisting of:

(i) a decrease in sulfation of the mucin;

(ii) a decrease in sulfation of the mucin coupled with a decrease in fucosylation of the mucin;

(iii) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin;

(iv) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin and a decrease in fucosylation of the mucin; (v) a decrease in the ratio sulfate: sialic acid in the mucin; and

(vi) an increase in the ratio sialic acid: sulfate in the mucin.

Preferably, the mucin is MUC5B.

A second aspect of the present invention provides prognostic methods for determining the course of an inflammatory condition or infection of the respiratory tract in a subject over time.

It will be apparent to the skilled artisan that the course of the disease state over time is suitably performed by a process comprising performing the diagnostic assay of the invention at different time points (eg., at the time of primary diagnosis of the inflammatory condition or infection and at least on additional time point thereafter, such as, for example, following treatment) and comparing the results obtained, wherein no change in the assay result indicates that the subject has not improved or recovered, or has deteriorated. Alternatively, a reversion of one or more diagnostic indicators may indicate that the subject has improved or responded to treatment.

In accordance with this aspect of the invention, one embodiment provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising detecting in a biological sample from said subject an enhanced or reduced level of a MUC5B apoprotein relative to the level of said MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, said MUC5B apoprotein comprising an amino acid sequence selected from the group consisting of: (i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID NO: 1.

In a related embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising detecting in a biological sample from said subject an enhanced or reduced level of a MUC5B apoprotein relative to the level of said MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, said MUC5B apoprotein consisting of the amino acid sequence set forth in SEQ ID NO: 1.

Preferably, the MUC5B apoprotein is detected by a process comprising contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of SEQ ID NO: 1 and wherein the presence of the complex indicates the presence of the MUC5B apoprotein. Even more preferably, the antigen-antibody complex formed was absent from the subject at diagnosis of the inflammation or infection.

In a further embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising detecting in a biological sample from said subject an enhanced or reduced level of a modified MUC5B apoprotein relative to the level of said modified MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said modified MUC5B apoprotein has a sequence that does not comprise an amino acid sequence selected from the group consisting of:

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and (ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID NO: 1.

In a related embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising detecting in a biological sample from said subject an enhanced or reduced level of a modified MUC5B apoprotein relative to the level of said modified MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said modified MUC5B apoprotein has a sequence an amino acid sequence consisting of residues from about 2345 to about 4922 of SEQ ID NO: 1.

Preferably, the modified MUC5B apoprotein is detected by a process comprising: (i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of

SEQ ID NO: 1 ; (ii) contacting a reference sample from the subject obtained at diagnosis with the antibody for a time and under conditions sufficient to form an antigen- antibody complex and detecting the complex formed; and

More preferably, the antigen-antibody complex formed at (i) is absent.

In accordance with these embodiments for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, the detection of a native or unmodified form of the MUC5B apoprotein, or alternatively, a reduced level of the modified form of MUC5B relative to that present in the sputum of the subject at the point of diagnosis of the inflammatory condition or infection, indicates that the subject is recovering from the inflammatory condition or infection. As will be apparent to the skilled artisan, both the native form and the modified form of the MUC5B apoprotein may be present in the sample, such as, for example, if the subject has not fully recovered. In this case, the relative proportions of the two isoforms of the protein are used to assess recovery eg., by comparing the changes over time since the primary diagnosis.

In a further embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂FucιNeuAc-ι; HexNac₃Hexι; HexNac₃Hex₃Fuc₂NeuAcι; HexNac₃Hex₃FucιNeuAc₂Sulfι; HexNac₃Hex₃Fuc₂NeuAc2; HexNac₃Hex₂Fuc₂; HexNac₃Hex₂Fuc₂Sulfι ; HexNac₂Hex₂FucιNeuAcιSulf-ι; HexNac₂Hex₂FucιNeuAc₂; and HexNac₃Hex₃FucιNeuAcι and wherein a similar or elevated level of the oligosaccharide relative to the level at diagnosis indicates that the subject has not recovered and a reduced level of the oligosaccharide relative to the level at diagnosis indicates that the subject has recovered. Preferably, the oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂FucιNeuAcι; HexNac₃Hex₃FucιNeuAc₂Sulf₁; HexNac₃Hex₃Fuc₂NeuAc₂; HexNac₃Hex₂Fuc₂Sulf₁; and HexNac₂Hex₂FucιNeuAcιSulfι, and more preferably, HexNac₂Hex₂FucιNeuAc₂.

Alternatively, or in addition, the level of the oligosaccharide is determined by reference to an internal control for the sample, in particular an oligosaccharide comprising a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; HexNac₂Hex₂Fuc-|NeuAcι; and HexNac₂Hex₂NeuAc₂, more preferably HexNac₂Hex2NeuAc₂. In a particularly preferred embodiment, the ratio of the level of an oligosaccharide comprising the composition HexNac₂Hex₂FucιNeuAc₂ to the level of an oligosaccharide comprising the composition HexNac₂Hex₂NeuAc₂ is determined, wherein a similar or higher ratio for the test sample relative to the ratio for the subject at diagnosis indicates that the subject has not recovered, and a reduced ratio indicates that the subject has recovered.

In an alternative embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B- containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₃Hex₂FucιSulfι; HexNac₃Hex₂Fuc₂Sulf₂; HexNac₃Hex₃FucιSulf₂; HexNac₃Hex2Fuc2Sulf₂; HexNac₄Hex₃FuciSulf₂; HexNac₂HexιNeuAcι; HexNac4Hex₃Fuc₂Sulf₂;

HexNac4Hex₃Fuc₃Sulf₂; HexNac₄Hex₄Fuc₂Sulf₂; HexNac₃Hex₂Sulf₁;

HexNac Hex4Fuc₃Sulf₂; HexNac₂Hex2NeuAcιSulfι; HexNac₃Hex₂FucιSulfι; and HexNac₂Hex₂FucιNeuAcιSulfι.and wherein a reduced or similar level of the oligosaccharide relative to the level at diagnosis indicates that the subject has not recovered and an elevated or enhanced level of the oligosaccharide relative to the level at diagnosis indicates that the subject has recovered. Preferably, the oligosaccharide comprises a composition selected from the group consisting of HexNac3Hex₂Sulf-ι; and HexNac₂Hex₂FucιNeuAcιSulfι.

Alternatively, or in addition, the level of the oligosaccharide is determined by reference to an internal control for the sample, in particular an oligosaccharide comprising a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; HexNac₂Hex₂FucιNeuAcι; and HexNac₂Hex₂NeuAc₂, more preferably HexNac₂Hex₂NeuAc₂.

In a further embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of:

(i) a decrease in sulfation of the mucin coupled with an increase in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin and an increase in fucosylation of the mucin; (v) a decrease in the ratio sulfate: fucose in the mucin; and (vi) an increase in the ratio fucose: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has improved or recovered from the inflammatory condition of infection.

In an alternative embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B- containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of:

(i) a decrease in sulfation of the mucin coupled with a decrease in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin and a decrease in fucosylation of the mucin; (v) a decrease in the ratio sulfate: sialic acid in the mucin; and (vi) an increase in the ratio sialic acid: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has not improved or recovered from the inflammatory condition of infection or has deteriorated.

It is to be understood that the prognostic methods referred to herein are also useful for determining a subject that has or has not responded to treatment with one or more therapeutic compounds eg., an antibiotic regime, for the condition. Accordingly, a third aspect of the present invention provides methods for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection. In one embodiment, there is provided a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting in a biological sample from said subject an amount of a modified MUC5B apoprotein following treatment and comparing the amount of the modified MUC5B apoprotein to the amount of said modified MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said modified MUC5B apoprotein has a sequence that does not comprise an amino acid sequence selected from the group consisting of:

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

NO: 1 and wherein an amount of the modified MUC5B apoprotein following treatment that is not reduced compared to the amount at diagnosis indicates that the subject has not responded to treatment.

In a related embodiment, there is provided a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting in a biological sample from said subject an amount of a modified MUC5B apoprotein following treatment and comparing the amount of the modified MUC5B apoprotein to the amount of said modified MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said modified

MUC5B apoprotein has an amino acid sequence consisting of residues from about 2345 to about 4922 of SEQ ID NO: 1 and wherein an amount of the modified MUC5B apoprotein following treatment that is not reduced compared to the amount at diagnosis indicates that the subject has not responded to treatment. Preferably, the modified MUC5B apoprotein is detected by a process comprising: (i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of

SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of

(iii) comparing the amount of complex formed at (i) and (ii).

In a further embodiment, there is provided a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting in a biological sample from said subject an amount of a MUC5B apoprotein following treatment and comparing the amount of the MUC5B apoprotein to the amount of said MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said MUC5B apoprotein comprises an amino acid sequence selected from the group consisting of: (i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and (ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID NO: 1 and wherein an amount of the MUC5B apoprotein following treatment that is enhanced following treatment compared to the amount at diagnosis indicates that the subject has responded to treatment.

In a related embodiment, there is provided a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting in a biological sample from said subject an amount of a MUC5B apoprotein following treatment and comparing the amount of the MUC5B apoprotein to the amount of said MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said MUC5B apoprotein consists of the amino acid set forth in SEQ ID NO: 1 and wherein an amount of the MUC5B apoprotein following treatment that is enhanced following treatment compared to the amount at diagnosis indicates that the subject has responded to treatment.

Preferably, the MUC5B apoprotein is detected using an antibody that binds specifically to the native isoform of the protein, rather than to the cleaved form, such as, for example, an antibody that binds to the N-terminal and/or C-terminal portion of MUC5B. In accordance with this embodiment, the native MUC5B apoprotein can be detected by a process comprising: (i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of

SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of SEQ ID NO: 1 ;

(ii) contacting a reference sample from the subject obtained at diagnosis with the antibody for a time and under conditions sufficient to form an antigen- antibody complex and detecting the complex formed; and (iii) comparing the amount of complex formed at (i) and (ii).

In a further embodiment, the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide comprises a composition selected from the group consisting of: HexNac2Hex2NeuAc₂; HexNac2Hex₂FucιNeuAcι; HexNac₃Hexι; HexNac3Hex₃Fuc₂NeuAcι; HexNacsHexaFuCiNeuA Sulfi; HexNac₃Hex3Fuc₂NeuAc₂; HexNac3Hex₂Fuc₂; HexNac₃Hex₂Fuc₂Sulfι; HexNac₂Hex₂FucιNeuAcιSulfι; HexNac₂Hex₂FucιNeuAc₂; and HexNac3Hex₃FucιNeuAcι and wherein a similar or elevated level of the oligosaccharide relative to the level at diagnosis indicates that the subject has not responded to treatment and a reduced level of the oligosaccharide relative to the level at diagnosis indicates that the subject has responded to treatment.

Preferably, the oligosaccharide comprises a composition selected from the group consisting of: HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂FucιNeuAcι;

HexNac₃Hex3Fucι NeuAc₂Sulfι ; HexNac₃Hex₃Fuc₂NeuAc₂;

HexNac₃Hex₂Fuc₂Sulfι; and HexNac₂Hex2FucιNeuAcιSulfι, and more preferably selected from the group consiting of: HexNac₃Hex₃Fuc₂NeuAc₂; HexNac₂Hex₂NeuAcι; and HexNac₂Hex₂FucιNeuAc₂

Alternatively, or in addition, the level of the oligosaccharide is determined by reference to an internal control for the sample, in particular an oligosaccharide comprising a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; HexNac₂Hex₂FucιNeuAcι; and HexNac₂Hex₂NeuAc₂, more preferably HexNac₂Hex₂NeuAc₂. In a particularly preferred embodiment, the ratio of the level of an oligosaccharide comprising the composition HexNac₂Hex₂FucιNeuAc2 to the level of an oligosaccharide comprising the composition HexNac₂Hex₂NeuAc₂ is determined, wherein a similar or higher ratio for the test sample relative to the ration for the subject at diagnosis indicates that the subject has not responded to treatment, and a reduced ratio indicates that the subject has responded to treatment.

In an alternative embodiment, the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₃Hex₂FucιSulfι; HexNac₃Hex₂Fuc2Sulf₂; HexNac₃Hex₃FucιSulf₂; HexNac₃Hex₂Fuc₂Sulf₂; - HexNac4Hex₃FucιSulf₂; HexNac₂HexιNeuAcι;

HexNac₄Hex₃Fuc₂Sulf₂; HexNac Hex3Fuc₃Sulf₂; HexNac4Hex4Fuc₂Sulf2; HexNac₃Hex₂Sulfι; HexNac₄Hex4Fuc₃Sulf2; HexNac Hex₂NeuAcιSulfι; HexNac₃Hex₂FucιSulf-ι; and HexNac₂Hex₂FucιNeuAcιSulfι and wherein a reduced or similar level of the oligosaccharide relative to the level at diagnosis indicates that the subject has not responded to treatment and an elevated or enhanced level of the oligosaccharide relative to the level at diagnosis indicates that the subject has responded to treatment. Preferably, the oligosaccharide comprises a composition selected from the group consisting of HexNac₃Hex₂Sulfι; and HexNac₂Hex2Fuc-iNeuAc-iSulfi.

In a further embodiment, the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of: (i) a decrease in sulfation of the mucin coupled with an increase in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin and an increase in fucosylation of the mucin; (v) a decrease in the ratio sulfate: fucose in the mucin; and (vi) an increase in the ratio fucose: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has responded to treatment.

In an alternative embodiment, the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of:

(i) a decrease in sulfation of the mucin coupled with a decrease in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin and a decrease in fucosylation of the mucin; (v) a decrease in the ratio sulfate: sialic acid in the mucin; and (vi) an increase in the ratio sialic acid: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has not responded to treatment. In a further embodiment of the diagnostic/prognostic methods described herein, the biological sample is obtained previously from the subject. In accordance with such an embodiment, the prognostic or diagnostic method is performed ex vivo.

In yet another embodiment, the subject diagnostic/prognostic methods further comprise processing the sample from the subject to produce a derivative or extract that comprises the analyte (eg., protein).

As will be apparent to the skilled person, assays for determining modifications to the MUC5B apoprotein or glycosylation of a mucin in the MUC5B-containing fraction of sputum relative to the point of diagnosis will clearly indicate no improvement to the subject's clinical condition if there is no change to the parameter being tested. The present invention clearly encompasses diagnostic tests wherein the patient's condition does not alter over time as being indicative of no improvement or response to treatment.

The present invention further encompasses any suitable assay format for determining the changes to MUC5B apoprotein and/or modifications to glycosylation of mucins in the MUC5B-containing fraction of mucins. The diagnostic and prognostic assays described herein are performed using standard assay formats appropriate to the detection of proteins or antibodies, or alternatively, for the detection of monosaccharide or oligosaccharide residues. Immunoassay formats, such as, for example, for the detection of protein or sugars, are particularly preferred. Affinity ligands, such as, for example, lectins that bind specific sugars, can also be used in place of, or alongside, antibodies. Alternatively, or in addition, total carbohydrate content of samples is measured using, for example, Periodic Acid-Schiffs reagent (PAS), whilst the presence of acidic residues is measured using, for example, acetic acid Alcian Blue (aAB) and/or sulfuric acid Alcian Blue (sAB). In this respect, aAB detects sialic acid and sulfate and sAB is specific for sulfate. High throughput assay formats are also particularly preferred, and immunoassay formats, or detection systems using lectins, or combinations of PAS, aAB and sAB, or mass spectrometry, are particularly useful for this purpose.

A still further aspect of the present invention provides a method of treatment of a subject suffering from an inflammatory condition or infection of the respiratory tract comprising performing a diagnostic method or prognostic method as described herein.

A still further aspect of the present invention further encompasses any synthetic or recombinant peptides derived from MUC5B or a modified form thereof of a MUC5B apoprotein referred to herein, or antibodies thereto, suitable for use in the assays described herein, in particular, any fragment of a native or unmodified MUC5B apoprotein consisting essentially of residues 1 to about 2344 of SEQ ID NO: 1 or residues from about 4922 to about 5703 of SEQ ID NO: 1 , and more particularly exemplified by a peptide fragment set forth in any one of SEQ ID Nos: 2-17.

Antibodies or fragments thereof are useful in therapeutic, diagnostic and research applications, including the purification and study of the diagnostic/prognostic proteins, identification of cells expressing the isoform of MUC5B, or for sorting or counting of such cells. Thus, the present invention clearly encompasses the use of an antibody or fragment thereof described herein (e.g., monoclonal antibodies or an antigen-binding fragment thereof) in therapy, including prophylaxis, diagnosis, or prognosis, and the use of such antibodies or fragments for the manufacture of a medicament for use in treatment of an inflammatory condition or infection of the respiratory tract.

A still further aspect of the present invention provides a method for diagnosing cystic fibrosis (CF) or a past or present acute pulmonary exacerbation in a CF subject said method comprising determining a reduced sulfation of sputum mucins relative to the level of sulfation in a non-CF subject. Brief description of the drawings

Figure 1 is a photographic representation of one-dimensional (1 D) SDS-AgPAGE of high molecular weight glycoproteins from sputum following PAS staining for carbohydrates. Lane I - Cystic Fibrosis subject #1 with acute pulmonary exacerbation; Lane II - Cystic Fibrosis subject #1 recovered after antibiotic/antiinflammatory treatment; Lane III - Normal subject; Lane IV - Cystic Fibrosis subject #2 with acute pulmonary exacerbation; and Lane V - Cystic Fibrosis subject #2 non-responsive to antibiotic/anti-inflammatory treatment. Numbering at the left of the figure indicates molecular weight of mucins.

Figure 2a is a representation of a mass spectrum showing the glycosylation profile of O-linked oligosaccharides released from a MUC5B-containing mucin fraction of sputum from a healthy non-CF subject. Numbering indicates the m/z ions of different glycans present in the MUC5B-containing mucin fraction.

Figure 2b is a representation of a mass spectrum showing the glycosylation profile of O-linked oligosaccharides released from a MUC5B-containing mucin fraction of sputum from a CF patient suffering from a clinical exacerbation. Numbering indicates the m/z ions of different glycans present in the MUC5B- containing mucin fraction.

Figure 2c is a representation of a mass spectrum showing the glycosylation profile of O-linked oligosaccharides released from a MUC5B-containing mucin fraction of sputum from the CF subject indicated in Figure 2b, following successful treatment for the clinical exacerbation. Numbering indicates the m/z ions of different glycans present in the MUC5B-containing mucin fraction.

Figure 3 is a representation of a single ion chromatograph of the m/z 1331.3 ± 1.0 ion in Figures 2a (left) and 2b (right) following reversed phase HPLC separation of glycans. Differences are observed between the samples. For example, the normal sputum sample shows three distinct isomers of the m/z 1331.3 ± 1.0 ion, whereas the cystic fibrosis sputum sample shows only two isomers.

Figure 4 is a representation showing 1D SDS-AgPAGE profiles of induced sputum and corresponding oligosaccharide mass profiles for each separated mucin band, for the non-CF control subject CYFB1-22 and the exacerbated CF subject CYFB 1-37.

Figure 5 is a graphical representation showing the average oligosaccharide composition of sputum mucins from exacerbated CF (open boxes) and non-CF control subjects (filled boxes) in the cohort of subjects studied. Monosaccharide residues identified are indicated on the x-axis, as follows: HexNAc - N- acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - Λ/-acetylneuraminic acid (Sialic acid); Sulf- sulfate. Error bars show standard error of the mean.

Figure 6 is a graphical representation showing the results of principal components analysis of mucin oligosaccharide composition data for non-CF healthy controls (samples marked H followed by a numeric indicator) and exacerbated CF subjects (samples marked CF followed by a numeric indicator). Compositional arrows for sialic acid, sulfate, N-acetyl hexosamine and fucose are also indicated. Data show strong correlations between an acute pulmonary exacerbation and either sialic acid or sulfate content, and a moderate correlation between an acute pulmonary exacerbation and fucose content, of MUC5B- containing fraction mucins.

Figure 7 is a graphical representation showing the results of principal components analysis of mucin oligosaccharide composition data for a cohort of CF subjects suffering from an acute pulmonary exacerbation (samples marked CF followed by a numeric indicator), and after treatment (samples marked CF followed by a numeric indicator and the identifier B). Compositional arrows for sialic acid, sulfate, N-acetyl hexosamine and fucose are also indicated. Three isolated and roughly grouped post-discharge CF subjects are circled. Figure 8 is a representation showing 1 D SDS-AgPAGE profiles of induced sputum, and corresponding oligosaccharide mass profiles for each separated MUC5B-containing mucin band, for a CF subject during an acute pulmonary exacerbation (CYFB1-11) and following treatment (CYFB1-11B).

Figure 9 is a representation showing 1 D SDS-AgPAGE profiles of induced sputum, and corresponding oligosaccharide mass profiles for each separated MUC5B-containing mucin band, for a CF subject during an acute pulmonary exacerbation (CYFB1-41) and following treatment (CYFB1-41B).

Figure 10a is a graphical representation showing average oligosaccharide composition of sputum mucins at pulmonary exacerbation and post-discharge from hospitalization, for CF subject CYFB1-11 , who displayed marked improvement in clinical signs after treatment. Monosaccharide residues identified are indicated on the x-axis, as follows: HexNAc - Λ/-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - Λ/-acetylneuraminic acid (Sialic acid); Sulf - sulfate.

Figure 10b is a graphical representation showing average oligosaccharide composition of sputum mucins at pulmonary exacerbation and post-discharge from hospitalization, for CF subject CYFB1-41 , who displayed marked improvement in clinical signs after treatment. Monosaccharide residues identified are indicated on the x-axis, as follows: HexNAc - Λ/-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - Λ/-acetylneuraminic acid (Sialic acid); Sulf - sulfate.

Figure 10c is a graphical representation showing average oligosaccharide composition of sputum mucins at pulmonary exacerbation and post-discharge from hospitalization, as the average of data for the two CF subjects CYFB1-11 and CYFB1-41 , who both displayed marked improvement in clinical signs after treatment. Monosaccharide residues identified are indicated on the x-axis, as follows: HexNAc - Λ/-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - Λ/-acetylneuraminic acid (Sialic acid); Sulf - sulfate. Error bars indicate standard error of the mean.

Figure 11 is a representation showing 1 D SDS-AgPAGE profiles of induced sputum, and corresponding oligosaccharide mass profiles for each separated MUC5B-containing mucin band, for a CF subject during an acute pulmonary exacerbation (CYFB1-37) and following treatment (CYFB1-37B). The subject acquired a viral infection during treatment and did not recover from the pulmonary infection at the time the post-treatment sample was taken.

Figure 12 is a graphical representation showing the average oligosaccharide composition of sputum mucins at pulmonary exacerbation and post-discharge from hospitalization, for CF subject CYFB1-37, who did not respond to treatment. Monosaccharide residues identified are indicated on the x-axis, as follows: HexNAc - Λ/-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - N- acetylneuraminic acid (Sialic acid); Sulf - sulfate.

Detailed-description ofthe^referred-embodiments Diagnostic and prognostic assays for modified forms of MUC5B apoprotein

One aspect of the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising detecting in a biological sample from said subject a modified MUC5B apoprotein that does not comprise an amino acid sequence selected from the group consisting of:

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

NO: 1.

A second aspect of the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising detecting in a biological sample from said subject an enhanced or reduced level of a MUC5B apoprotein relative to the level of said MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, said MUC5B apoprotein comprising an amino acid sequence selected from the group consisting of:

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

NO: 1.

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID NO: 1. In a related embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising detecting in a biological sample from said subject an enhanced or reduced level of a modified MUC5B apoprotein relative to the level of said modified MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said modified MUC5B apoprotein has a sequence an amino acid sequence consisting of residues from about 2345 to about 4922 of SEQ ID NO: 1.

A third aspect of the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting in a biological sample from said subject an amount of a modified MUC5B apoprotein following treatment and comparing the amount of the modified MUC5B apoprotein to the amount of said modified MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said modified MUC5B apoprotein has a sequence that does not comprise an amino acid sequence selected from the group consisting of: (i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID NO: 1 and wherein an amount of the modified MUC5B apoprotein following treatment that is not reduced compared to the amount at diagnosis indicates that the subject has not responded to treatment or has deteriorated.

MUC5B apoprotein has an amino acid sequence consisting of residues from about 2345 to about 4922 of SEQ ID NO: 1 and wherein an amount of the modified MUC5B apoprotein following treatment that is not reduced compared to the amount at diagnosis indicates that the subject has not responded to treatment or has deteriorated.

In a further embodiment, there is provided a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting in a biological sample from said subject an amount of a MUC5B apoprotein following treatment and comparing the amount of the MUC5B apoprotein to the amount of said MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said MUC5B apoprotein comprises an amino acid sequence selected from the group consisting of: (i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1; and (ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

NO: 1 and wherein an amount of the MUC5B apoprotein following treatment that is enhanced following treatment compared to the amount at diagnosis indicates that the subject has responded to treatment.

The term "about" in the present context shall be taken to mean that the position of the stated amino acid residue may be varied by an amount that extends N- terminally along SEQ ID NO: 1 from position 2344 to the nearest lysine or arginine residue in said sequence, or alternatively, that extends C-terminally along SEQ ID NO: 1 from position 4923 to the nearest lysine or arginine residue in said sequence. Peptides that are not defined by the specific stated amino acid positions are said to "consist essentially of the amino acid residues at those stated positions.

As used herein the term "modified" with respect to a MUC5B apoprotein is to be taken to mean a cleaved or processed or degraded form of a native MUC5B protein that comprises the full-length sequence set forth in SEQ ID NO: 1 , or alternatively, a modified form of MUC5B that differs by one or more glycosylations from the form of the protein present in the saliva or sputum of a subject that does not suffer from cystic fibrosis and has no external or internal symptoms associated with a respiratory infection.

As used herein the term "MUC5B apoprotein " shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of set forth in SEQ ID NO: 1. The term "MUC5B apoprotein" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of MUC5B in sputum. As used herein the term "known biological properties" shall be understood to mean any physico-chemical properties by which a particular peptide, polypeptide, or protein may be characterised, such as, for example molecular weight, post-translational modifications, amino acid composition, or isoelectric point, amongst others.

Preferably, a modified MUC5B apoprotein will be missing residues 1 to about

2344 of SEQ ID NO: 1 and/or residues from about 4923 to 5703 of SEQ ID NO: 1 , as consequence of enhanced proteolytic cleavage of MucδB in the sputum of the subject. Accordingly, a modified MUC5B apoprotein preferably consists essentially of residues from about 2345 to about 4922 of SEQ ID NO: 1.

In a related embodiment, the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising detecting in a biological sample from said subject a modified MUC5B apoprotein having an amino acid sequence consisting of residues from about

2345 to about 4922 of SEQ ID NO: 1.

Preferably, the percentage identity to SEQ ID NO: 1 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. In determining whether or not two amino acid sequences fall within the defined percentage identity limits supra, those skilled in the art will be aware that it is possible to conduct a side-by-side comparison of the amino acid sequences. In such comparisons or alignments, differences will arise in the positioning of non- identical residues depending upon the algorithm used to perform the alignment. In the present context, references to percentage identities and similarities between two or more amino acid sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard algorithm known to those skilled in the art. In particular, amino acid identities and similarities are calculated using software of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America, eg., using the GAP program of Devereaux et al., Nucl. Acids Res. 12, 387-395, 1984, which utilizes the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 443-453, 1970. Alternatively, the CLUSTAL W algorithm of Thompson et al., Nucl. Acids Res. 22, 4673-4680, 1994, is used to obtain an alignment of multiple sequences, wherein it is necessary or desirable to maximise the number of identical/similar residues and to minimise the number and/or length of sequence gaps in the alignment. Amino acid sequence alignments can also be performed using a variety of other commercially available sequence analysis programs, such as, for example, the BLAST program available at NCBI.

In one embodiment, the detection of full length and/or cleaved MUC5B proteins are used to diagnose or prognose a clinical exacerbation. It will be apparent from the preceding description that the detection of N-terminal and/or C-terminal peptides of MUC5B is negatively correlated with the presence of the modified MUC5B apoprotein and, as a consequence, is also negatively correlated with the infectious or inflammatory state. Accordingly, the absence of N-terminal and/or C-terminal fragments of MUC5B in the sputum or saliva of a subject is indicative of the infectious or inflammatory state, whereas the disappearance of these fragments is also indicative of a recovery or successful treatment. Assay formats for quantitation of proteins

The amount of a MUC5B protein or a modified form thereof is determined using any one or more of a variety of techniques known to the skilled artisan such as, for example, a technique selected from the group consisting of, an immunoblot, a Western blot, a dot blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), isotope-coded affinity tags (ICAT), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), Mass spectrometry (including tandem mass spectrometry, eg LC MS/MS), biosensor technology, evanescent fiber-optics technology or protein chip technology.

In one embodiment the assay used to determine the amount or level of MUC5B protein is a semi-quantitative assay.

In another embodiment the assay used to determine the amount or level of a MUC5B protein or modified form thereof is a quantitative assay.

In one embodiment, the amount of MUC5B or a modified form thereof in a biological sample derived from a patient is compared to the amount of the same protein detected in a biological sample previously derived from the same patient. As will be apparent to a person skilled in the art, this method may be used to continually monitor a patient with CF. In this way a patient may be monitored for the onset of an acute clinical exacerbation, with the goal of commencing treatment for said exacerbation prior to it becoming established and causing damage to the lungs of said patient.

Alternatively, or in addition, the amount of a MUC5B protein or a modified form thereof detected in a biological sample derived from a subject with CF may be compared to a reference sample, wherein the reference sample is derived from one or more CF patients that do not suffer from an acute clinical exacerbation or alternatively, one or more CF patients that have recently received successful treatment for an acute exacerbation, and/or one or more subjects that do not have CF and that do not suffer from an acute clinical exacerbation.

In one embodiment, a modified form of MUC5B is detected in a reference sample, however said modified form is not detected in a patient sample, indicating that the patient from whom the sample was derived is suffering from or will develop an acute exacerbation state.

Alternatively, the amount of modified MUC5B is detected at enhanced levels in a biological sample isolated from a CF patient that is suffering from and developing an exacerbated state, when said level is compared to the level detected in a reference sample. Again, this indicates that the patient from whom the biological sample was isolated is suffering from or will develop an acute exacerbated state.

Standard solid-phase ELISA formats are particularly useful in determining the concentration of a protein from a variety of patient samples.

Preferably, the MUC5B apoprotein is detected using an antibody that binds specifically to the native isoform of the protein, rather than to the cleaved form, such as, for example, an antibody that binds to the N-terminal and/or C-terminal portion of MUC5B. Preferably, the MUC5B apoprotein is detected by a process comprising contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of SEQ ID NO: 1 and wherein the presence of the complex indicates the presence of the MUC5B apoprotein. Detection of this protein is positively correlated with the healthy state of a subject, whereas absence of this native protein is indicative of the disease state. In an alternative embodiment, the native MUC5B apoprotein can be detected by a process comprising:

(i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of SEQ ID NO: 1;

(ii) contacting a reference sample from the subject obtained at diagnosis with the antibody for a time and under conditions sufficient to form an antigen- antibody complex and detecting the complex formed; and

(iii) comparing the amount of complex formed at (i) and (ii).

Preferably, the modified MUC5B apoprotein is detected by a process comprising: (i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of SEQ ID NO: 1 ;

(ii) contacting a reference sample from the subject obtained at diagnosis with the antibody for a time and under conditions sufficient to form an antigen- antibody complex and detecting the complex formed; and (iii) comparing the amount of complex formed at (i) and (ii). The comparison of amount of complex formed permits the user to assess whether or not the amount of native MUC5B is reduced or enhanced in the patient test sample. As will be apparent form the preceding description, a reduced amount of native MUC5B indicates that the patient has not recovered or repsonded to treatment form infection or inflammation, as the case may be.

SEQ ID NO: 1 ;

(ii) contacting a reference sample from the subject obtained at diagnosis with the antibody for a time and under conditions sufficient to form an antigen- antibody complex and detecting the complex formed; and (iii) comparing the amount of complex formed at (i) and (ii) wherein a modifed amount of complex at (i) compared to (ii) indicates a modified level of the modified MUC5B apoprotein.

Alternatively, the modified MUC5B apoprotein is detected by a process comprising:

(i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of

SEQ ID NO: 1 ; (ii) contacting a reference sample from a healthy subject with the antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed; and (iii) comparing the amount of complex formed at (i) and (ii) wherein a modifed amount of complex at (i) compared to (ii) indicates a modified level of the modified MUC5B apoprotein.

More preferably, the antigen-antibody complex formed at (i) is absent from the sample that typifies an infected state. In one form, such immunoassays involve immobilising a biological sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).

An antibody that specifically binds to the N-terminal portion or C-terminal portion of MUC5B and not to the modified MUC5B is brought into direct contact with the immobilised biological sample, and forms a direct bond with any of its target protein present in said sample. This antibody is generally labelled with a detectable reporter molecule, such as for example, a fluorescent label (e.g. FITC or Texas Red) or an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or β-galactosidase, or alternatively a second labelled antibody can be used that binds to the first antibody. Following washing to remove any unbound antibody the label may be detected either directly, in the case of a fluorescent label, or through the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D- galaotopyranoside (x-gal).

Such ELISA based systems are particularly suitable for quantification of the amount of native MUC5B protein in a sample, by calibrating the detection system against known amounts of a protein standard to which the antibody binds, such as for example, an isolated recombinant isoform of MUC5B or a peptide that binds to the antibody.

In another form, an ELISA consists of immobilizing an antibody that specifically binds native MUC5B but not modified MUC5B on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support. A patient sample is then brought into physical relation with said antibody, and the MUC5B apoprotein is bound or 'captured'. The bound protein can then be detected using a labelled antibody. For example, if the isoform of MUC5B is captured from a human sample, an anti- human antibody is used to detect the captured protein. Alternatively, a third labelled antibody can be used that binds the second (detecting) antibody. It will be apparent to the skilled person that the assay formats described herein are amenable to high throughput formats, such as, for example automation of screening processes, or a microarray format as described in Mendoza et al, Biotechnigues 27(4): 778-788, 1999. Furthermore, variations of the above described assay will be apparent to those skilled in the art, such as, for example, a competitive ELISA.

Alternatively, the presence of an enhanced level of MUC5B or a modified form thereof is detected using a radioimmunoassay (RIA). The basic principle of the assay is the use of a radiolabelled antibody or antigen to detect antibody antigen interactions. An antibody that specifically binds to MUC5B or a modified form thereof is bound to a solid support and a biological sample brought into direct contact with said antibody. In order to detect the of bound antigen, an isolated and/or recombinant form of the antigen is radiolabelled is brought into contact with the same antibody. Following washing the amount of bound radioactivity is detected. As any antigen in the biological sample inhibits binding of the radiolabelled antigen the amount of radioactivity detected is inversely proportional to the amount of antigen in the sample. Such an assay may be quantitated by using a standard curve using increasing known concentrations of the isolated antigen.

As will be apparent to the skilled artisan, such an assay may be modified to use any reporter molecule, such as, for example, an enzyme or a fluorescent molecule, in place of a radioactive label.

Western blotting is also useful for detecting an enhanced level of a protein. In such an assay protein from a biological sample is separated using sodium doedecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using techniques well known in the art and described in, for example, Scopes (jI v Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994). Separated proteins are then transferred to a solid support, such as, for example, a membrane or more specifically PVDF membrane, using methods well known in the art, for example, electrotransfer. This membrane may then be blocked and probed with a labelled antibody or ligand that specifically binds MUC5B or a modified form thereof. Alternatively, a labelled secondary, or even tertiary, antibody or ligand can be used to detect the binding of a specific primary antibody.

To determine the amount of MUC5B or a modified form thereof, the amount detected may be determined using methods well known in the art, such as, for example, densitometry. For example, the amount of a protein species can be determined by densitometry of stained proteins is SDS-PAGE. The intensity of a protein band or spot is normalised against the total amount of protein loaded on a SDS-PAGE gel using methods well known in the art. Alternatively, an amount of MUC5B detected may be normalised against the amount of a control/reference protein. The expression of such a control protein should not be affected by the clinical state of a patient from whom a biological sample is isolated. Such control proteins are well known in the art, and include, actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β2 microglobulin, hydroxy-methylbilane synthase, hypoxanthine phosphoribosyl-transferase 1 (HPRT), ribosomal protein L13c, succinate dehydrogenase complex subunit A and TATA box binding protein (TBP). Following normalisation, a relative amount of MUC5B as detected in a biological sample from a CF patient can be compared to the relative amount of the same protein detected in a reference sample.

The detection of a MUC5B apoprotein or a modified form thereof using a method such as, for example, mass spectrometry, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionisation (ESI), protein chip, biosensor technology, evanescent fiber optics, isotope-coded affinity tags (ICAT) or fluorescence resonance energy transfer, is clearly contemplated in the present invention. High-throughput methods for detecting the presence or absence of a protein are particularly preferred.

In one embodiment, MALDI-TOF is used for the rapid identification of a protein that has been separated by either one- or two-dimensional gel electrophoresis. Accordingly, there is no need to detect the proteins of interest using an antibody or ligand that specifically binds to the protein of interest. Rather, proteins from a biological sample are separated using gel electrophoresis using methods well known in the art and those proteins at approximately the correct molecular weight and/or isoelectric point are analysed using MALDI-TOF to determine the presence or absence of a protein of interest.

Alternatively, MALDI or ESI or a combination of approaches is used to determine the concentration of a particular protein in a biological sample, such as, for example sputum. Such proteins are preferably well characterised previously with regard to parameters such as molecular weight and isoelectric point.

Biosensor devices generally employ an electrode surface in combination with current or impedance measuring elements to be integrated into a device in combination with the assay substrate (such as that described in U.S. Patent No. 5,567,301). An antibody or ligand that specifically binds to a protein of interest is preferably incorporated onto the surface of a biosensor device and a biological sample isolated from a patient (for example sputum that has been solubilised using the methods described herein) contacted to said device. A change in the detected current or impedance by the biosensor device indicates protein binding to said antibody or ligand. Some forms of biosensors known in the art also rely on surface plasmon resonance to detect protein interactions, whereby a change in the surface plasmon resonance surface of reflection is indicative of a protein binding to a ligand or antibody (U.S. Patent No. 5,485,277 and 5,492,840).

Biosensors are of particular use in high throughput analysis due to the ease of adapting such systems to micro- or nano-scales. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This permits the simultaneous detection of several proteins or peptides in a small amount of body fluids.

Evanescent biosensors are also preferred as they do not require the pretreatment of a biological sample prior to detection of a protein of interest. An evanescent biosensor generally relies upon light of a predetermined wavelength interacting with a fluorescent molecule, such as for example, a fluorescent antibody attached near the probe's surface, to emit fluorescence at a different wavelength upon binding of the diagnostic protein to the antibody or ligand.

To produce protein chips, the proteins, peptides, polypeptides, antibodies or ligands that are able to bind specific antibodies or proteins of interest are bound to a solid support such as for example glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, metal or silicon nitride. This immobilization is either direct (e.g. by covalent linkage, such as, for example, Schiffs base formation, disulfide linkage, or amide or urea bond formation) or indirect. Methods of generating a protein chip are known in the art and are described in for example U.S. Patent Application No. 20020136821 , 20020192654, 20020102617 and U.S. Patent No. 6,391 ,625. In order to bind a protein to a solid support it is often necessary to treat the solid support so as to create chemically reactive groups on the surface, such as, for example, with an aldehyde-containing silane reagent. Alternatively, an antibody or ligand may be captured on a microfabricated polyacrylamide gel pad and accelerated into the gel using microelectrophoresis as described in, Arenkov et al. Anal. Biochem. 278:123-131 , 2000.

A protein chip is preferably generated such that several proteins, ligands or antibodies are arrayed on said chip. This format permits the simultaneous screening for the presence of several proteins in a sample. Alternatively, a protein chip may comprise only one protein, ligand or antibody, and be used to screen one or more patient samples for the presence of one polypeptide of interest. Such a chip may also be used to simultaneously screen an array of patient samples for a polypeptide of interest.

Preferably, a protein sample to be analysed using a protein chip is attached to a reporter molecule, such as, for example, a fluorescent molecule, a radioactive molecule, an enzyme, or an antibody that is detectable using methods well known in the art. Accordingly, by contacting a protein chip with a labelled sample and subsequent washing to remove any unbound proteins the presence of a bound protein is detected using methods well known in the art, such as, for example using a DNA microarray reader.

Alternatively, biomolecular interaction analysis-mass spectrometry (BIA-MS) is used to rapidly detect and characterise a protein present in complex biological samples at the low- to sub-fmole level (Nelson et al. Electrophoresis 21: 1155- 1163, 2000). One technique useful in the analysis of a protein chip is surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI- TOF-MS) technology to characterise a protein bound to the protein chip. Alternatively, the protein chip is analysed using ESI as described in U.S. Patent Application 20020139751.

As will be apparent to the skilled artisan, protein chips are particularly amenable to multiplexing of detection reagents. Accordingly, several antibodies or ligands each able to specifically bind a different peptide or protein may be bound to different regions of said protein chip. Analysis of a biological sample using said chip then permits the detecting of multiple proteins (or isoforms of a protein) of interest. Multiplexing of diagnostic and prognostic markers described herein, in particular multiplexing of MUC5B determination with sugar identification, is particularly contemplated in the present invention. In a further embodiment, the samples are analysed using ICAT, essentially as described in US Patent Application No. 20020076739. This system relies upon the labelling of a protein sample from one source (i.e. a healthy individual) with a reagent and the labelling of a protein sample from another source (i.e. a CF patient) with a second reagent that is chemically identical to the first reagent, but differs in mass due to isotope composition. It is preferable that the first and second reagents also comprise a biotin molecule. Equal concentrations of the two samples are then mixed, and peptides recovered by avidin affinity chromatography. Samples are then analysed using mass spectrometry. Any difference in peak heights between the heavy and light peptide ions directly correlates with a difference in protein abundance in a biological sample. The identity of such proteins may then be determined using a method well known in the art, such as, for example MALDI-TOF, or ESI.

In a particularly preferred embodiment, a MUC5B protein or a modified form thereof in a biological sample is detected using 2-dimensional gel electrophoresis. In accordance with this embodiment, it is preferable to remove certain particulate matter from the sample prior to electrophoresis, such as, for example, by centrifugation, filtering, or a combination of centrifugation and filtering. Proteins in the biological sample are then separated. For example, the proteins may be separated according to their charge using isoelectric focussing and/or according to their molecular weight. Two-dimensional separations allow various isoforms of proteins to be identified, as proteins with similar molecular weight are also separated by their charge. Using image analysis software it is possible to determine whether or not a protein of interest is present in a patient sample.

As will be apparent from the preceding discussion, it is particularly preferred to employ a detection system that is antibody or ligand based. Immunoassay formats are even more particularly preferred. Clearly, any antibody or ligand of use in such an assay is encompassed by the instant invention. Methods for the production of such an antibody or ligand are well known in the art and described herein.

Antibodies that bind to N-terminal or C-terminal regions of MUC5B

As used herein the term "antibody" refers to intact monoclonal or polyclonal antibodies, immunoglobulin (IgA, IgD, IgG, IgM, IgE) fractions, humanized antibodies, or recombinant single chain antibodies, as well as fragments thereof, such as, for example Fab, F(ab)2, and Fv fragments.

Antibodies referred to herein are obtained from a commercial source, or alternatively, produced by conventional means. Commercial sources will be well known to those skilled in the art.

Wickstrom et al., J. Biol. Chem., 276, 47116-47121 , 2001 disclose several antisera against peptides within the D1 , D2, D3, and the first Cys domain of MUC5B. Those antibodies bind specifically to the N-terminal protein of native MUC5B that is cleaved from the apoprotein during clinical exacerbation as described herein and, as a consequence, are useful in the immunoassays described herein. Antibodies that bind to 12 amino acids (RNREQVGKFKMC) located in 4 of the cysteine domains of the tandem repeat region, have been publicly disclosed to recognize full-length MUC5B in sputum (Wickstrom et al., Biochem J. 334, 685-693, 1998). Based upon the information disclosed herein, this antibody binds downstream of the N-terminal cleavage site of MUC5B and will therefore also recognize cleaved MUC5B and, as a consequence, can serve as a useful control in these assays.

High titer antibodies are preferred, as these are more useful commercially in kits for diagnostic or therapeutic applications. By "high titer" is meant a titer of at least about 1 :10³ or 1 :10⁴ or 1 :10⁵. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art, and are described, for example in, Harlow and Lane (In: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the antigenic polypeptide is initially injected into any one of a wide variety of mammals (e.g., mice, rats, rabbits, sheep, humans, dogs, pigs, chickens and goats). The immunogen is derived from a natural source, produced by recombinant expression means, or artificially generated, such as by chemical synthesis (e.g., BOC chemistry or FMOC chemistry). In this step, the polypeptides or fragments thereof of this invention may serve as the immunogen without modification. Alternatively, a peptide, polypeptide or protein is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen and optionally a carrier for the protein is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and blood collected from said the animals periodically. Optionally the immunogen may be injected in the presence of an adjuvant, such as, for example Freund's complete or incomplete adjuvant, lysolecithin and dinitrophenol to enhance the immune response to the immunogen. Monoclonal or polyclonal antibodies specific for the polypeptide may then be purified from the blood isolated form an animal by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest may 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 the polypeptide of interest). 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 syngenic 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 or electrofused and then grown in 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 growth media in which the cells have been grown is tested for the presence of binding activity against the polypeptide (immunogen). Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies using methods such as, for example, affinity purification. 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. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

It is preferable that an immunogen used in the production of an antibody is one which is sufficiently antigenic to stimulate the production of antibodies that will bind to the immunogen and is preferably, a high titer antibody. In one embodiment, an immunogen may be an entire protein.

In another embodiment, an immunogen may consist of a peptide representing a fragment of a peptide. Preferably an antibody raised to such an immunogen also recognises the full-length protein from which the immunogen was derived, such as, for example, in its native state or having native conformation.

Alternatively, or in addition, an antibody raised against a peptide immunogen will recognise the full-length protein from which the immunogen was derived when the protein is denatured or reduced. By "denatured" is meant that conformational epitopes of the protein are disrupted under conditions that retain linear B cell epitopes of he protein. As will be known to a skilled artisan linear eptitopes and conformational epitopes may overlap.

In one embodiment, a peptide immunogen is determined using the method described by Hopp Peptide Research 6, 183-190 (1993), wherein a hydrophilic peptide is selected as it is more likely to occur at the surface of the native protein. However, a peptide should not be too highly charged, as this may reduce the efficiency of antibody generation.

In another embodiment, a peptide immunogen is determined using the method described by Palfreyman et al J. Immunol. Meth. 75, 383-393 (1984), wherein the amino- and/or carboxy- terminal amino acids are used to generate a peptide against which specific antibodies are raised.

In yet another embodiment, a peptide immunogen is predicted using an algorithm such as for example that described in Kolaskar and Tongaonkar FEBS Lett. 276(1-2) 172-174 (1990). Such methods are based upon determining the hydrophilicity of regions of a protein, usually 6 amino acids, and determining those hydrophilic regions that are associated with turns in proteins, surface flexibility, or secondary structures, and are unlikely to be modified at the post- translational level, such as, for example by glycosylation. Such regions of a protein are therefore likely to be exposed, that is, at the surface of the three- dimensional structure of the protein. Furthermore, as these regions are not modified, they are likely to remain constant and as such offer a likely site of antibody recognition.

In yet another embodiment, overlapping peptides spanning the entire protein of interest, or a region of said protein may be generated by synthetic means, using techniques well known in the art. Alternatively, a relatively short protein of low abundance or a portion of a protein that is difficult to purify from a natural source, can be produced chemically (e.g. by BOC chemistry or FMOC chemistry). Synthetic peptides are then optionally screened to determine linear B cell epitopes, using techniques well known in the art. In one embodiment, the peptides are screened using an ELISA based screen to determine those against which a CF patient with a clinical exacerbation has raised specific antibodies. Particularly preferred peptides are those against which a CF patient with a clinical exacerbation has raised specific antibodies, but a CF patient not suffering from an exacerbated state, or a healthy individual has not. Any peptide identified in such a screen is of particular use in a peptide based diagnostic or prognostic test.

Alternatively, or in addition, such an immunogenic peptide is used to generate a monoclonal or polyclonal antibody using methods well known in the art, such as, for example, those described herein. The antibody is then tested to determine its specificity and sensitivity using, for example, an ELISA based assay. An antibody that specifically detects an antigen in a CF patient suffering from an acute exacerbation, but not a healthy CF patient, or a normal healthy individual is particularly preferred. More preferable is an antibody that is able to detect an antigen in a CF patient that progresses to develop an acute exacerbation, and thus is an early diagnostic of such a state.

In a particulariy preferred embodiment, the peptide immunogen comprises amino acids consisting essentially of residues 1 to about 2344 of SEQ ID NO: 1 (ie., an N-terminal fragment), or residues from about position 4923 to about position 5703 of SEQ ID NO: 1 (ie., a C-terminal fragment). In an alternative embodiment, any one or more of the tryptic peptides set forth in SEQ ID Nos: 2-17 can be used to generate antibodies that bind specifically to native MUC5B and not to the cleaved form thereof.

Modified glycosylation of mucins in the MUCδB-containing fraction of sputum a) Mass spectrometry assays for detecting specific oligosaccharide structures One aspect of the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction an elevated level of an oligosaccharide relative to the level of the oligosaccharide in a healthy control subject, wherein said oligosaccharide produces an ion by mass spectrometry having a m/z selected from the group consisting of m/z 665.2 ± 1.0, m/z 728.8 ± 1.0, m/z 738.3 ± 1.0, m/z 790.2 ± 1.0, m/z 848.1 ± 1.0, m/z 947.3 ± 1.0, m/z 960.7 ± 1.0, m/z 993.8 ± 1.0, m/z 1040.5 ± 1.0, m/z 1146.2 ± 1.0, m/z 1167.5 ± 1.0, m/z 1186.4 ± 1.0, m/z 1244.4 ± 1.0, m/z 1322.1 ± 1.0, m/z 1331.3 ± 1.0, m/z 1438.2 ± 1.0, m/z 1469.2 ± 1.0, m/z 1477.4 ± 1.0, m/z 1551.3 ± 1.0, m/z 1619.2 ± 1.0, m/z 1695.0 ± 1.0, m/z 1743.0 ± 1.0, m/z 1803.3 ± 1.0, m/z 1828.6 ± 1.0, m/z 1940.9 ± 1.0 and m/z 1970.5 ± 1.0.

Preferably, the ion has an m/z following mass spectrometry selected from the group consisting of m/z 728.8 ± 1.0,. m/z 848.1 ± 1.0, m/z 947.3 ± 1.0, m/z 960.7 ± 1.0, m/z 993.8 ± 1.0, m/z 1146.2 ± 1.0, m/z 1167.5 ± 1.0, m/z 1244.4 ± 1.0, m/z 1322.1 ± 1.0, m/z 1438.2 ± 1.0, m/z 1469.2 ± 1.0, m/z 1477.4 ± 1.0, m/z 1551.3 ± 1.0, m/z 1619.2 ± 1.0, m/z 1695.0 ± 1.0, m/z 1743.0 ± 1.0, m/z 1803.3 ± 1.0, m/z 1828.6 ± 1.0, m/z 1940.9 ± 1.0 and m/z 1970.5 ± 1.0. Even more preferably, the ion has an m/z following mass spectrometry selected from the group consisting of m/z 993.8 ± 1.0, m/z 1244.4 ± 1.0, and m/z 1477.4 ± 1.0, and still more preferably m/z 1477.4 ± 1.0.

Alternatively, or in addition, an elevated level of the oligosaccharide is determined by reference to an internal control for the sample, in particular a ion having an m/z selected from the group consisting of m/z 1040.5 ± 1.0 and m/z 1186.4 ± 1.0 and m/z 1331.3 ± 1.0, more preferably m/z 1186.4 ± 1.0 or m/z 1331.3 ± 1.0 and still more preferably m/z 1331.3 ± 1.0.

In a particularly preferred embodiment, the ratio of the level of the m/z 1477.4 ± 1.0 ion to the m/z 1331.3 ± 1.0 ion is determined, wherein a higher level of this ratio in the subject compared to the healthy control indicates that the subject is suffering from an inflammatory condition or infection of the respiratory tract. In an alternative embodiment, the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction a reduced level of an oligosaccharide relative to the level of the oligosaccharide in a healthy control subject, wherein said oligosaccharide produces an ion by mass spectrometry having a m/z selected from the group consisting of m/z 628.9 ± 1.0, m/z 701.9 ± 1.0, m/z 710.1 ± 1.0, m/z 782.8 ± 1.0, m/z 811.4 ± 1.0, m/z 878.1 ± 1.0, m/z 884.5 ± 1.0, m/z 957.6 ± 1.0, m/z 965.3 ± 1.0, m/z 1032.3 ± 1.0, m/z 1038.4 ± 1.0, m/z 1120.2 ± 1.0, m/z 1178.2 ± 1.0 and m/z 1266.1 ± 1.0. Preferably, the ion has an m/z selected from the group consisting of m/z 782.8 ± 1.0, m/z 878.1 ± 1.0, m/z 1038.4 ± 1.0, and m/z 1266.1 ± 1.0.

A second aspect of the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide produces an ion by mass spectrometry having an m/z selected from the group consisting of m/z 728.8 ± 1.0, m/z 848.1 ±

1.0, m/z 947.3 ± 1.0, m/z 960.7 ± 1.0, m/z 993.8 ± 1.0, m/z 1146.2 ± 1.0, m/z

1167.5 ± 1.0, m/z 1244.4 ± 1.0, m/z 1322.1 ± 1.0, m/z 1438.2 ± 1.0, m/z 1469.2 ±

1.0, m/z 1477.4 ± 1.0, m/z 1551.3 ± 1.0, m/z 1619.2 ± 1.0, m/z 1695.0 ± 1.0, m/z

1743.0 ± 1.0, m/z 1803.3 ± 1.0, m/z 1828.6 ± 1.0, m/z 1940.9 ± 1.0 and m/z 1970.5 ± 1.0 and wherein a similar or elevated level of the ion relative to the level at diagnosis indicates that the subject has not recovered and a reduced level of the ion relative to the level at diagnosis indicates that the subject has recovered.

Preferably, the ion has an m/z selected from the group consisting of m/z 993.8 ± 1.0, m/z 1244.4 ± 1.0, and m/z 1477.4 ± 1.0, and more preferably, the ion has m/z 1477.4 ± 1.0. Alternatively, or in addition, the level of the oligosaccharide is determined by reference to an internal control for the sample, in particular a ion having an m/z selected from the group consisting of m/z 1040.5 ± 1.0 and m/z 1186.4 ± 1.0 and m/z 1331.3 ± 1.0, more preferably m/z 1186.4 ± 1.0 or m/z 1331.3 ± 1.0 and still more preferably m/z 1331.3 ± 1.0.

In a particularly preferred embodiment, the ratio of the level of the m/z 1477.4 ± 1.0 ion to the m/z 1331.3 ± 1.0 ion is determined, wherein a similar or higher ratio for the test sample relative to the ration for the subject at diagnosis indicates that the subject has not recovered, and a reduced ratio indicates that the subject has recovered.

In an alternative embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B- containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide produces an ion by mass spectrometry having an m/z selected from the group consisting of m/z

628.9 ± 1.0, m/z 701.9 ± 1.0, m/z 710.1 ± 1.0, m/z 782.8 ± 1.0, m/z 811.4 ± 1.0, m/z 878.1 ± 1.0, m/z 884.5 ± 1.0, m/z 957.6 ± 1.0, m/z 965.3 ± 1.0, m/z 1032.3 ±

1.0, m/z 1038.4 ± 1.0, m/z 1120.2 ± 1.0, m/z 1178.2 ± 1.0 and m/z 1266.1 ± 1.0 and wherein a reduced or similar level of the ion relative to the level at diagnosis indicates that the subject has not recovered and an elevated or enhanced level of the ion relative to the level at diagnosis indicates that the subject has recovered.

Preferably, the ion has an m/z selected from the group consisting of m/z 782.8 ± 1.0, m/z 878.1 ± 1.0, m/z 1038.4 ± 1.0, and m/z 1266.1 ± 1.0.

Alternatively, or in addition, the level of the oligosaccharide is determined by reference to an internal control for the sample, in particular a ion having an m/z selected from the group consisting of m/z 1040.5 ± 1.0 and m/z 1186.4 ± 1.0 and m/z 1331.3 ± 1.0, more preferably m/z 1186.4 ± 1.0 or m/z 1331.3 ± 1.0 and still more preferably m/z 1331.3 ± 1.0.

A third aspect of the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide produces an ion by mass spectrometry having an m/z selected from the group consisting of m/z 728.8 ± 1.0, m/z 848.1 ± 1.0, m/z 947.3 ± 1.0, m/z 960.7 ± 1.0, m/z 993.8 ± 1.0, m/z 1146.2 ± 1.0, m/z 1167.5 ± 1.0, m/z 1244.4 ± 1.0, m/z 1322.1 ± 1.0, m/z 1438.2 ± 1.0, m/z 1469.2 ± 1.0, m/z 1477.4 ± 1.0, m/z 1551.3 ± 1.0, m/z 1619.2 ± 1.0, m/z 1695.0 ± 1.0, m/z 1743.0 ± 1.0, m/z 1803.3 ± 1.0, m/z 1828.6 ± 1.0, m/z 1940.9 ± 1.0 and m/z 1970.5 ± 1.0 and wherein a similar or elevated level of the ion relative to the level at diagnosis indicates that the subject has not responded to treatment and a reduced level of the ion relative to the level at diagnosis indicates that the subject has responded to treatment.

Preferably, the ion has an m/z selected from the group consisting of m/z 993.8 ± 1.0, m/z 1244.4 ± 1.0, and m/z 1477.4 ± 1.0, and more preferably, the ion has m/z 1477.4 ± 1.0.

Alternatively, or in addition, the level of the oligosaccharide is determined by reference to an internal control for the sample, in particular a ion having an m/z selected from the group consisting of m/z 1040.5 ± 1.0 and m/z 1186.4 ± 1.0 and m/z 1331.3 ± 1.0, more preferably m/z 1186.4 ± 1.0 or m/z 1331.3 ± 1.0 and still more preferably m/z 1331.3 ± 1.0. In a particularly preferred embodiment, the ratio of the level of the m/z 1477.4 ± 1.0 ion to the m/z 1331.3 ± 1.0 ion is determined, wherein a similar or higher ratio for the test sample relative to the ration for the subject at diagnosis indicates that the subject has not responded to treatment, and a reduced ratio indicates that the subject has responded to treatment.

In an alternative embodiment, the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide produces an ion by mass spectrometry having an m/z selected from the group consisting of m/z 628.9 ± 1.0, m/z 701.9 ± 1.0, m/z 710.1 ± 1.0, m/z 782.8 ± 1.0, m/z 811.4 ± 1.0, m/z 878.1 ± 1.0, m/z 884.5 ± 1.0, m/z 957.6 ± 1.0, m/z 965.3 ± 1.0, m/z 1032.3 ± 1.0, m/z 1038.4 ± 1.0, m/z 1120.2 ± 1.0, m/z 1178.2 ± 1.0 and m/z 1266.1 ± 1.0 and wherein a reduced or similar level of the ion relative to the level at diagnosis indicates that the subject has not responded to treatment and an elevated or enhanced level of the ion relative to the level at diagnosis indicates that the subject has responded to treatment. Preferably, the ion has an m/z selected from the group consisting of m/z 782.8 ± 1.0, m/z 878.1 ± 1.0, m/z 1038.4 ± 1.0, and m/z 1266.1 ± 1.0.

Those skilled in the art will be aware that the term "m/z" refers to the mass-to- charge ratio obtained by dividing the mass of an ion by its charge number. In accordance with the various embodiments described herein, any mass spectrometry (MS) format may be employed, such as, for example, electrospray LC-MS, MALDI, MALDI-TOF, or tandem MS (for general reviews see Karas and Hillenkam, Anal. Chem. 60, 2299-2301 1988; Fenn et al. Science 246, 64-71, 1989; and Patterson and Aebersold, Electrophoresis 16, 1791-1814, 1995).

Electrospray ionization (ESI) methods are most commonly employed, due in part to the simplicity of their implementation.

ESI may also be coupled to LC. However, parameters for coupling LC and ESI mass spectrometry impose several undesirable limitations, making this technique less suitable for glycoproteome determination. Specifically, the separation system and mass spectrometer employed are coupled directly in real time, making the construction of parallel analysis systems difficult or costly, and often preventing the mass spectrometer from continually collecting data due to the equilibration and washing periods typical of separation techniques.

One- or two-dimensional electrophoresis, or multi-dimensional chromatography, can also be combined with MS and/or tandem MS methods (see, for example, Yates Trends. Genet. 16, 5-8, 2000; Aebersold and Goodlett Chem. Rev. 101, 269-295, 2001). Samples are partially purified and separated by one or more liquid chromatographic techniques, the fractions from which are then analyzed and identified by separating gaseous ions of the substances according to their mass-to-charge ratio. The chromatographic separations serve to disperse the complexity of the initial sample, preferably partially purifying the MUC5B fraction of mucin.

b) Assays for detecting modified glycosylations of MUC5B One aspect of the present invention provides a method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin in a healthy control subject, wherein the modified glycosylation is selected from the group consisting of: (i) a decrease in sulfation of the mucin;

(ii) a decrease in sulfation of the mucin coupled with a decrease in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin and a decrease in fucosylation of the mucin; (v) a decrease in the ratio sulfate: sialic acid in the mucin; and (vi) an increase in the ratio sialic acid: sulfate in the mucin.

In a further embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of: (i) a decrease in sulfation of the mucin coupled with an increase in fucosylation of the mucin;

(iii) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin;

(iv) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin and an increase in fucosylation of the mucin;

(v) a decrease in the ratio sulfate: fucose in the mucin; and

(vi) an increase in the ratio fucose: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has improved or recovered from the inflammatory condition of infection. In an alternative embodiment, the present invention provides a method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B- containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of: (i) a decrease in sulfation of the mucin coupled with a decrease in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin and a decrease in fucosylation of the mucin;

(v) a decrease in the ratio sulfate: sialic acid in the mucin; and (vi) an increase in the ratio sialic acid: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has not improved or recovered from the inflammatory condition of infection or has deteriorated.

A third aspect of the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of:

(i) a decrease in sulfation of the mucin coupled with an increase in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin and an increase in fucosylation of the mucin; (v) a decrease in the ratio sulfate: fucose in the mucin; and (vi) an increase in the ratio fucose: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has responded to treatment.

In an alternative embodiment, the present invention provides a method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of: (i) a decrease in sulfation of the mucin coupled with a decrease in fucosylation of the mucin;

(iii) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin and a decrease in fucosylation of the mucin; (v) a decrease in the ratio sulfate: sialic acid in the mucin; and (vi) an increase in the ratio sialic acid: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has not responded to treatment.

Preferably, the mucin according to any one or more of the preceding embodiments is MUC5B. In accordance with this embodiment, notwithstanding that the MUC5B-containing fraction may comprise additional mucins, such as, for example, MUC1 , MUC2, MUC4, MUC5AC, MUC7 or MUC8, or other high molecular weight glycoproteins such as, for example, gp340, the fraction may contain MUC5B in a substantially pure form sufficient to permit identification of glycosyl structures thereon. Such a degree of purity may be obtained by fractionation of the sputum mucins, such as, for example, using one- or two- dimensional electrophoresis, multi-dimensional or liquid chromatographic techniques.

Any suitable assay format can be used to determine modifications to the sialic acid, fucose or sulfate content of the mucins in the MUC5B-contianing fraction of sputum or saliva.

Immunoassay formats, such as, for example, for the detection of sugars, are particularly preferred. In this respect, several antibodies are available publicly that bind to sialic acid, sulfate or fucose. For example, antibodies, such as, for example, monoclonal antibody F2 that recognizes the Sθ₃-3Galβ1-3GlcNAc moiety of the sulfo-Lewis^a antigen (Veerman et al., Glycobiol. 7, 37-44, 1997), or the monoclonal antibody INES that binds to less-acidic mucins (Rathman et al., J. Biol. Buccale 181, 19-27, 1990), or monoclonal antibody C241 that binds specifically to sial-Lewis^a (Nilsson et al., J., Dermatol. Surg. Oncol. 1, 49-51, 1987), may be useful for antibody-based determinations of sulfate and/or sialic acid contents.

Additional antibodies that bind to sialic acid, sulfated oligosaccharides or fucosylated oligosaccharides, are prepared by standard means. In general, this involves the chemical synthesis of specific oligosaccharides, oligosaccharide substructures, or oligosaccharide epitopes, linkage to a hapten, and immunization of an animal for a time and under conditions sufficient to generate antibodies against the oligosaccharide moiety of the hapten conjugate.

Affinity ligands, such as, for example, selectins or lectins that bind specific sugars, can also be used in place of, or alongside, antibodies. Additionally, the specificity of selectins toward carbohydrates has been extensively reviewed (Rosen et al., Curr. Opin. Cell Biol. 6, 663-673, 1994; Varki, J. Clin. Invest. 99, 158-162, 1997). Various independent research groups have disclosed selectins that recognize carbohydrates incorporating either the sialyl-Lexis^a, sialyl-Lewis^x, sulfated-Lewis^x or sulfated Lewis³ structures.

Many lectins are available commercially, such as, for example, from Sigma Chemical Company. For example, a Helix pomantia lectin detects O-linked oligosaccharides such as galNAc; a Maackia amerensis lectin binds to sialic acid; Pseudomonas aeruginosa lectin PA-1 binds to galactose; and Ulex europaeeus lectin binds to α-L-fucose.

Alternatively, or in addition, Periodic Acid-Schiff s reagent (PAS) can be used to measure total carbohydrate content of samples, whilst acetic acid Alcian Blue (aAB) is suitable for determining the presence of acidic oligosaccharides such as, for example, sialic acid and sulfate, and sulfuric acid Alcian Blue (sAB) is suitable for determining sulfate content specifically.

For example, reduced/ alkylated sputum from a subject being assayed can be transferred in replica to a suitable matrix eg., PVDF membrane, and each sample stained independently with the following stains (i) Direct Blue to determine total protein content of the sample; (ii) PAS to determine total oligosaccharide content of the sample; (iii) aAB to determine sialic acid and sulfate content of the sample; and (iv) sAB to determine sulfate content of the sample. Stained samples are then imaged according to standard procedures, and the intensity of staining determined. Protein content, oligosaccharide content, acidic oligosaccharide content, and sulfate content per microlitre of sample are determined. The ratio of acidic oligosaccharides to total oligosaccharides (ratio=aAB/PAS), the ratio of sulfate to total oligosaccharide (sAB/PAS), and the ratio of sulfate to acidic oligosaccharide (sAB/aAB) are also calculated directly from the staining obtained.

Optionally, the intensity of staining for each of (i) to (iv) is normalized to the intensity of staining of a MUC2 standard. Accordingly, the sulfate content, sialic acid/sulfate ratio, total protein and total oligosaccharide content can be rapidly determined for any MUC5B-containig sample, by simple colorimetric means.

High throughput assay formats are also particularly preferred, and immunoassay formats, or detection systems using lectins, or combinations of PAS, aAB and sAB, or mass spectrometry, are particularly useful for this purpose.

Biological samples and reference samples Unless otherwise specified, it is preferred that the biological sample that forms the basis of the assays described herein comprises a tissue selected from the group consisting of lung, lymphoid tissue associated with the lung, paranasal sinuses, bronchi, a bronchiole, alveolus, ciliated mucosal epithelia of the respiratory tract, mucosal epithelia of the respiratory tract, squamous epithelial cells of the respiratory tract, a mast cell, a goblet cell, a pneumocyte (type 1 or type 2), broncheoalveolar lavage fluid (BAL), alveolar lining fluid, an intra epithelial dentritic cell, sputum, mucus, saliva, blood, serum, plasma, a PBMC, a neutrophil and a monocyte.

Sputum and saliva are preferred for performance of the diagnostic/prognostic assays of the invention. Sputum can be isolated from lung of a patient using, for example the method described in Gershman, N.H. et al, J Allergy Clin Immunol, 10(4): 322-328, 1999.

In another preferred embodiment a biological sample is plasma that has been isolated from blood collected from a patient using a method well known in the art.

In one embodiment a biological sample is obtained previously from a patient.

In one embodiment a biological sample is obtained from a subject by a method selected from the group consisting of surgery or other excision method, aspiration of a body fluid such as hypertonic saline or propylene glycol, broncheoalveolar lavage, bronchoscopy, saliva collection with a glass tube, salivette (Sarstedt AG, Sevelen, Switzerland), Ora-sure (Epitope Technologies Pty Ltd, Melbourne, Victoria, Australia), omni-sal (Saliva Diagnostic Systems, Brooklyn, NY, USA) and blood collection using any method well known in the art, such as, for example using a syringe.

In yet another embodiment, a biological sample is treated prior to use in a diagnostic or prognostic assay.

In one embodiment, a biological sample is treated to lyse a cell in said sample. Such methods include the use of detergents, enzymes, repeatedly freezing and thawing said cells, sonication or vortexing said cells in the presence of glass beads, amongst others.

In another embodiment, a biological sample is treated to denature a protein present in said sample. Methods of denaturing a protein include heating a sample, treatment with 2-mercaptoethanol, or treatment with detergents and other compounds such as, for example, guanidinium or urea.

In yet another embodiment, a biological sample is treated to concentrate a protein is said sample. Methods of concentrating proteins include precipitation, freeze drying, use of funnel tube gels (TerBush and Novick, Journal of Biomolecular Techniques, 10(3); 1999), ultrafiltration or dialysis.

As will be apparent, the diagnostic and prognostic methods provided by the present invention require a degree of quantification to determine either, the amount of a protein that is diagnostic or prognostic of an acute clinical exacerbation in a CF patient, or alternatively, the amount of a modified protein that is diagnostic or prognostic of an acute exacerbation in a CF patient.

Furthermore, certain diagnostic and prognostic methods described herein require the detection of the amount of both an unmodified and modified form of a protein that is diagnostic or prognostic of an acute clinical exacerbation in a CF patient. Such quantification can be determined by the inclusion of appropriate reference samples in the assays described herein, wherein said reference samples are derived from healthy or normal individuals.

In one embodiment, the reference sample comprises for example cells, tissue, plasma, serum, whole blood, sputum, saliva, or BAL fluid derived from the same subject when the individual was not suffering from an acute clinical exacerbation. In another embodiment, the reference sample comprises for example cells, tissue, plasma, serum, whole blood, sputum, or BAL fluid derived from another CF patient that was not suffering from an acute exacerbation. In yet another embodiment, the reference sample comprises (cells, tissue, plasma, serum, whole blood, sputum, saliva, or BAL fluid) derived from a normal healthy individual.

Accordingly, a reference sample and a test (or patient) sample are both processed, analysed or assayed and data obtained for a reference sample and a test sample are compared. In one embodiment, a reference sample and a test sample are processed, analysed or assayed at the same time. In another embodiment, a reference sample and a test sample are processed, analysed or assayed at a different time.

In an alternate embodiment, a reference sample is not included in an assay. Instead, a reference sample may be derived from an established data set that has been previously generated. Accordingly, in one embodiment, a reference sample comprises data from a sample population study of healthy individuals, such as, for example, statistically significant data for the healthy range of the integer being tested. Data derived from processing, analysing or assaying a test sample is then compared to data obtained for the sample population.

Data obtained from a sufficiently large number of reference samples so as to be representative of a population allows the generation of a data set for determining the average level of a particular parameter. Accordingly, the amount of a protein that is diagnostic or prognostic of an acute clinical exacerbation in a CF patient, or the amount of an unmodified and/or modified protein that is diagnostic or prognostic of an acute clinical exacerbation in a CF patient can be determined for any population of individuals, and for any sample derived from said individual, for subsequent comparison to levels of the expression product determined for a sample being assayed. Where such normalized data sets are relied upon, internal controls are preferably included in each assay conducted to control for variation.

Multiplexed assays

As will be apparent to those skilled in the art a diagnostic or prognostic assay may simultaneously measure several parameters that characterize the acute pulmonary exacerbation or recovery phase of infection in a CF subject. Such multiplexed assays are useful in increasing the specificity and accuracy of a prognostic or diagnostic assay.

As used herein the term "multiplex", shall be understood not only to mean the detection of two or more diagnostic or prognostic markers or analytes (eg., sialic acid, sulfated oligosaccharide, fucosylated oligosaccharide or MUC5B apoprotein) in a single sample simultaneously, but also to encompass consecutive detection of two or more diagnostic or prognostic markers in a single sample, or the simultaneous detection of two or more diagnostic or prognostic markers in distinct but matched samples, or the consecutive detection of two or more diagnostic or prognostic markers in distinct but matched samples. As used herein the term "matched samples" shall be understood to mean two or more samples derived from the same initial biological sample, or two or more biological samples isolated at the same point in time.

In the case of analysis of two biological samples isolated at the same point in time, these samples may be the same type of biological sample, eg sputum, or different biological samples, eg sputum and plasma. Accordingly, a multiplexed assay may comprise an assay that detects the amount of a native MUC5B apoprotein and an oligosaccharide epitope in the same reaction and simultaneously. As will be apparent to the skilled artisan, if such an assay is antibody or ligand based, both of these antibodies must function under the same conditions.

Alternatively, or in addition, a multiplexed assay may comprise first detecting the amount of a native MUC5B apoprotein, followed by the detection of the oligosaccharide epitope in the biological sample. Accordingly, based on the result of the first step in this process it may be unnecessary to proceed to the second step, eg if the first assay indicates that a patient is not suffering from an acute clinical exacerbation.

A multiplexed assay may also comprise the detection of analytes in separate reactions. This may consist of each analyte being detected in a separate reaction or one or more of these analytes may be detected in one reaction and one or more analytes in another reaction. Again, such an assay may detect all of these analytes simultaneously, that is all reactions including all samples proceed at the same time. Alternatively, these reactions may be consecutive, with each reaction proceeding following either the commencement or completion of another assay. Accordingly, based on the result of the first step in this process it may be unnecessary to proceed to the second step, eg if the first assay indicates that a patient is not suffering from an acute clinical exacerbation.

In a preferred embodiment, the multiplexed assay comprises capturing the MUC5B protein in the sample using an antibody that binds MUC5B and then detecting of one or more oligosaccharides on the captured high molecular weight glycoprotein selected from the group consisting of sialylated oligosaccharide, fucosylated oligosaccharide and sulfated oligosaccharide using and antibody or lectin that binds specifically to the oligosaccharide. Alternatively, a colorimetric assay as described herein for determining the oligosaccharide moieties can be used in place of antibodies or lectins.

In an alternative embodiment, the multiplexed assay comprises capturing the high molecular weight glycoproteins in the sample using and antibody or lectin that binds specifically to oligosaccharide residues, and then detecting MUC5B protein in the sample using an antibody that binds MUC5B. Optionally, specific oligosaccharides are captured eg., an oligosaccharide selected from the group consisting of sialylated oligosaccharide, fucosylated oligosaccharide and sulfated oligosaccharide, using specific affinity ligands, such as, for example, antibodies or lectins. Alternatively, or in addition, the captured glycoprotein may be deglycosylated or reduced following capture to facilitate subsequent binding of the antibody to the MUC5B apoprotein.

In a particularly preferred embodiment, a multiplex assay is performed by a process comprising:

(i) obtaining a sample of induced sputum, expectorated sputum, whole saliva, submandibular or sublingual saliva;

(ii) applying the sample to an antibody immobilized on a supporting matrix (eg., PVDF membrane) that binds the MUC5B apoprotein for a time and under conditions sufficient for an antibody-antigen complex to form;

(iii) contacting the matrix with an antibody that binds to an oligosaccharide epitope selected from the group consisting of sialylated oligosaccharide (eg., sialyl-Lewis^x) and sulfated oligosaccharide (eg., sulfo-Lewis^a) for a time and under conditions sufficient for an antibody-antigen complex to form; and

(iv) detecting the complex formed at (iii).

Through multiplexing the diagnostic or prognostic assays of the present invention the general systemic state of a patient is determined.

As used herein, the term "general systemic state" shall be understood to mean that a multiplexed assay indicates the general pulmonary health of a CF patient. Accordingly, such an assay may determine the presence of an infection of the respiratory tract of a CF patient, the level of inflammation of the respiratory tract of a CF patient and the degree of damage to the respiratory tract of said patient. Accordingly, such a diagnostic assay permits the skilled artisan to continually monitor a CF patient thereby facilitating the correct treatment of said patient for inflammation or infection of the respiratory system.

Chronic or repeated episodes of acute inflammation and infection damage the lungs of CF patients resulting in bronchiectasis and eventually respiratory failure requiring a lung transplant. Accordingly, a diagnostic or prognostic method that can effectively monitor both inflammation and infections and allow the early detection and treatment of these complications will result in a reduction of the damage caused to the respiratory tract of a CF patient.

In another embodiment, a diagnostic or prognostic assay of the present invention is multiplexed with another assay or marker that is diagnostic or prognostic of an acute clinical exacerbation of CF.

In one embodiment, an additional assay is the measurement of forced expiration volume in one second (FEVi). Such an assay will be known to those skilled in the art. FEVi is measured by, for example a spirometer. Other such measurement that may be of use in a multiplexed assay include, peak expiratory flow (PEF), vital capacity (VC) which is the maximum volume of air that can be inhaled or exhaled, forced expiratory flow at 50% of FEV-i (FEF₅₀o_/0), and forced expiratory time. Methods of measuring such are known in the art.

In yet another embodiment, an assay or marker of the present invention is multiplexed with an assay that detects the presence of a bacterial infection, such as a P. aeruginosa infection. Such assays include the detection of IgG in a CF subject that is specific to the core lipopolysaccharide of P. aeruginosa (US Patent No. 5,179,001), or IgA specific to P. aeruginosa cells (as described by Brett et al, J. Clin. Pathol. 41(10), 1130-1134, 1988), or an antibody specific to sodium alginate exoploysaccharide of P. aeruginosa (as described in Bryan et al, J. Clin. Microbiol. 18(2), 276-282, 1983). Alternatively, this assay detects a type-Ill secretory protein of P. aeruginosa (as described by Roy-Burman et al, J. Infect. Dis. 183(12), 1767-1774, 2001 ).

Diagnostic assay kits

Another aspect of the present invention relates to an antibody, ligand or synthetic or recombinant peptide that is generated for use and/or used in a diagnostic or prognostic assay as described herein. Methods of isolating such an antibody or ligand are well known in the art and/or described herein.

Clearly this aspect of the present invention also relates to the use of any novel or previously undescribed antibody, ligand or synthetic or recombinant peptide in other therapeutic or diagnostic applications or for research. Such applications include, the purification and study of the diagnostic/prognostic proteins, identification of cells expressing said proteins, and sorting or counting cells. Accordingly, the present invention encompasses the use of a novel antibody or fragment thereof, ligand or synthetic or recombinant peptide in therapy, including, prophylaxis, diagnosis, prognosis, or the use of such agents in the manufacture of a medicament for use in treatment of an acute clinical exacerbation in a CF patient.

Optionally, the kit further comprises means for the detection of the binding of an antibody, fragment thereof or a ligand to MUC5B or a modified form thereof. Such means include a reporter molecule such as, for example, an enzyme (such as horseradish peroxidase or alkaline phosphatase), a substrate, a cofactor, an inhibitor, a dye, a radionucleotide, a luminescent group, a fluorescent group, biotin or a colloidal particle, such as colloidal gold or selenium. Preferably such a reporter molecule is directly linked to the antibody or ligand.

In another embodiment, the binding of an antibody, or ligand to MUC5B or a modified form thereof is detected by another antibody, fragment thereof or ligand. Preferably, this antibody, fragment thereof or ligand is directly linked to a reporter molecule.

In yet another embodiment, a kit may additionally comprise a reference sample. Such a reference sample may for example, be a protein sample derived from a biological sample isolated from one or more CF subjects suffering from a clinical exacerbation. Alternatively, a reference sample may comprise a biological sample isolated from one or more CF subjects that do not suffer from a clinical exacerbation, or one or more normal healthy individuals. Such a reference sample is optionally included in a diagnostic or prognostic assay. Results obtained from a biological sample are compared to results obtained from a reference sample facilitating a diagnosis or prognosis of the clinical status of the patient from whom the biological sample was isolated.

In another embodiment, a reference sample comprises a peptide that is detected by an antibody or a ligand. Preferably, the peptide is of known concentration. Such a peptide is of particular use as a standard. Accordingly various known concentrations of such a peptide may be detected using a prognostic or diagnostic assay described herein. Accordingly, these results may be used to determine concentration of MUC5B or a modified form thereof in a biological sample derived from a subject, facilitating a diagnosis or prognosis of the clinical state of said subject.

In a related embodiment, a peptide is the peptide against which an antibody was raised. Such a peptide is of particular use in control samples in an assay. In such samples saturating amounts of the peptide is added to a sample in addition to an antibody that binds MUC5B or a modified form thereof. Accordingly, this will block the binding of said antibody. Such a sample acts as a negative control, in which the specific binding of said antibody is determined.

In yet another embodiment, a kit optionally comprises means for sample preparations. Preferably such means are means of solubilizing sputum, such as, for example, a detergent (eg tributyl phosphine, C7BZO, dextran sulfate, or Polyoxyethylenesorbitan monolaurate

In a related embodiment, sample preparation means optionally comprise means for cell lysis. Methods of cell lysis are well known in the art and are described, for example, in Scopes (! Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994).

In yet another embodiment, a kit comprises means for protein isolation. Methods of protein preparation are well known in the art and are described, for example, in Scopes (jIv Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994).

A further aspect of the present invention relates to a method of treatment of a CF subject suffering from an acute clinical exacerbation comprising performing a diagnostic or prognostic method described herein and if a marker of exacerbation is detected, treating said subject with a therapeutic compound for said exacerbation.

In another embodiment of the present invention a method of treatment of a CF subject suffering from an acute clinical exacerbation comprises performing a diagnostic or prognostic method described herein and if a marker of exacerbation is detected, identifying the source of said exacerbation using a method well known in the art and commencing treatment with a therapeutic compound and monitoring the effectiveness of said treatment using methods described herein.

The present invention is further described with reference to the following non- limiting examples

Example 1 Comparison of O-linked glycosylation profile of high molecular weight glycoproteins from a non-CF subject, and a CF subject during and following an acute pulmonary infection.

Materials and methods

Sputum collection

Sputum was obtained from healthy and cystic fibrosis patients

Separation of mucins by SDS-AGPAGE SDS-AGPAGE gels were made by boiling two solutions with 0.5 % agarose and 0.375 M Tris-HCI pH 8.1 , one also containing 6 % T, 2.5 % C (piperazine diacrylamide) and 10 % glycerol. The 0-6% gradient polyacryIamide/0.5% agarose gradient gels were cast in the mini-Protean gel casting apparatus (Bio- Rad, Hercules, CA) at 50°C after adding N,N,N',N'-tetramethylethylenediamine (0.0125%) and ammonium persulphate (0.005%) to each solution. The gels were polymerised for 1 hour at 50°C and the agarose was then allowed to set at room temperature over-night in a humidified environment. The anode and cathode buffer was 192 mM tris-borate pH 7.6 with 1 mM EDTA and 0.1 % SDS.

The sputum was reduced and alkylated in sample loading buffer (Tris-HCI pH 8.1 ) as described above and sample equivalent to 100 μg Muc2 and 20 μl saliva were loaded onto SDS-AGPAGE gels, and electrophoresed at 100 V for 2-3 hours, until the dye front migrated out of the gel. Proteins were then electroblotted as above, with methanol excluded from the anode buffer. Gels were stained using PAS or Alcian Blue (0.125 % Alcian Blue in 25% ethanol and 10 % acetic acid for 10 min and destained in 100 % methanol for 20 min.

Reductive Alkaline β-Elimination of Oligosaccharides

Oligosaccharides attached to glycoproteins separated by SDS-PAGE or SDS- AGPAGE and blotted to membrane were released by reductive β-elimination.

Direct blue or Alcian Blue stained bands were excised from the membrane, wetted with methanol, and incubated at 50°C for 16 hours in 20 μL 50 mM NaOH and 0.5 M NaBH₄. The resultant solutions were neutralised by the addition of 1 μL glacial acetic acid, before being desalted with 25 μL AG50WX8 cation exchange resin (Bio-Rad) in a zip-tip (Millipore), and dried in a Savant SpeedVac. Borate was removed as its methyl-ester by repeated (5 times) addition and evaporation of 50 μL 1 % acetic acid in methanol to each sample. Finally the samples were resuspended in 10 μL MilliQ water for LC-MS analysis.

Mass spectrometric identification of released oligosaccharides

Desalted oligosaccharides were analysed by LC-MS/MS on a home-made graphitised carbon column 7 μm Hypercarb particles (Thermo-Hypersil, Runcorn UK) in a 250 μm ID column, after introduction using a Surveyor autosampler. A solvent rate through the column of 5 μL/min was provided by a Surveyor LC pump (ThermoFinnigan, San Jose, CA) with flow splitting from 100 μL/min. Oligosaccharides were eluted with a H₂0-acetonitrile gradient (0-40 % acetonitrile in 30 min, followed by a 3 min wash with 90 % acetonitrile) with constant 10 mM NH₄HC0₃. Mass spectrometry was performed on an LCQ Deca (ThermoFinnigan) in negative ion mode, with three scan events: Full scan with mass range 320-2000 m/z, dependent zoom scan of the most intense ions in each scan, and dependent MS/MS scan after collision induced fragmentation. The capillary temperature was 180°C, the capillary voltage was 32.0 V and the electrospray voltage was 2.5 kV. Collision conditions used were a normalised collision energy of 40%, and an activation time of 30 ms. Dynamic exclusion of ions for zoom scan for 30 s was introduced after 3 selections within 30 s. For MS/MS the normalised collision energy was 35 % with an activation time of 30 ms.

Results and Discussion Analysis of Mucin Oligosaccharides.

As compared to smaller glycoproteins, mucins (> 200 kDa) are predominantly glycosylated with O-linked oligosaccharides, with up to 80 % of the weight.

Mucins found on mucosal surfaces are supposed to be important interaction molecules due to their glycosylation. Traditionally, characterisation of oligosaccharides from mucin is carried out after isolation of mucin fractions with isopycnic centrifugation, followed by gel- and anion exchange chromatography (ref). As a final step the oligosaccharides are released and characterised using mass spectrometry, monosaccharide composition analysis, and ¹H-NMR. The approach taken here is using a highly resolving agarose-polyacrylamide composite gel for isolation of mucin fraction, since high molecular weight of most mucins make them unsuitable for traditional SDS-PAGE. Limited characterisation of released oligosaccharides using high resolving LC-MS, provides sufficient information for identifying glycosylation from well defined mucin subpopulations. Another advantage of the methodology is that a lower amount of mucin will be consumed for the analysis i.e. less than 100 μg of a crude mucin fraction, compared to several milligram of purified mucin for the traditional analysis. The utility of the method described here for glycosylation analysis of gel separated mucins has been validated by comparison with previously reported glycosylation profiles of rat mucins.

The method described here profiles the oligosaccharide structures found on a mucin glycoprotein species from healthy and cystic fibrosis patient sputum.

O-linked glycosylation profiling of oligosaccharides released from mucin separated by 1-D AgPAGE gel electrophoresis shows marked differences between CF and normal sputum in both the gel pattern and the glycosylation of the high molecular weight glycoproteins. Figure 1 shows that acute pulmonary exacerbation of two cystic fibrosis patients (Lanes I and IV) results in the high molecular weight glycoprotein bands of sputum separating at a apparent lower molecular mass on AgPAGE gels than that of non-CF sputum (Lane III). Interestingly, the successful treatment of the pulmonary infection with antibiotics and anti-inflammatories results in the glycoprotein bands appearing in the CF patient sputum sample at the same high molecular weight as those of non-CF sputum (Lane II). In addition, the sputum of a CF patient who did not respond to treatment still maintains the appearance of lower molecular mass glycoproteins (Lane V). Correspondingly, the total ion mass spectra of the O-linked oligosaccharides released from the high molecular weight glycoproteins was determined (Figures 2a-2c). Data presented in Figures 2a-2c show differences in the relative amounts of the different oligosaccharide ions present in MUC5B and MUC 5AC-containing bands from 1-D AgPAGE profiles of sputa from a non-CF subject compared to a CF patient with an acute pulmonary infection.

The MUC5B-containing fraction from non-CF subjects routinely showed abundant structures having, for example, m/z 628.9 ± 1.0, m/z 701.9 ± 1.0, m/z 710.1 ± 1.0, m/z 782.8 ± 1.0, m/z 811.4 ± 1.0, m/z 878.1 ± 1.0, m/z 884.5 ± 1.0, m/z 957.6 ± 1.0, m/z 965.3 ± 1.0, m/z 1032.3 ± 1.0, m/z 1038.4 ± 1.0, m/z 1120.2 ± 1.0, m/z 1178.2 ± 1.0 and m/z 1266.1 ± 1.0, which were either not present, or present at a relatively low level compared to other structures, in the MUC5B-containing fraction of sputum from the CF subject during a pulmonary exacerbation (Table 1 and Table 2). The level of the sulfated oligosaccharides having m/z 1032.3 ± 1.0 and m/z 1266.1 ± 1.0 were not reproducibly present at significant levels in sputum of CF subjects during a clinical exacerbation (Table 1 ).

Structures having m/z 665.2 ± 1.0, m/z 738.3 ± 1.0, m/z 790.2 ± 1.0, m/z 848.1 ± 1.0, m/z 960.7 ± 1.0, m/z 993.8 ± 1.0, m/z 1040.5 ± 1.0, m/z 1186.4 ± 1.0, m/z 1244.4 ± 1.0, m/z 1322.1 ± 1.0, m/z 1331.3 ± 1.0, m/z 1469.2 ± 1.0, m/z 1477.4 ± 1.0 and m/z 1551.3 ± 1.0 were relatively high in abundance during pulmonary exacerbation in the CF subject (Figure 2b; Table 1) compared to the non-CF healthy subject (Figure 2a; Table 2). Of these structures, those having m/z 665.2, m/z 738.3 ± 1.0, m/z 960.7 ± 1.0, m/z 993.8 ± 1.0 and m/z 1469.2 ± 1.0, were also not reproducibly detectable at significant levels above background following treatment (Figure 2c; Table 3), suggesting that they may be related to the course of infection. Those structures having m/z 993.8 ± 1.0, m/z 1244.4 ± 1.0, and m/z 1477.4 ± 1.0 were amongst the most abundant species in the MUC5B-containing fraction from the CF patient during infection. Interestingly, the level of m/z 1040.5 ± 1.0 and m/z 1186.4 ± 1.0 and m/z 1331.3 ± 1.0 were high in the CF subject during exacerbation and following treatment, and may reflect an overall CF-related modification to MUC5B, or alternatively, genetic differences between the individuals tested.

Yet other oligosaccharides of MUC5B in the CF- subject following treatment of infection with antibiotics and anti-inflammatories were increased in relative abundance, without reference to the non-CF subject altered (compare Figures 2b and 2c

Analysis of single ion chromatographs from the reversed phase HPLC separation of the glycans also shows differences between samples. For example, the m/z 1331.3 ± 1.0 ion the normal sputum sample showed three distinct isomers (Figure 3a), while only two isomers were detected from the CF sputum sample (Figure 3b).

Those skilled in the art of mass spectrometry are aware that the but the observered m/z, z, mass_nβutrai and mass_predicted may depend on the analysis technique used, for example, sample preparation and mass spectrometry, and, as a consequence, the reported mass-to-charge ratios reported herein may not arise if different analytical techniques are used to those described. On the other hand, oligosaccharide compositions for each ion compositions would remain the same independent of the analysis technique employed. Accordingly, Tables 1-3 describe the massesoligosaccharide compositional data for each ion identified in the present studies. The compositions provided in Tables 1 to 3 include the reducing end, GalNacol, as a HexNac group.

Proceeding on this basis, structures that were routinely enhanced in the MUC5B- containing fraction from non-CF subjects, relative to exacerbated CF subjects, had the following compositions: HexNac₃Hex₂FucιSulfι (m/z 628.9 ± 1.0); HexNac₃Hex₂Fuc₂Sulf₂ (m/z 701.9 ± 1.0); HexNac₃Hex₃FucιSulf₂ (m/z 710.1 ± 1.0); HexNac₃Hex₂Fuc₂Sulf₂ (m/z 782.8 ± 1.0); HexNac₄Hex₃FucιSulf₂ (m/z 811.4 ± 1.0); HexNac₂HexιNeuAcι (m/z 878.1 ± 1.0); HexNac₄Hex₃Fuc₂Sulf₂ (m/z 884.5 ± 1.0); HexNac₄Hex3Fuc₃Sulf2 (m/z 957.6 ± 1.0); HexNac₄Hex4Fuc₂Sulf₂ (m/z 965.3 ± 1.0); HexNac₃Hex₂Sulfι (m/z 1032.3 ± 1.0); HexNac₄Hex₄Fuc₃Sulf₂ (m/z 1038.4 ± 1.0); HexNac2Hex₂NeuAdSulfι (m/z 1120.2 ± 1.0); HexNac₃Hex₂Fuc₁Sulfι (m/z 1178.2 ± 1.0) and HexNac₂Hex₂FucιNeuAcιSulf₁ (m/z 1266.1 ± 1.0). The level of the sulfated oligosaccharides having compositions HexNac₃Hex2Sulfι and HexNac₂Hex2FucιNeuAcιSulfι were not reporducibly present at significant levels in sputum of CF subjects during a clinical exacerbation (Table 1 ).

On the other hand, structures that were routinely and significantly enhanced in abundance during pulmonary exacerbation in CF subjects, relative to non-CF subjects, had the following compositions: HexNac₂Hex₂NeuAc₂ (m/z 665.2 ± 1.0); HexNac₂Hex₂FuCiNeuAcι (m/z 738.3 ± 1.0); HexNac₃Hexι (m/z 790.2 ± 1.0); HexNac₃Hex₃Fuc₂NeuAcι (m/z 848.1 ± 1.0), HexNac₃Hex₃FucιNeuAc₂Sulfι (m/z 960.7 ± 1.0); HexNac₃Hex₃Fuc₂NeuAc₂ (m/z 993.8 ± 1.0); HexNac₂Hex₂NeuAcι (m/z 1040.5 ± 1.0), HexNac₂Hex₂FucιNeuAcι (m/z 1186.4 ± 1.0); HexNac₃Hex₂Fuc₂ (m/z 1244.4 ± 1.0); HexNac₃Hex₂Fuc₂Sulfι (m/z 1322.1 ± 1.0); HexNac₂Hex₂NeuAc₂ (m/z 1331.3 ± 1.0); HexNac₂Hex₂FucιNeuAcιSulfι (m/z 1469.2 ± 1.0); HexNac₂Hex₂FucιNeuAc₂ (m/z 1477.4 ± 1.0) and m/z HexNac₃Hex₃FucιNeuAcι (1551.3 ± 1.0). Of these structures, those the following compositions were also not reproducibly detectable at significant levels above background following treatment: HexNac2Hex₂NeuAc₂ (m/z 665.2 ± 1.0); HexNac₂Hex₂FucιNeuAcι (m/z 738.3 ± 1.0); HexNac₃Hex₃FucιNeuAc₂Sulfι (m/z 960.7 ± 1.0); HexNac₃Hex₃Fuc₂NeuAc₂ (m/z 993.8 ± 1.0); HexNac₃Hex₂Fuc₂Sulfι (m/z 1322.1 ± 1.0); and HexNac₂Hex₂FucιNeuAcιSulfι (m/z 1469.2 ± 1.0), were also not reproducibly detectable at significant levels above background following treatment, suggesting that they may be related to the course of infection. Those structures having the following compositions were amongst the most abundant species in the MUC5B-containing fraction from CF patients during infection: HexNac₃Hex₃Fuc₂NeuAc₂ (m/z 993.8 ± 1.0); HexNac₂Hex₂NeuAcι (m/z 1040.5 ± 1.0), and HexNac₂Hex₂FucιNeuAc₂(m/z 1477.4 ± 1.0).

The following compositions were present in CF subjects during exacerbation and following treatment: HexNac₂Hex₂NeuAcι (m/z 1040.5 ± 1.0) and HexNac₂Hex₂FucιNeuAcι (m/z 1186.4 ± 1.0) and HexNac₂Hex₂NeuAc₂(m/z 1331.3 ± 1.0).

These representations of the glycosylation profiles of specific separated proteins, or protein mixtures, can be used separately or in combination to compare different sample types or disease states.

Diagnosis or prognosis of pulmonary infections/inflammations based on the compositional data provided compare the measured masses/compositions for a test sample with the masses/compositions listed for each of the groups in Tables 1 to 3. Alternatively, or in addition, a weighted average oligosaccharide composition is calculated based upon the data provided.

Example 2

Comparison of O-linked glycosylation profile of high molecular weight glycoproteins from a cohort of non-CF subjects and CF subjects during an acute pulmonary infection.

Materials and methods

Cohort samples

In this series of experiments, sputa from a cohort of adult subjects were analysed.

The cohort consisted of 19 CF subjects suffering from acute pulmonary exacerbations, 13 of the same CF subjects post-discharge from hospital, and 19 non-CF healthy control subjects.

Table 4 summarizes the clinical data on samples processed for glycoproteomic analysis of sputum. These samples were also analysed by 2D PAGE. No analysis was performed on sputum from exacerbated CF subject CYFB1-12 and non-CF healthy control subject CYFB1-23 due to insufficient sample volumes.

Sample treatment

As outlined in Example 1 , the methodology for glycoproteomic analysis of the high molecular weight MUC5B-containing fraction of sputum includes sample preparation, one-dimensional gel electrophoresis, oligosaccharide release, protein digestion, mass spectrometry and analysis of these data using integrated bioinformatic tools.

Briefly, aliquots of saline-induced sputum samples liquefied by gelsolin, endonuclease and dithiothreitol (DTT) were reduced and alkylated with DTT/iodoacetamide (IAA) in sample loading buffer, concentrated using 100 kDa MW cutoff spin columns and separated by 1 dimensional sodium dodecylsulfate- agarose polyacrylamide gel electrophoresis (1 D SDS-AgPAGE). Equal volumes of processed sputum (equivalent to 10 L of whole sputum) were separated by 1 D SDS-AgPAGE for each sample. Duplicate gels were then either stained for the presence of total oligosaccharides (PAS), or electroblotted to PVDF membrane and stained for the presence of acidic oligosaccharides (Alcian Blue).

Oligosaccharides were released from protein bands off PVDF membrane and analysed in the second dimension by liquid chromatography coupled to electrospray ionisation mass spectrometry (LC-ESI-MS) with an LCQ DECA XP (ThermoFinnigan, San Jose, CA). Protein bands on PVDF membrane were digested with trypsin, and the peptide mixture analysed using a Axima CFR (Kratos, Manchester, UK) for matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) and post source decay (PSD), and an LCQ DECA for tandem mass spectrometry (LC-ESI-MS/MS).

Peptide Mass Fingerprinting (PMF), post source decay (MALDI-PSD), and LC- ESI-MS/MS were performed to identify the high molecular weight glycoproteins present in one-dimensional gels. Tools within BioinformatlQ™ (Proteome Systems' web-based proteomic data management system), lonlQ 2™ (peptide mass fingerprinting) and FragmentastlQ™ (fragmented peptide ion matching), in addition to SEQUEST (ThermoFinnigan) (fragmented peptide ion pattern matching) were used for database searching and protein identification.

Oligosaccharide 2D data analysis was based on the oligosaccharide mass profiles of gel-separated mucins.

The oligosaccharide mass profiles of 9 exacerbated CF subjects and 11 non-CF healthy controls were dramatically different. Several differences in the profiles between the two subject groups were investigated. Preliminary data suggested that characteristics of the glycosylation of exacerbated CF sputum mucins included increased sialylation and decreased sulfation, compared with non-CF healthy control sputum.

Following production of oligosaccharide mass profiles, the oligosaccharide LC- MS/MS data were analysed to produce semi-quantitative characterisation of oligosaccharide composition and diversity for gel-separated glycoproteins. For each oligosaccharide mass profile, oligosaccharide ions with intensities greater than 10% of the most abundant ion were included in the analysis. This limit included approximately 10-100 different oligosaccharides, dependent on the characteristics of the particular subjects and glycoproteins. The base-peak chromatogram intensity was summed for the entire isotopic distribution of each selected ion. Ions with the same m/z (± 1.0) but differing retention times represent different oligosaccharide species and so were summed independently. The monosaccharide composition of each oligosaccharide was determined using GlycoMod (http://www.expasv.ch/tools/glvcomod). in combination with MS/MS data. GlycoMod provides a list of potential monosaccharide compositions matching an observed oligosaccharide mass, and marker fragment ions from MS/MS data can be diagnostic of particular monosaccharide components. More detailed interpretation of MS/MS data, to provide details of oligosaccharide structure and linkage, in addition to monosaccharide composition, is also carried out using art-recognized procedures.

The intensities obtained for each oligosaccharide ion isomer were normalised within each oligosaccharide mass spectrum, and the weighted average monosaccharide composition was determined.

Results and discussion

One-dimensional SDS-AgPAGE Glycoprotein Profiles of Sputa Sputum samples from 19 exacerbated CF and 19 non-CF healthy control subjects were subjected to 1-D SDS-AgPAGE, and PAS-stained for total oligosaccharide content. Duplicate gels were also electro-blotted to PVDF membrane and stained with Alcian Blue, to determine acidic oligosaccharide content.

Overall, 1-D SDS-AgPAGE protein profiles were reproducible between different samples within each group and confirmed the trend shown in Figure 1 for single samples. That is, non-CF healthy control subjects reproducibly have distinct major mucin bands having a molecular weight of about 4 MDa, with some subjects displaying additional less intense mucin bands of about 2 MDa to about 3 MDa (Figure 1). These lower molecular weight bands are typically more intense when stained with Alcian Blue suggesting that they are charged species.

In contrast, CF subjects with acute pulmonary exacerbation display several major mucin bands having a molecular weight range from about from 1 MDa to about 4 MDa, with the bands at about 2 MDa being typically the most intense (Figure 1 ). Several non-CF and exacerbated CF subjects also display glycoprotein bands of about 200 kDa molecular weight.

Protein Identification

Identification of the proteins present in the >100 kDa fraction of 10 exacerbated

CF and 8 non-CF healthy control induced adult sputum samples suggested the presence of several mucins and mucin-like proteins in the various Alcian Blue staining bands, as summarised in Table 5. In particular, MUC5B and MUC 5AC were identified in the 4 MDa band excised from 1-D gels of non-CF control subjects, and in the high molecular weight glycoproteins in the molecular weight range from about 1 MDa to about 4 MDa obtained from CF subjects during pulmonary exacerbation. In contrast, the mucin-like protein gp340 was identified in both the 200 kDa and 1 MDa bands for both groups.

With regard to the lower apparent molecular weight of mucins in CF sputum during a clinical exacerbation, compared to CF sputa post-treatment or non-CF sputa (eg., Figure 1) , trypsin digestion of high molecular weight mucin bands in non-CF sputa and sputa from CF subjects during a clinical respiratory exacerbation revealed differences in the MUC5B tryptic digests produced (not shown). In particular, a typical tryptic peptide map of MUC5B protein detected from the 1 MDa mucin band in the sputa of exacerbated CF subjects lacked no peptide masses originating from the non-glycosylated terminal regions of the MUC5B apoprotein. More particularly, there were no tryptic digests produced from the MUC 5B apoprotein that were N-terminal of position 2344 or C-terminal of position 4922, suggesting that the protein was degraded during a clinical exacerbation to an extent that leaves intact only a sequence comprising residues 2345-4922 of SEQ ID NO: 1. In contrast, the ~4 MDa mucin band in the sputa of non-CF subjects produced tryptic digests corresponding to regions of the MUC5B apoprotein from residue 292 to residue 5649, as expected for an intact MUC5B protein. These data indicate that the inability to detect appropriate N-terminal and/or C-terminal regions of MUC5B protein is a useful diagnostic of a clinical exacerbation in a CF subject, in particular the absence of detectable amino acid sequences that are N-terminal to residue 2344 of SEQ ID NO: 1 or C-terminal to residue 4922 of SEQ ID NO: 1. The absence of detectable tryptic peptide fragment of MUC 5B in these N-terminal and/or C-terminal regions is particularly useful and, in this respect, the following MUC5B peptide fragments were actually identified in the sputum of non-CF controls but absent from the sputum of CF patients suffering from a clinical exacerbation:

1. N-terminal fragment CRCPTCPCATFVEYSR (SEQ ID NO: 2);

2. N-terminal fragment GVQLSDWR (SEQ ID NO: 3);

3. N-terminal fragment YAYWDACQPTCR (SEQ ID NO: 4);

4. N-terminal fragment IVTENIPCGTTGTTCSK (SEQ ID NO: 5);

5. N-terminal fragment QCSILHGPTFAACR (SEQ ID NO: 6);

6. N-terminal fragment CPPSQPFFNEDQMK (SEQ ID NO: 7);

7. N-terminal fragment GYQVCPVLADIECR (SEQ ID NO: 8);

8. N-terminal fragment GLMCANSQQSPPLCHDYELR (SEQ ID NO: 9);

9. N-terminal fragment AAGGHLCQQPK (SEQ ID NO: 10);

10. N-terminal fragment VHCDVHFGLVCR (SEQ ID NO: 11);

11. N-terminal fragment MCYNYR (SEQ ID NO: 12);

12. C-terminal fragment LEVPCQSLEAYAELCR (SEQ ID NO: 13);

13. C-terminal fragment GVCSDWR (SEQ ID NO: 14);

14. C-terminal fragment PCGPIQPATCNSR (SEQ ID NO: 15);

15. C-terminal fragment TGCCYSCEEDSCQVR (SEQ ID NO: 16); and

16. C-terminal fragment YSAEAQAMQHQCTCCQER (SEQ ID NO: 17). Without being bound by any theory or mode of action, the differences in tryptic peptide maps for exacerbated CF subjects compared to non-CF subjects suggests that the mucin bands with lower apparent molecular weight are partially degraded or digested mucins, still containing at least a portion of the original highly glycosylated central domain, but without sections of the sparsely glycosylated N- and C-terminal regions. The several distinct mucin bands across a large apparent molecular weight range may therefore correspond to cleavage fragments containing varying numbers of glycosylated MUC5B domains. This would also be in agreement with the similarity of the glycosylation profiles of the different MUC5B-containing bands from the same subjects (eg., Figure 4 below). Moreover, as not all potential trypsin cleavage sites actually produce peptide fragments, it is possible that the large, highly glycosylated mucin-domains may efficiently or partially protect the apoprotein from enzymatic cleavage, thereby explaining why the central core region of the apoprotein is left intact during a clinical exacerbation. The decreased pH in the secretory pathway and in the extracellular milieu of a CF lung, arising as a consequence of the mutation in the CFTR, may also contribute to enhanced mucin degradation. As the 1-D SDS- AgPAGE gel profiles of non-CF healthy controls also contain these lower molecular weight bands, albeit at a low abundance, there may be a low or background level of mucin protein degradation in normal healthy sputum. An alternative explanation is that non-CF sputum comprises other mucins or mucin- like proteins that are not readily identified with the approach used here.

O-Linked Oligosaccharide Composition of Sputum Mucins a) Oligosaccharide mass spectrometry

O-linked oligosaccharides were released by reductive alkaline β-elimination from the Alcian Blue staining bands from sputum for each subject in the cohort, after separation by 1 D SDS-AgPAGE and electroblotting to PVDF. These oligosaccharides were then analysed by LC-ESI-MS/MS. Oligosaccharide mass profiles were obtained for each gel-separated mucin band from each subject, and the weighted average oligosaccharide composition was determined for the major mucin band from each subject, as described above. Figure 4 shows the 1D SDS-AgPAGE images, with accompanying oligosaccharide mass spectrometry profiles for mucins in the molecular weight range from about 2 MDa to about 4MDa, in the sputum of (i) the non-CF control subject CYFB1-22; and (ii) the exacerbated CF subject CYFB1-37. High molecular weight glycoproteins in this molecular weight range were excised from 1 D SDS-AgPAGE as gel slices and subjected to mass spectrometry as described supra. Glycoproteins having a molecular weight of about 200 kDa were also analysed. In Figure 4, the oligosaccharide mass spectrometry profiles are shown for each excised glycoprotein band.

The oligosaccharide mass spectrometry profiles shown in Figure 4 were typical of all cohort subjects analysed. MUC5B-containing bands dominate the 1 D protein profile in the high molecular weight range from about 1 MDa to about 4 MDa. The corresponding oligosaccharide mass profiles derived from the MUC5B bands differ strikingly between the cohort of sputa from exacerbated CF patients and those from non-CF control subjects. On the other hand, the oligosaccharide mass profiles for each of the excised MUC5B-containing bands in the 1 MDa to 4 MDa molecular weight range were very similar for each subject analysed.

As with the single subject data presented in Figures 2a and 2b, data obtained for the cohort of subjects (eg., Figures 4, 8 and 9; Tables 3 and 4) indicate that three structures present in the sputum of healthy non-CF subjects and having m/z 628.9 ± 1.0, m/z 70.1.99 ± 1.0 and m/z 782.8 ± 1.0 are either not present, or present at a relatively low level compared to other structures, in the MUC5B- containing fraction of sputum from CF subjects during a pulmonary exacerbation.

On the other hand, data obtained for the cohort confirm that m/z 1186.4 and m/z 1331.3 are high in CF subjects during exacerbation and following treatment, however not present at high levels in the sputa of non-CF subjects and, as a consequence, may reflect an overall CF-related modification to MUC5B or other mucin in the MUC5B-containing fraction. Similarly, data obtained for the cohort of subjects (eg., Figures 4, 8 and 9; Tables 3 and 4) confirm that structures having m/z 665.2 ± 1.0, m/z 1186.4 ± 1.0, m/z 1244.4 ± 1.0, m/z 1331.3 ± 1.0, and m/z 1477.4 ± 1.0 were relatively high in abundance during pulmonary exacerbation in CF subjects compared to non-CF healthy subjects. Again, data obtained from CF patients following recovery confirm that the relative abundance of the m/z 1477.41 structure decreases following successful treatment, however does not decline in relative abundance in CF subjects that are not successfully treated (eg., subject CYFB1-37), suggesting that this structure at least may be diagnostic of the course of infection. Data obtained for the cohort also confirm that the m/z 1477.4 ion was amongst the most abundant species in the MUC5B-containing fraction from CF patients during infection.

Many oligosaccharide structures are isomers, with the same mass but different elution times on graphitised carbon HPLC. These isomers most likely represent very different oligosaccharide structures.

b) Detailed glycosylation

Further detailed glycosylation analysis was performed on the most intense MUC5B-containing band for each subject. Oligosaccharide diversity, composition, and relative abundance for non-CF healthy control subject CYFB1-22 and exacerbated CF subject CYFB1-37 are shown in Table 6 and Table 7.

A summary of the resulting weighted average O-linked oligosaccharide compositions for all subjects in the cohort are shown in Table 8, and are graphically represented in Figure 5. Results of student's t-tests comparing exacerbated CF and non-CF healthy control subjects and fold differences for the abundance of each monosaccharide are shown in Table 9. There were significant differences at the 95% confidence interval (c.i.) for the average amounts of all monosaccharides from sputum mucins from exacerbated CF and non-CF healthy control subjects. Differences in the relative abundances of terminal residues in sputum mucins from exacerbated CF and non-CF healthy control sputum were especially apparent, with exacerbated CF subjects showing, at the 95% confidence interval, a 1.5-fold decrease in fucosylated MUC5B- containing mucin, a 2.8-fold increase in sialylated MUC5B-containing mucin, and a 4.3-fold decrease in sulfated MUC5B-containing mucin, relative to the non- CF healthy controls. Small decreases in the contents of N-acetyl hexosamine (HexNAc) and hexose in the MUC5B-containing mucins of exacerbated CF subjects were also apparent. Subjects within each group displayed a moderate variation in monosaccharide composition, with the exception of sulfate content. Sulfate content was highly variable within the non-CF healthy control subjects, but was consistently very low in exacerbated CF subjects.

Principal components analysis was performed on the data to allow simultaneous comparison of multiple components of the mucin oligosaccharide compositions of sputum from exacerbated CF and non-CF healthy control subjects. The results of this principal components analysis are represented graphically in Figure 6. Data presented in Figure 6 indicate that there is dramatic separation between exacerbated CF subjects and non-CF healthy control subjects, on the basis of mucin oligosaccharide composition. The directional arrows representing sialic acid and sulfate content are the largest, indicating that these two parameters are the most important in determining the separation between non-CF subjects and CF subjects suffering from an acute clinical exacerbation. Fucose content is also of some importance in determining separation.

All of these differences are indicative of substantially different glycosylation patterns in the MUC5B-containing mucin fraction of non-CF healthy controls compared to CF subjects suffering from a clinical respiratory exacerbation. In summary, the data presented herein indicate that measurements of the absolute amounts of fucosylated and/or sialylated and/or sulfated MUC5B-containing mucins, or alternatively, measurements of the ratio(s) of any one or more of these glycosyl groups in the MUC5B-containing mucin fraction, is a suitable diagnostic of CF or a clinical exacerbation in a CF subject.

These changes may also suggest dramatic differences in mucin-mediated interactions in the lung. The glycosylation differences between the two groups are especially apparent in the abundances of sialic acid, fucose and sulfate, all of which are predominantly terminal monosaccharides. These are most likely to be involved in oligosaccharide-mediated interactions between host mucins and pathogens, and are therefore potential therapeutic targets.

The dramatic 4.3-fold reduction in sulfation of exacerbated CF sputum mucins relative to non-CF controls is very striking. The mucin glycosylation profiles described in this report may be inherently biased towards the mucins separated by 1 D SDS-AgPAGE, and those oligosaccharides detected by LC-ESI-MS. Notwithstanding that this might be the case, processed sputum samples directly dot-blotted to PVDF membrane before oligosaccharide release showed equivalent glycosylation profiles to the dominant gel separated mucins, suggesting that no bias was introduced by electrophoretic separation (Data not shown). Comparison of dot blots of exacerbated CF and non-CF healthy control sputum stained with low pH alcian blue (specific for sulfate groups) also supported the LC-ESI-MS results, suggesting that non-CF healthy control sputum mucins are much more heavily sulfated than exacerbated CF subjects (Data not shown).

The increases in sialic acid and decreases in fucose content of exacerbated CF sputum mucins tend to suggest that sialyl-Lewis type structures are not increased relative to non-CF MUC5B or other mucin in the MUC5B-containing fraction, as these epitopes require both monosaccharides in combination. Detailed analysis of oligosaccharide MS/MS data, as well as additional non-MS based analysis, is used to assign such oligosaccharide structures. Summary

Together, the protein profile, protein expression and oligosaccharide compositional data show substantial differences between non-CF healthy control and exacerbated CF sputum. These differences are principally related to the modifications of the major mucins present, rather than the presence of completely new proteins. Specifically, in CF subjects with acute pulmonary exacerbation, MUC5B-containing mucin fractions are present over a wide molecular weight range from -1-4 MDa, and largely contain moderately fucosylated, highly sialylated and sparsely sulfated oligosaccharide structures. In healthy controls, the same mucins are present mainly at ~4 MDa, and contain moderately fucosylated, lowly sialylated and highly sulfated oligosaccharides. These specific differences are potential prognostic markers of lung function, and due to the effects of oligosaccharides on mucin-mediated interactions in the lung, are also potential drug targets.

Example 3 Comparison of Sputum from CF Subjects with Acute Pulmonary Exacerbation and at Post-Discharge from Hospital

Materials and methods Cohort samples

CF subjects with acute pulmonary exacerbation (Table 4) were hospitalised for treatment, including intravenous antibiotics and anti-inflammatory drugs. Subjects were discharged typically after two weeks of treatment, when sputum samples were also obtained, allowing comparison of individual changes in sputum protein and glycosylation profiles after treatment. Ten (10) additional exacerbated CF, and 9 additional discharged CF subjects were also included in this study. The CF subjects post-discharge from hospital had recovered to varying extents as determined by changes in FEVi and other clinical measures and, as a consequence, were clinically heterogeneous. Sample treatment

Sputa were obtained and analysed as described in examples 1 and 2 supra.

Results and discussion 1 D SDS-AgPA GE Protein Profiles and Protein Identification

Because the subjects displayed varying degrees of response to treatment as determined by changes in FEVi (Table 4 and Table 13), the glycoproteomic profiles of CF subjects post-discharge from hospital were compared individually with their profiles upon acute pulmonary exacerbation.

On discharge from hospital, the 1D SDS-AgPAGE protein profiles of sputum from CF subjects that had recovered from an acute pulmonary exacerbation showed the same general changes as indicated in Figure 1 for a single sample. The recovery profiles typically consisted of several MUC5B-containing mucin bands having molecular weights in the range from about 1 MDa to about 4 MDa, with lower intensity bands containing gp340 at about 1 MDa molecular weight. Subject CYFB1-11 showed a dramatic change in 1D gel profile at post-discharge from hospital, displaying an intense MUC5B-containing band at about 4 MDa, as typically seen in non-CF healthy controls, with lower intensity MUC5B-containing bands in the range from about 2 MDa to about 4 MDa.

Average O-Linked Oligosaccharide Composition of Sputum Mucins The average mucin oligosaccharide composition of CF subjects with acute pulmonary exacerbations and CF subjects upon discharge from hospital are shown in Table 10.

Sulfate content was significantly different in the post-discharge group compared to CF patients during an acute pulmonary exacerbation, at the 94% confidence interval. More particularly, the sulfate content of MUC5B-containing mucin fractions from 1-D gels, which was already reduced compared to non-CF control subjects, decreased about 2.3-fold on discharge, compared to the level at exacerbation. This represents a large average change in sulfate content. The levels of other monosaccharides were not significantly altered at this confidence interval.

Principal components analysis (PCA) was performed on the data from Table 10. The PCA data are represented graphically in Figure 7. PCA revealed three outlying discharged CF subjects, CYFB1-11B, CYFB1-41B and CYFB1-44B that were distinguished from the remainder of the cohort on the basis of both reduced sulfation and sialylation, with some increase in fucosylation. The large sulfate compositional arrow in Figure 6 also supported the large difference in sulfate content of discharged subjects relative to CF subjects at exacerbation.

Individual CF Subject Comparisons upon Exacerbation and Post-Discharge As a consequence of the clinical heterogeneity of the post-discharge group, as indicated by changes in FEVi values (Table 4 and Table 13), total sputum protein concentration and sputum protein profiles, and average sputum mucin characteristics, comparisons were made of the changes in mucin and glycosylation profiles for each individual. Representative samples are described herein below. Data for each sample are available from the inventors on request.

CF subjects CYFB1-11 and CYFB1-41 showed clear differences in lung biological state at exacerbation and post-discharge, as determined by measuring their mucin apoprotein and glycosylation profiles (described below). These subjects also showed the largest return to sputum and plasma proteomes resembling those of non-CF healthy controls. These two subjects were therefore chosen as representative of CF subjects that exhibit a good recovery after hospital treatment for an acute clinical exacerbation. Gel profiles and glycosylation analysis for CF subjects CYFB1-11 and CYFB1-41 are shown in Figure 8 and Figure 9, respectively. The same individual analysis was performed for all 19 CF subjects at exacerbation and 13 CF subjects at discharge.

Data shown in Figure 8 show large differences between the oligosaccharide mass profiles of subject CYFB1-11 at exacerbation and post-discharge. However, each of the separated MUC5B mucin bands from each sputum sample for this subject showed similar oligosaccharide mass profiles. In particular, data obtained for CYFB1-11 confirm that m/z 1186.4 ± 1.0 and m/z 1331.3 ± 1.0 ions were high both during exacerbation and following treatment. Similarly, data shown in Figure 8 confirm that the relative abundance of the m/z 1477.41 ± 1.0 structure decreased following successful treatment of CYFB1-11. Data obtained for this patient also confirmed that the m/z 1477.4 ± 1.0 ion was amongst the most abundant species in the MUC5B-containing fraction during infection.

Data shown in Figure 9 also show large differences between the oligosaccharide mass profiles of subject CYFB1-41 at exacerbation and post-discharge, with similar oligosaccharide mass profiles for each of the separated MUC5B mucin bands from each sputum sample for this subject. Data obtained for CYFB1-41 also confirm that the m/z 1331.3 ± 1.0 ion was high both during exacerbation and following treatment. Again, data in Figure 9 confirm that the relative abundance of the m/z 1477.41 ± 1.0 structure decreased following successful treatment of CYFB1-41. Data obtained for this patient again confirmed that the m/z 1477.4 ± 1.0 ion was amongst the most abundant species in the MUC5B-containing fraction during infection.

Thus, data obtained for several subjects in this study support the use of the m/z 1477.41 ± 1.0 structure as a diagnostic for clinical exacerbation and recovery in a CF patient, optionally referenced against the level of the m/z 1186.4 ± 1.0 and/or m/z 1331.3 ± 1.0 ions. For example, a high ratio of m/z 1477.41 ± 1.0 relative to the level of m/z 1186.4 ± 1.0 and/or m/z 1331.3 ± 1.0 in the MUC5B-containing mucin fraction can indicate a clinical exacerbation, whereas a reduction in this ratio can indicate a recovery or successful treatment.

The data presented in Figures 8 and 9 also suggest that the changes in glycosylation are not primarily responsible for the changes observed in mucin apparent molecular weight, because mucins having the same apparent molecular weight at exacerbation and post-discharge display different oligosaccharide mass profiles. Changes in mucin molecular weight and glycosylation profile are therefore largely independent, whilst both are potentially related to improvements in the overall health of the lung.

Figures 10a-10c show graphical representations of detailed oligosaccharide compositional analyses for subjects CYFB1-11 (Figure 10a) and CYFB1-41 (Figure 10b), and averages thereof (Figure 10c). The data presented in Figures 10a-10c are derived from the most intense-staining MUC5B-containing mucin band from each sample. The average data and fold differences for this analysis is shown in Table 11.

Data presented in Figures 10a-10c and Table 11 indicate that there were large and consistent differences in the composition of sputum mucin oligosaccharides from the CF subjects CYFB1-11 and CYFB1-41 , at exacerbation and post- discharge. The most marked modification is a 1.8-fold increase in the fucose content and a 2-fold decrease in the sialic acid content of the MUC5B-containing fraction in recovered CF subjects (i.e, the "post-discharge" group) relative to the acute pulmonary exacerbation phase. Small increases in the hexose and HexNAc contents of the MUC5B-containing fraction were also apparent for the post- discharge subjects. While a 1.7-fold decrease in sulfate content was also observed, this value is biased by the small absolute values of sulfate content in the subjects, both during acute pulmonary exacerbation and post-discharge. These two post-discharge subjects show glycosylation profiles that closely resemble non-CF healthy subjects, with the exception of the continuing very low sulfate content.

Subject CYFB1-37 was also of particular interest in this study, as this subject suffered from an overall deterioration in lung condition during hospitalisation, as a consequence of a viral infection. This deterioration also manifested as a reduced FEVi and little change in sputum and plasma proteomes during treatment. Gel profiles and glycosylation analysis for subject CYFB1-37 at exacerbation and post-hospital treatment are presented in Figure 11. Data presented in Figure 11 indicate that, whilst CYFB1-37 displayed disperse mucin bands at exacerbation, the apparent molecular weight of these bands was further reduced after discharge, thereby indicating of a deterioration in lung state. Differences were also apparent in the O-linked oligosaccharide mass profiles displayed in Figure 11. In particular, the mucin oligosaccharide composition of the most intense MUC5B-containing band for CYFB1-37 at exacerbation and discharge failed to show the characteristic reduction in relative abundance of the m/z 1477.41 ± 1.0 structure, compared to, for example, the level of the m/z 1331.3 ± 1.0 ion.

Figure 12 and Table 12 show the levels of various monosaccharides in the MUC5B-containing fraction of sputum mucins for subject CYFB1-37 during an acute pulmonary exacerbation and post-discharge. While the proportion of hexose and N-acetyl hexosamine in the sputum mucin oligosaccharides stayed relatively constant during hospitalisation, the amount of fucosylated MUC5B or other mucin in the MUC5B-containing fraction decreased 1.7-fold, and the sialic acid content increased 1.7-fold, in apposition to the changes observed for the successfully-treated subjects CYFB1-11 and CYFB1-41. Similar to the changes observed for CYFB1-11 and CYFB1-41 , the sulfate content of MUC5B-containing mucins decreased 1.8-fold following treatment, however as with those other samples, the data are biased by the low overall sulfate content of the sputum mucins in CF subjects.

Summaries of mucin protein and glycosylation profiles for CF subjects at exacerbation and post-discharge from hospital are shown in Table 13. Data presented in Table 13 show a correlation between the changes observed in mucin protein, mucin glycosylation, and 2-DE sputum protein profiles, in individuals between exacerbation and post-discharge. Summary

The changes in mucin oligosaccharide composition reported herein suggest that changes in mucin protein and glycosylation profiles are indicative of recovery following treatment for an acute pulmonary exacerbation, or alternatively, a further deterioration in clinical indicators following an acute pulmonary exacerbation.

These changes in mucin protein and glycosylation profile could be used as prognostic markers to monitor pulmonary infections in CF. More particularly, these data indicate that measurements of the absolute amounts of fucosylated and/or sialylated MUC5B-containing mucins, or alternatively, measurements of the ratio(s) of these glycosyl groups in the MUC5B-containing mucin fraction, is a suitable diagnostic for recovery from a clinical exacerbation in a CF subject. The low sulfate content of MUC5B-containing mucins compared to non-CF subjects may also be diagnostic of a past or present clinical exacerbation in a CF subject (i.e. a general diagnostic of pulmonary condition).

The data suggest further that the mucin and high molecular weight glycoprotein composition and characteristics of sputum are directly and dynamically related to changes in aspects of pulmonary health or infection status. This potentially provides the opportunity for improving methods of monitoring pulmonary infections by direct measurement of particular oligosaccharide epitopes or modified protein isoforms from patients' sputum.

Table 1

Weighted average mucin oligosaccharide composition for peaks detected in the sputum of CF subjects with acute pulmonary exacerbations (n=19)

Composition m/z Mass theoretical

Mass neutral HexNac Hex Fuc NeuAc Sulf

513.2 1 514.2 514.2 1

530.2 1 531.2 531.2 1 1

587.1 1 588.1 588.2 2 1

675.2 1 676.2 676.2 1 1

716.2 1 717.2 717.3 2

733.2 1 734.2 734.3 2 1

749.1 1 750.1 750.3 2 2

790.2 1 790.2 791.3 3 1

821.3 1 822.3 822.3 1 1

829.3 1 830.3 830.2 2 2

878.1 < 879.1 879.3 2 1

895.2 896.2 896.3 2 2

936.2 937.2 937.4 3 1

952.2 953.2 953.4 3 2

958.1 959.1 959.3 2 1

966.2 967.2 967.3 1 1

975.3 976.3 976.3 2 2

1024.2 1025.2 1025.4 2 1

1040.5 1041.5 1041.4 2 2

1041.4 1042.4 1042.4 2 2

1081.3 I 1082.3 1082.4 3 1

1082.3 I 1083.3 1083.4 3 1 2

1098.3 I 1099.3 1099.4 3 2 1

1104.3 I 1105.3 1105.3 2 1 1

1114.4 I 1115.4 1115.4 3 3

1120.2 I 1121.2 1121.3 2 2

1121.3 I 1122.3 1122.3 2 2 2

1139.4 I 1,140.4 1140.4 4 1 1

1178.2 I 1179.2 1179.4 3 2 1

1186.4 I 1187.4 1187.4 2 2 1

1187.3 I 1188.3 1188.4 2 2 3

1227.4 I 1228.4 1228.5 3 1 1

1243.2 I 1244.2 1244.4 3 2

1244.4 I 1245.4 1245.5 3 2 2

628.9 ; 2 1259.8 1259.3 3 2 1

1260.3 1 1261.3 1261.5 3 3 1

633.0 : 2 1268.0 1267.4 2 2 1

1266.1 1 1267.1 1267.4 2 2 1

1267.3 1 1268.3 1268.4 2 2 3

1301.4 1 1302.4 1302.5 4 2 1

1315.3 1 1316.3 1316.5 2 1 1

1322.1 1 1323.1 1325.4 3 2 2

665.2 2 1332.4 1332.5 2 2 2

1331.3 1 1332.3 1332.5 2 2 2 1332.4 1 1333.4 1333.5 2 2 2 1

1340.3 1 1341.3 1341.4 3 3 1 1

1373.5 1 1374.5 1374.5 3 1 2 1

1381.6 1 1382.6 1382.4 4 2 1 1

694.5 2 1391.0 1390.5 3 2 1 1

1389.4 1 1390.4 1390.5 3 2 1 1

1390.3 1 1391.3 1391.5 3 2 3

701.9 2 1405.8 1405.4 3 2 2 2

705.3 2 1412.6 1412.4 2 2 2 1

1411.4 1 1412.4 1412.4 2 2 2 1

705.8 2 1413.6 1413.4 2 2 2 1 1

709.9 2 1421.8 1421.4 3 3 1 2

710.1 2 1422.2 1421.4 3 3 1 2

1447.4 1 1448.4 1448.5 4 2 2

1469.2 1 1470.2 1470.5 3 2 1 1 1

1470.3 1 1471.3 1471.5 3 2 3 1

738.3 2 1478.6 1478.5 2 2 1 2

1477.4 1 1478.4 1478.5 2 2 1 2

1479.4 1 1480.4 1480.5 4 4

1486.3 1 1487.3 1487.5 3 3 2 1

1527.3 1 1528.3 1528.5 4 2 2 1

767.1 2 1536.2 1535.5 3 2 2

1534.4 1 1535.4 1535.5 3 2 2

1535.4 1 1536.4 1536.6 3 2 2 1

1535.7 1 1536.7 1536.6 3 2 2 1

775.1 2 1552.2 1552.6 3 3 1 1

1551.3 1 1552.3 1552.6 3 3 1 1

778.2 2 1558.4 1558.5 2 2 1 2 1

782.8 2 1567.6 1567.4 3 3 2 2

784.0 2 1570.0 1569.6 3 4 2

1592.5 1 1593.5 1593.6 4 2 1 1

807.5 2 1617.0 1616.5 3 2 2 1 1

811.4 2 1624.8 1624.5 4 3 1 2

815.6 2 1633.2 1632.5 3 3 1 1 1

1639.3 1 1640.3 1640.5 4 4 2

824.8 2 1651.6 1651.6 5 2 2

1650.5 1 1651.5 1651.6 5 2 2

840.0 2 1682.0 1681.6 3 2 1 2

1680.2 1 1681.2 1681.6 3 2 1 2

848.4 2 1698.8 1697.6 3 3 2

848.1 2 1698.2 1698.6 3 3 2 1

857.0 2 1716.0 1715.6 3 4 3

876.8 2 1755.6 1755.6 4 3 1 1

884.5 2 1771.0 1770.5 4 3 2 2

887.9 2 1777.8 1777.6 3 3 2 1

898.0 2 1798.0 1797.7 5 2 3

912.5 2 1827.0 1826.6 3 2 3

913.0 2 1828.0 1827.7 3 2 2 2

920.7 2 1843.4 1843.7 3 3 1 2

921.0 2 1844.0 1843.7 3 3 1 2

950.1 2 1902.2 1901.7 4 3 2 1

957.6 2 1917.2 1916.6 4 3 3 2 960.7 2 1923.4 1923.6 3 3 1 2 1

965.3 2 1932.6 1932.6 4 4 2 2

993.8 2 1989.6 1989.7 3 3 2 2

998.2 2 1998.4 1998.7 4 4 3 1

1002.4 2 2006.8 2006.7 3 4 3 1

1004.7 2 2011.4 2011.5 4 4 1 3

1005.1 2 2012.2 2012.5 4 4 2 3

1011.0 2 2024.0 2023.7 3 5 4

1013.2 1 1014.2 2023.7 3 5 4

1031.1 2 2064.2 2063.8 4 4 2 1

1033.7 2 2069.4 2069.7 3 3 2 2 1

1038.4 2 2078.8 2078.6 4 4 3 2

1062.4 2 2126.8 2126.7 4 3 1 2 1

1078.3 2 2158.6 2158.6 4 4 3 3

1080.6 2 _, 2163.2 2162.8 6 3 3

1095.3 2^' 2192.6 2191.8 4 3 3

1103.6 2 2209.2 2208.8 4 4 1 2

1103.9 2 2209.8 2209.8 4 4 3 1

1110.9 2 2223.8 2224.8 4 5 2

1134.9 2 2271.8 2271.7 4 3 3 1

1176.7 2 2355.4 2280.8 3 3 2 3

1143.6 2 2289.2 2288.7 4 4 1 2 1

1176.2 2 2354.4 2353.8 4 4 3

1183.4 2 2368.8 2368.7 4 4 1 2 2

1216.5 2 2435.0 2434.8 4 4 2 2 1

1249.0 2 2500.0 2499.9 4 4 1 3

1255.4 2 2512.8 2513.7 4 4 3 2

1289.6 2 2581.2 2580.9 4 4 3 2 1

1321.4 2 2644.8 2644.9 4 4 4

1321.9 2 2645.8 2645.9 4 4 2 3

1432.3 2 2866.6 2866.0 5 5 3 2

1504.5 2 3011.0 3010.1 5 5 4

1505.1 2 3012.2 3011.1 5 5 2 3

1577.2 2 3156.4 3156.1 5 5 1 4

Table 2

Weighted average mucin oligosaccharide composition for peaks detected in healthy non-CF subjects (n=19)

_-— /-— Composition m/z z MaSS theoretical

MaSS neutral HexNac Hex Fuc NeuAc Sulf

530.2 1 531.2 531.2 1 1 1

555.9 2 1113.8 1113.3 3 2 2

587.1 1 588.1 588.2 2 1

628.9 2 1259.8 1259.3 3 2 1 2

667.2 1 668.2 668.2 2 1 1

669.9 2 1341.8 1341.4 3 3 1 1

675.2 1 676.2 676.2 1 1 1

701.9 2 1405.8 1405.4 3 2 2 2

709.9 2 1421.8 1421.4 3 3 1 2

710.1 2 1422.2 1421.4 3 3 1 2

733.2 1 734.2 734.3 2 1 1

734.5 2 1471.0 1470.5 3 2 1 1 1

742.9 2 1487.8 1487.5 3 3 2 1

755.1 1 756.1 756.2 1 1 1 1

774.2 2 1550.4 1550.4 3 2 1 1 2

775.1 2 1552.2 1552.6 3 3 1 1

782.8 2 1567.6 1567.4 3 3 2 2

807.1 2 1616.2 1616.5 3 2 2 1 1

811.4 2 1624.8 1624.5 4 3 1 2

813.3 2 1628.6 1624.5 4 3 1 2

829.2 1 830.2 830.2 2 2 1

844.5 2 1691.0 1690.6 4 3 2 1

855.2 2 1712.4 1712.5 3 3 1 1 2

856.1 2 1714.2 1713.5 3 3 3 2

878.1 1 879.1 879.3 2 1 1

884.5 2 1771.0 1770.5 4 3 2 2

888.5 2 1779.0 1778.6 3 3 2 1 1

892.5 2 1787.0 1786.5 4 4 1 2

895.2 1 896.2 896.3 2 2 1

928.3 2 1858.6 1858.5 3 3 2 1 2

932.1 2 1866.2 1866.5 4 4 1 3

932.2 2 1866.4 1858.5 3 3 2 1 2

952.3 1 953.3 953.4 3 2

957.6 2 1917.2 1916.6 4 3 3 2

958.1 1 959.1 959.3 2 1 1 1

965.3 2 1932.6 1932.6 4 4 2 2

966.2 1 967.2 967.3 1 1 2

975.3 1 976.3 976.3 2 2 1 1

998.2 2 1998.4 1998.7 4 4 3 1

1005.1 2 2012.2 2012.5 4 4 2 3

1024.2 1 1025.2 1025.4 2 1 1 1

1030.5 2 2063.0 2062.7 4 4 2

1032.3 1 1033.3 1033.3 3 2 1

1038.4 2 2078.8 2078.6 4 4 3 2 1038.4 2 2078.8 2078.6 4 4 3 2

1040.5 1 1041.5 1041.4 2 2 1

1041.4 1 1042.4 1042.4 2 2 2

1066.9 2 2135.8 2135.6 5 4 2 2

1067.0 2 2136.0 2135.6 5 4 2 2

1078.3 2 2158.6 2158.6 4 4 3 3

1086.3 2 2174.6 2174.6 4 5 2 3

1098.3 1 1099.3 1099.4 3 2 1

1100.2 2 2202.4 2201.7 5 4 3 1

1104.2 1 1105.2 1105.3 2 1 1 1 1

1110.9 2 2223.8 2224.8 4 5 2

1119.4 2 2240.8 2240.7 4 5 3 2

1120.2 1 1121.2 1121.3 2 2 1 1

1121.3 1 1122.3 1122.3 2 2 2 1

1140.1 2 2282.2 2281.7 5 4 3 2

1178.2 1 1179.2 1179.4 3 2 1 1

1183.8 2 2369.6 2368.7 4 4 1 2 2

1192.7 2 2387.4 2386.7 4 5 4 2

1213.1 2 2428.2 2427.8 5 4 4 2

1221.1 2 2444.2 2443.8 5 5 3 2

1244.4 1 1245.4 1245.5 3 2 2

1260.3 1 1261.3 1261.5 3 3 1

1266.1 1 1267.1 1267.4 2 2 1 1 1

1267.3 1 1268.3 1268.4 2 2 3 1

1283.3 1 1284.3 1284.4 2 3 2 1

1294.0 2 2590.0 2589.8 5 5 4 2

1324.3 1 1325.3 1325.4 3 2 2 1

1331.3 1 1332.3 1332.5 2 2 2

1333.7 2 2669.4 2668.8 5 5 2 1 3

1340.3 1 1341.3 1341.4 3 3 1 1

1366.4 2 2734.8 2733.8 5 5 1 2 2

1389.4 1 1390.4 1390.5 3 2 1 1

1403.5 2 2809.0 2808.9 6 6 3 2

1469.2 1 1470.2 1470.5 3 2 1 1 1

1477.4 1 1478.4 1478.5 2 2 1 2

1486.3 1 1487.3 1487.5 3 3 2 1

1565.8 2 3133.6 3133.1 6 4 4 2 1

1632.7 1 1633.7 1633.5

Table 3

Weighted average mucin oligosaccharide composition for peaks detected in CF subjects after recovery from acute pulmonary exacerbation (n=3)

Composition m/z Mass theoretical

Mass neutral HexNac Hex Fuc NeuAc Sulf

5 51133..22 ^• I 551144..22 514.2 1 1

5 53300..22 ' I 553311..22 531.2 1 1 1

5 58877..11 I 558888..11 588.2 2 1

6 67755..22 ' I 667766..22 676.2 1 1 1

771166..22 ' I 771177..22 717.3 2 1

733.2 734.2 1 1

775.5 2 1553.0 1552.6 3 3 1 1

783.9 2 1569.8 1569.6 3 4 2

790.2 1 790.2 791.3 3 1

795.7 2 1593.4 1593.6 4 2 1 1

824.8 2 1651.6 1651.6 5 2 2

836.1 2 1674.2 1674.6 4 2 3

848.1 2 1698.2 1698.6 3 3 2 1

856.9 2 1715.8 1715.6 3 4 3

895.2 1 896.2 896.3 2 2 1

898.1 2 1798.2 1797.7 5 2 3

905.8 2 1813.6 1813.7 5 3 2

928.8 2 1859.6 1859.7 3 4 2

936.2 1 937.2 937.4 3 1 1

949.9 2 1901.8 1901.7 4 3 2 1

978.8 2 1959.6 1959.7 5 3 3

1002.0 2 2006.0 2005.7 3 4 1 2

1031.5 2 2065.0 2064.8 4 4 4

1039.3 2 2080.6 2080.8 4 5 3

1040.5 1 1041.5 1041.4 2 2 1

1041.2 1 1042.2 1042.4 2 2 2

1051.9 2 2105.8 2105.8 5 3 4

1080.1 2 2162.2 2162.8 6 3 3

1081.3 1 1082.3 1082.4 3 1 1

1082.3 1 1083.3 1083.4 3 1 2

1098.3 1 1099.3 1099.4 3 2 1

1103.6 2 2209.2 2209.8 4 4 3 1

1139.4 1 1140.4 1140.4 4 1 1

1161.2 2 2324.4 2324.9 6 4 3

1162.5 1 1163.5 1163.4 3 1 2

1186.4 1 1187.4 1187.4 2 2 1 1

1244.2 1 1245.2 1245.5 3 2 2

1266.2 1 1267.2 1267.4 2 2 1 1

1285.5 1 1286.5 1286.5 4 1 2

1301.4 1 1302.4 1302.5 4 2 1

1315.3 1 1316.3 1316.5 2 1 1 2

1331.3 1 1332.2 1332.5 2 2 2

1332.4 1 1333.4 1333.5 2 2 2 1

1381.6 1 1382.6 1382.4 4 2 1

1389.4 1 1390.4 1390.5 3 2 1 1 1390.3 1 1391.3 1391.5 3 2 3

1406.4 1 1407.4 1407.5 3 3 2

1432.3 2 2866.6 2866.0 5 5 3 2

1447.4 1 1448.4 1448.5 4 2 2

1477.4 1 1478.4 1478.5 2 2 1 2

1527.5 1 1528.5 1528.5 4 2 2 1

1534.4 1 1535.4 1535.5 3 2 2

1535.7 1 1536.7 1536.6 3 2 2 1

1551.3 1 1552.3 1552.6 3 3 1 1

1568.4 1 1569.4 1569.6 3 4 2

1592.5 1 1593.5 1593.6 4 2 1 1

1593.4 1 1594.4 1594.6 4 2 3

1650.5 1 1651.5 1651.6 5 2 2

1697.2 1 1698.2 1698.6 3 3 2 1

Table 4

Forced expiratory volumes (FEV^ of subjects processed for glycoproteomic analysis of sputum. FEVi (% Predicted) values are indicated.

Sputum

CF: Acute

CF: Post-

Pulmonary Non-CF Control Hospitalisation

Exacerbation

Subject FEV, Subject FEV_! Subject FEV_!

CYFBl-4 52.3 CYFB1-7 90.2

CYFB1-6 50.3 CYFB1-8 96.3

CYFB1-10 17.8 CYFB1-9 98.7

CYFB1-11 59.5 CYFB1-11B 81.3 CYFB1-13 92.6

CYFB1-12B 62.3 CYFB1-17 114.5

CYFB1-15 50.4 CYFB1-18 122.4

CYFB1-16 46.7 CYFB1-16B 46.7 CYFB1-19 103.5

CYFB1-20 28.3 CYFB1-20B 35.4 CYFB1-21 97

CYFB1-35 59.1 CYFB1-22 114.5

CYFB1-37 47.8 CYFB1-37B 40.6 CYFB1-24 102.2

CYFB1-38 14.3 CYFB1-38B 27.3 CYFB1-25 92.2

CYFB1-39 65.1 CYFB1-39B 85.2 CYFB1-26 105.1

CYFB1-40 18.4 CYFB1-40B 25.8 CYFB1-27 108.8

CYFB1-41 60.7 CYFB1-41B 66.5 CYFB1-28 102.7

CYFB1-43 46.4 CYFB1-43B 69.4 CYFB1-29 108.7

CYFB1-44 56.9 CYFB1-44B 76.0 CYFB1-30 107.5

CYFB1-45 30.7 CYFB1-45B 26.2 CYFB1-32 86.6

CYFB1-46 55.6 CYFB1-46B 60.5 CYFB1-34 94.6

CYFB1-47 39.4 CYFB1-36 98.2

CYFB1-48

19 13 19 Table 5

Summary of Proteins Identified in the >100 kDa fraction of Sputum from Exacerbated CF Subjects and Non-CF Healthy Controls

Molecular Exacerbated CF Non-CF Healthy

Weight Subjects Controls

4 MDa MUC5B and MUC5AC

1-4 MDa MUC5B and MUC5AC

1 MDa GP340 GP340

200 kDa GP340 GP340

Table 6

Oligosaccharide diversity and compositional analysis for the most intense MUC5B-containing mucin band from non-CF healthy control subject CYFB1-22.

CYFB1M22

Composition m/z X Mass _neutra. - Elution Time % Abundance

HexNac Hex Fuc NeuAc Sulf

587.1 588.2 2 1 12.9 2.2

667.2 668.2 2 1 14.4 0.9

667.2 668.2 2 1 15.2 1.8

958.1 959.3 2 1 1 16.5 2.9

975.3 976.3 2 2 1 14.0 4.2

1032.4 871.3 3 1 17.0 2.3

1032.4 871.3 3 1 18.5 6.9

555.9 2 1113.3 3 2 2 19.3 1.3

555.9 2 1113.3 3 2 2 21.4 2.6

1178.2 1 1179.4 3 2 1 1 17.0 9.1

628.9 2 1259.3 3 2 1 2 18.2 1.3

628.9 2 1259.3 3 2 1 2 19.7 6.9

701.9 2 1405.4 3 2 2 2 15.6 0.9

701.9 2 1405.4 3 2 2 2 17.3 6.2

710.1 2 1421.4 3 3 1 2 17.4 2.4

710.1 2 1421.4 3 3 1 2 18.5 2.8

774.2 2 1550.4 3 2 1 1 2 21.5 2.1

774.2 2 1550.4 3 2 1 1 2 22.6 2.0

782.8 2 1567.4 3 3 2 2 16.8 9.3

811.4 2 1624.5 4 3 1 2 21.5 3.7

884.5 2 1770.5 4 3 2 2 20.3 8.9

957.6 2 1916.6 4 3 3 2 17.5 5.3

1005.1 2 2012.5 4 4 2 3 20.4 5.6

1078.3 2 2158.6 4 4 3 3 18.2 3.5

1078.3 2 2158.6 4 4 3 3 19.1 4.7

Weighted Average: 3.20 2.43 1.37 0.07 1.81

m/z - observed ion; x - ion charge state; Mass_neutrai - calculated neutral oligosaccharide mass; HexNAc - Λ/-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - N- acetylneuraminic acid (Sialic acid); Sulf - sulfate; Elution time - Graphitised carbon HPLC elution time; % abundance - percent abundance relative to detected oligosaccharides; Weighted Average - weighted average abundance of each monosaccharide over all oligosaccharides detected. Table 7

Oligosaccharide diversity and compositional analysis for the most intense

MUC5B-containing mucin band for exacerbated CF subject CYFB1-37.

CYFB1M37

Composition m/z X Mass _neutral - Elution Time % Abundance

HexNac Hex Fuc NeuAc Sulf

530.2 1 531.2 1 1 21.2 1.0

675.2 1 676.2 1 1 13.4 1.2

675.2 1 676.2 1 1 11.5 2.2

733.2 1 734.3 2 1 15.4 1.3

821.3 1 822.3 1 1 1 23.1 0.9

936.2 1 937.4 3 1 18.3 1.4

966.2 1 967.3 1 2 14.6 2.9

975.3 1 976.3 2 2 1 1 23.1 2.3

1040.5 1 1041.4 2 2 1 16.2 1.0

1040.5 1 1041.4 2 2 1 18.8 1.3

1041.4 1 1042.4 2 2 2 22.9 2.3

1081.3 1 1082.4 3 1 1 19.8 1.2

1121.3 1 1122.3 2 2 2 1 23.1 3.6

1178.2 1 1179.4 3 2 1 1 18.7 3.2

1186.4 1 1187.4 2 2 1 1 16.4 1.9

1186.4 1 1187.4 2 2 1 1 23.8 2.3

1187.3 1 1188.4 2 2 3 22.9 1.9

1244.4 1 1245.5 3 2 2 22.6 0.9

1244.4 1 1245.5 3 2 2 19.7 3.0

1266.1 1 1267.4 2 2 1 1 1 25.2 1.3

1266.1 1 1267.4 2 2 1 1 1 17.3 1.7

1267.3 1 1268.4 2 2 3 1 23.1 1.9

1324.3 1 1325.4 3 2 2 1 17.4 1.4

1324.3 1 1325.4 3 2 2 1 18.0 1.4

1331.3 1 1332.5 2 2 2 17.1 2.4

1331.3 1 1332.5 2 2 2 20.6 11.4

1332.4 1 1333.5 2 2 2 1 22.9 2.0

1359.2 2 1341.4 3 3 1 1 20.8 1.2

1389.4 1 1390.5 3 2 1 1 20.9 2.5

1390.3 1 1391.5 3 2 3 19.6 1.6

1470.3 1 1471.5 3 2 3 1 17.1 1.2

1477.4 1 1478.5 2 2 1 2 17.4 1.7

1477.4 1 1478.5 2 2 1 2 17.8 6.5

1535.7 1 1536.6 3 2 2 1 18.0 1.5

921.0 2 1843.7 3 3 1 2 20.5 0.9

921.0 2 1843.7 3 3 1 2 18.6 1.1

957.6 2 1916.6 4 3 3 2 18.2 1.0'

994.2 2 1989.7 3 3 2 2 18.1 1.3

1110.9 2 2224.8 4 5 2 18.0 0.9

1432.3 2 2866.0 5 5 3 2 21.0 2.0

Weighted Average: 2.32 2.00 1.14 0.95 0.26 Table 8

Weighted average oligosaccharide compositions for the most intense MUC5B- containing mucin band from sputum from 19 exacerbated CF and 19 non-CF healthy control subjects.

OH Exacerbated Non -cι- control

Monosaccharide omposition Monosaccnaπαe composition

Subject Subject

HexNAc Hex Fuc NeuAc Sulf HexNAc Hex FUC NeuAc Suit

4 2.63 2.31 1.30 0.62 0.85 t 4.63 2.24 2.16 0.00 0.01

6 3.11 1.95 1.08 1.08 0.05 8 3.21 2.68 1.32 0.27 1.47

10 2.42 2.31 0.96 0.85 0.09 9 2.57 2.01 1.21 0.16 0.76

11 2.52 2.08 0.80 1.15 0.04 13 3.38 2.94 1.99 0.09 1.78

15 2.33 2.29 0.67 1.58 0.36 17 2.06 1.75 1.24 0.61 0.58

16 2.93 2.29 0.74 1.26 0.05 18 2.77 2.43 1.06 0.59 0.13

20 2.14 2.02 0.42 1.83 0.00 19 3.16 2.58 1.76 0.19 1.63

35 2.93 2.40 1.19 1.00 0.43 21 3.24 2.90 1.70 0.14 1.82

37 2.32 2.00 1.14 0.95 0.26 22 3.20 2.43 1.37 0.07 1.81

38 2.75 2.58 0.70 1.74 0.18 24 3.36 3.03 1.71 0.20 1.66

39 2.76 2.17 0.78 1.46 0.14 25 2.68 2.22 1.49 0.17 1.13

40 2.37 1.96 0.82 1.02 0.29 26 3.64 2.35 1.74 0.00 1.32

41 2.84 1.72 0.86 0.76 0.05 27 2.48 2.09 1.26 0.23 1.05

43 2.62 2.05 0.83 1.11 0.20 28 3.05 2.96 1.78 0.04 1.38

44 2.34 1.82 0.89 0.52 0.20 29 2.33 2.11 0.84 1.06 0.60

45 2.44 2.03 1.01 0.98 0.07 30 2.53 2.20 1.20 0.83 0.90

46 2.46 ^• 1.99 1.07 0.93 0.15 32 3.07 2.71 1.70 0.29 1.65

47 2.85 1.86 1.22 0.51 0.48 34 3.20 2.81 1.81 0.26 1.64

48 2.25 2.03 0.63 1.71 0.06 36 2.86 2.39 1.52 0.72 0.79

Average 2.58 2.10 0.90 \M 0.21 Average 3.02 ^"2:46 1.52 0.31 1.16

Standard Error 0.27 0.22 0.23 0.40 0.21 Standard Error 0.56 0.37 0.34 0.30 0.57

HexNAc - Λ-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - N- acetylneuraminic acid (Sialic acid); Sulf - sulfate; Average - weighted average abundance of each monosaccharide over all oligosaccharides detected; Standard error - standard error of all data points.

Table 9

Fold differences between mucin oligosaccharide composition of sputum MUC5B- containing mucin fraction from exacerbated CF subjects and non-CF healthy controls. HexNAc Hex Fuc NeuAc Sulf Average ^* "147 1.17 1 W 3^57 ^: &sa

95% c.i. , 4_fϋ$ litt JΛA - 2.83 429

HexNAc - Λ/-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - N- acetylneuraminic acid (Sialic acid); Sulf - sulfate; Average - Fold difference in average values for each group. 95% c.i. - Fold differences at the 95% confidence interval between each group (all monosaccharides showed significant differences at 95% c.i.). Changes in levels of HexNAc, Hex, Fuc and Sulf were reductions in CF subjects compared to non-CF subjects. The change in NeuAc was an increase in CF subjects relative to non-CF subjects.

Table 10

Weighted average oligosaccharide compositions for MUC5B-containing mucin fraction of sputum from exacerbated and discharged CF subjects.

CF Exacerbated CF Discharged

Monosaccharide Composition Monosaccharide Composition

Subject Subject

HexNAc Hex Fuc NeuAc Sulf HexNAc Hex Fuc NeuAc Sulf

4 2.63 2.31 1.30 0.62 0.85

6 3.11 1.95 1.08 1.08 0.05

10 2.42 2.31 0.96 0.85 0.09

11 2.52 2.08 0.80 1.15 0.04 11B 2.86 2.81 1.46 0.57 0.00

12B 2.64 2.42 1.07 1.57 0.17

15 2.33 2.29 0.67 1.58 0.36

16 2.93 2.29 0.74 1.26 0.05 16B 2.75 2.₄0 1.14 1.30 0.05

20 2.14 2.02 0.42 1.83 0.00 20B 2.72 2.₄0 0.80 1.54 0.06

35 2.93 2.40 1.19 1.00 0.43

37 2.32 2.00 1.14 0.95 0.26 37B 2.15 2.12 0.66 1.62 0.15

38 2.75 2.58 0.70 1.74 0.18 38B 2.81 2.53 0.72 1.71 0.18

39 2.76 2.17 0.78 1.46 0.14 39B 2.81 2.33 0.90 1.43 0.09

40 2.37 1.96 0.82 1.02 0.29 40B 2.47 1.96 0.93 1.05 0.07

₄1 2.84 1.72 0.86 0.76 0.05 41 B 3.49 1.86 1.48 0.39 0.06

₄3 2.62 2.05 0.83 1.11 0.20 43B 2.36 1.90 0.62 1.24 0.11 ₄ 2.34 1.82 0.89 0.52 0.20 ₄B 2.47 1.84 1.10 0.66 0.00

45 2.44 2.03 1.01 0.98 0.07 45B 2.15 1.93 0.83 1.24 0.07

46 2.46 1.99 1.07 0.93 0.15 46B 2.62 2.01 1.22 0.87 0.16

₄7 2.85 1.86 1.22 0.51 0.48

₄8 2.25 2.03 0.63 1.71 0.06

Average 2.58 2.10 0.90 1.11 0.21 Average 2.64 2.19 1.00 1.17 0.09

Standard Error 0.27 0.22 0.23 0.40 0.21 Standard Error 0.35 0.31 0.28 0.43 0.06

HexNAc - Λ/-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - N- acetylneuraminic acid (Sialic acid); Sulf - sulfate; Average - weighted average abundance of each monosaccharide over all oligosaccharides detected; Standard error - standard error of all data points.

Table 11

Fold differences between mucin oligosaccharide composition of MUC5B- containing mucin fraction of sputum from CF subjects who showed large clinical improvements upon discharge from hospital compared to at exacerbation.

HexNAc Hex Fuc NeuAc Sulf

CF Exacerbated 2.68 1.90 0.83 0.96 0.05 CF Recovered 3.18 2.33 1.47 0.48 0.03

Fold Difference 1.18 1.23 1.77 i 1.64

HexNAc - Λ/-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - N- acetylneuraminic acid (Sialic acid); Sulf - sulfate.

Table 12

Fold differences between mucin oligosaccharide composition of MUC5B- containing mucin fraction of sputum from CF subject CYFB1-37 who showed a deterioration in clinical signs upon discharge from hospital compared to at exacerbation.

HexNAc Hex Fuc NeuAc Sulf

CYFB1-37 2.32 2.00 1.14 0.95 0.26 CYFB1-37B 2.15 2.12 0.66 1.62 0.15

Fold Difference W 1.06 1.71

HexNAc - /V-acetylhexosamine; Hex - Hexose; Fuc - Fucose; NeuAc - Λ7- acetylneuraminic acid (Sialic acid); Sulf - sulfate. Table 13

Summary of mucin protein and glycosylation profiles of MUC5B-containing mucin fraction of sputum from CF subjects at exacerbation and post-discharge from hospital, compared with changes in FE\ and sputum 2-dimensional electrophoresis (2-DE) proteome. Absolute and qualitative changes in fucosylation, sialylation, sulfation, and FE\ are shown.

t = increased = decrease. NC = No change. Discharge Δ glycosylation state is a qualitative summary of improvement (t) or deterioration (i) in lung state as judged by glycosylation changes, with improvement considered to be increased fucosylation, decreased sialylation and increased sulfation. Discharge sputum mucin and sputum 2- DE profile changes are relative to healthy control profiles. Example 4 Production of antibodies that detect N-terminal and/or C-terminal fragments of the MUC5B apoprotein It appears that MUC5B is cleaved in response to an acute clinical exacerbations. Accordingly, peptides spanning the N-terminal and/or C-terminal portions of this protein, including each of the tryptic peptide digests presented in SEQ ID Nos: 2- 19, are generated

Peptide antigens and peptide probes are synthesised essentially using the methods described in Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg and Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg.

Peptides are purified using HPLC and purity assessed by amino acid analysis.

Female BalB/c mice are immunised with a purified form of the peptide (SEQ ID NO: 4). Initially mice were sensitised by intraperitoneal injection of Hunter's Titermax adjuvant (CytRx Corp., Norcross, GA,). Three boosts of the peptide are administered at 2, 5.5 and 6.5 months post initial sensitisation. The first of these boosts is a subcutaneous injection while the remaining are administered by intraperitoneal injection. The final boost is administered 3 days prior to fusion.

The splenocytes of one of the immunised BALB/c mice is fused to X63-Ag8.653 mouse myeloma cells using PEG 1500. Following exposure to the PEG 1500 cells are incubated at 37°C for 1 hour in heat inactivated foetal bovine serum. Fused cells are then transferred to RPMI 1640 medium and incubated overnight at 37°C with 10% C0₂. The following day cells are plated using RPMI 1640 media that has been supplemented with macrophage culture supematants.

Two weeks after fusion, hybridoma cells are screened for antibody production by solid phase ELISA assay. Standard microtitre plates are coated with isolated peptides in a carbonate based buffer. Plates are then blacked with BSA, washed and then the test samples (ie supernatant from the fused cells) was added, in addition to control samples, (ie supernatant from an unfused cell). Antigen- antibody binding is detected by incubating the plates with goat-anti-mouse HRP conjugate (Jackson ImmunoResearch Laboratories) and then using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance was read on an automatic plate reader at a wavelength of 405 nm.

Any colonies that are identified as positive by this screen are continued to be grown and screened for several further weeks. Stable colonies are then isolated and stored at -80°C.

Positive stable hybridomas are then cloned by growing in culture for a short period of time and diluting the cells to a final concentration of 0.1 cells/well of a 96 well tissue culture plate. These clones are then screened using the previously described assay. This procedure is then repeated in order to ensure the purity of the clone.

Four different dilutions, 5 cells/well, 2 cells/well, 1 cell/well, 0.5 cells/well of the primary clone are prepared in 96-wells microtiter plates to start the secondary cloning. Cells are diluted in IMDM tissue culture media containing the following additives: 20% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 1% GMS-S, 0.075% NaHC0₃. To determine clones that secrete anti-MUC5B antibody, supernatants from individual wells of the 0.2 cells/well microtiter plate are withdrawn after two weeks of growth and tested for the presence of anti-MUC5B antibody by ELISA assay as described above.

All clones are then adapted and expanded in RPMI media containing the following additives: 10% FBS, 2 mM L-glutamine, 100 units/ml of penicillin, 100

.μg/ml of streptomycin, 1% GMS-S, 0.075% NaHC0₃, and 0.013 mg/ml of oxalaacetic acid. A specific antibody is purified by Protein A affinity chromatography from the supernatant of cell culture.

Example 5 Assessment of specificity of anti-MUC5B antibodies

Purified peptides are combined with P. aeruginosa (Calbiochem, San Diego, Ca 92121, USA) and incubated at 37°C. Following incubation samples are ethanol precipitated, supernatant fluid removed and the samples freeze-dried. Samples are then reconstituted and adsorbed onto a microtitre plate. Purified MUC5B peptides are adsorbed onto another microtitre plate.

Cell culture supernatant from the hybridomas generated in Example 8 are then screened to determine those that produce an antibody that is able to specifically recognise the uncleaved form of MUC5B.

Microtitre plates are blocked with BSA, washed and then the test samples (ie supernatant from a hybridoma) is added, in addition to control samples, (ie supernatant from an unfused cell). Antigen-antibody binding is detected by incubating the plates with goat-anti-mouse HRP conjugate (Jackson ImmunoResearch Laboratories) and then using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance was read on an automatic plate reader at a wavelength of 405 nm.

Example 6 Development of an assay to diagnose or prognose an acute clinical exacerbation in a CF patient.

The antibody of Example 5 is adsorbed to a microtitre plate at a concentration appropriate for detecting the presence of MUC5B. Samples are adsorbed for 1 hour at room temperature or overnight at 4°C. Plates are then washed with Tris buffered saline. Plates are blocked with BSA in TBS and then washed with TBS. A positive control, isolated MUC5B from a non-CF subject, is included in that assay at various concentrations. Additionally one or more biological samples, ie sputum samples, isolated from a normal healthy individual is also included, in order to determine the amount of uncleaved MUC5B that is observed in a control individual. These samples are added to a well of the microtitre plate.

A biological sample isolated from one or more CF patients is added to the microtitre plate. It is preferable that multiple concentrations of this sample is added to the plate.

Samples are then incubated for approximately 1 hour before being washed with TBS containing 0.01% Tween 20 (Sigma Aldrich). This wash step is repeated twice more.

A dilution (in TBS) of a secondary, goat-anti-human HRP conjugated antibody (Jackson ImmunoResearch Laboratories) is then added to each of the wells of the microtitre plate and incubated for approximately 1 hour. Samples are again washed three times with TBS and Tween, before binding of the secondary antibody is measured using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance is read on an automatic plate reader at a wavelength of 405 nm.

The absorbance detected in the control (normal healthy individual) sample/s are then compared to the CF subject test sample of known concentration in order to determine the approximate concentration of uncleaved MUC5B in these samples. The absorbance detected in the test sample is also compared to this standard in order to determine the amount of protein in this sample.

These results are then used to determine the clinical status of the CF subject. If there is less signal generated using antibodies that bind specifically to the N- terminal and/or C-terminal portion of MUC5B for the CF subject than for the positive control (ie., a non-CF healthy individual), and therefore less uncleaved MUC5B in the sample, the CF subject is suffering from, or will soon enter an acute pulmonary exacerbation.

Claims

WE CLAIM:

1. A method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising detecting in a biological sample from said subject a modified MUC5B apoprotein that does not comprise an amino acid sequence selected from the group consisting of:

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

NO: 1.

2. The method of claim 1 wherein the modified MUC5B apoprotein has an amino acid sequence consisting essentially of residues from about 2345 to about 4922 of SEQ ID NO: 1.

3. The method of claim 1 wherein the modified MUC5B apoprotein has an amino acid sequence consisting of residues from about 2345 to about 4922 of SEQ ID NO: 1.

4. The method according to any one of claims 1 to 3 wherein the modified MUC5B apoprotein is detected by a process comprising:

5. The method of claim 4 wherein the complex formed at (i) is absent.

6. The method according to any one of claims 1 to 5 wherein the biological sample comprises sputum or saliva.

7. A method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction an elevated level of an oligosaccharide relative to the level of the oligosaccharide in a healthy control subject, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂FucιNeuAcι; HexNac₃Hexι; HexNac₃Hex₃Fuc₂NeuAcι; HexNac₃Hex₃FucιNeuAc₂Sulfι; HexNac₃Hex₃Fuc₂NeuAc₂; HexNac₃Hex₂Fuc₂; HexNac₃Hex₂Fuc₂Sulf₁; HexNac₂Hex₂FucιNeuAcιSulf-ι; HexNac₂Hex₂FucιNeuAc₂; and HexNac₃Hex₃Fucι NeuAci .

8. The method according to claim 7 wherein the ioligosaccharide comprises a composition selected from the group consisting of: HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂FucιNeuAcι; HexNac₃Hex3FucιNeuAc₂Sulfι; HexNac₃Hex₃Fuc₂NeuAc₂; HexNac₃Hex₂Fuc₂Sul ι; and HexNac2Hex₂FucιNeuAcιSulfι.

9. The method according to claim 8 wherein the oligosaccharide comprises a composition selected from the group consisting of: HexNac₃Hex₃Fuc₂NeuAc2;

HexNac₂Hex₂NeuAcι; and HexNac₂Hex₂FucιNeuAc₂

10. The method of claim 9 wherein oligosaccharide comprises the composition HexNac₂Hex₂Fucι NeuAc₂.

11. The method according to any one of claims 7 to 10 wherein the elevated level of the oligosaccharide is determined by reference to an internal control oligosaccharide comprising a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; HexNac₂Hex₂FucιNeuAcι; and HexNac₂Hex₂NeuAc₂.

12. The method according to claim 11 wherein the internal control oligosaccharide comprises a composition selected from the group consisting of

HexNac₂Hex₂NeuAcι; and HexNac₂Hex₂NeuAc .

13. The method according to claim 12 wherein the internal control oligosaccharide comprises HexNac₂Hex₂NeuAc₂.

14. The method according to any one of claims 11 to 13 comprising determining the ratio of the level of an oligosaccharide comprising the composition HexNac2Hex₂Fuc₁NeuAc2 to the level of an oligosaccharide comprising the composition HexNac₂Hex₂NeuAc and wherein a higher level of this ratio in the subject compared to the healthy control indicates that the subject is suffering from an inflammatory condition or infection of the respiratory tract.

15. A method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction a reduced level of an oligosaccharide relative to the level of the oligosaccharide in a healthy control subject, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₃Hex2FucιSulfι; HexNac₃Hex2Fuc2Sulf2; HexNac₃Hex₃FucιSulf₂; HexNac₃Hex2Fuc₂Sulf2; HexNac₄Hex3FucιSulf2; HexNac₂HexιNeuAcι; HexNac Hex3Fuc2Sulf₂; HexNac₄Hex₃Fuc3Sulf2;

HexNac Hex₄Fuc₂Sulf₂; HexNac₃Hex₂Sulfι; HexNac4Hex4Fuc3Sulf₂;

HexNac₂Hex₂NeuAcιSulfι; HexNac₃Hex₂FucιSulfι; and HexNac₂Hex₂Fucι NeuAciSulfi .

16. The method according to claim 15 wherein the oligosaccharide comprises a composition selected from the group consisting of HexNac3Hex₂Sulfι; and HexNac₂Hex₂Fucι NeuAci Sulf ₁.

17. A method of diagnosing an inflammatory condition or infection of the respiratory tract in a subject comprising obtaining a MUC5B-containing fraction of sputum from said subject and detecting in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin in a healthy control subject, wherein the modified glycosylation is selected from the group consisting of: (i) a decrease in sulfation of the mucin;

(iii) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin and a decrease in fucosylation of the mucin; (v) a decrease in the ratio sulfate: sialic acid in the mucin; and (vi) an increase in the ratio sialic acid: sulfate in the mucin.

18. The method according to claim 17 wherein the mucin is MUC5B.

19. The method of claim 17 or 18 wherein a decrease in sulfation of the mucin and a decrease in fucosylation of the mucin are determined.

20. The method of claim 17 or 18 wherein a decrease in sulfation of the mucin is determined.

21. The method of claim 17 or 18 wherein a decrease in sulfation of the mucin and an increase in sialylation of the mucin are determined.

22. The method of claim 17 or 18 wherein a decrease in sulfation of the mucin and an increase in sialylation of the mucin and a decrease in fucosylation of the mucin are determined.

23. The method of claim 17 or 18 wherein a decrease in the ratio sulfate: sialic acid in the mucin is determined.

24. The method of claim 17 or 18 wherein an increase in the ratio sialic acid: sulfate in the mucin is determined.

25. The method according to any one of claims 17 to 24 wherein the glycosylation is determined by a process comprising performing mass spectrometry on the MUC5B-containing fraction.

26. The method according to any one of claims 17 to 24 wherein the glycosylation is determined by a process comprising contacting an antibody or lectin with the MUC5B-containing fraction for a time and under conditions sufficient for the antibody or lectin to bind to a sugar residue in the mucin.

27. The method according to any one of claims 17 to 24 wherein the glycosylation consists of sulfation and wherein the sulfation is determined by a process comprising staining the MUC5B-containing fraction with sulfuric acid Alcian Blue (sAB).

28. The method of claim 27 further comprising staining the MUC5B-containing fraction with Periodic Acid-Schiff s reagent (PAS).

29. The method of claim 27 or 28 further comprising staining the MUC5B- containing fraction with acetic acid Alcian Blue (aAB).

30. The method according to any one of claims 1 to 29 wherein the subject suffers from cystic fibrosis (CF).

31. The method according to claim 30 wherein the inflammatory condition or infection consists of an acute pulmonary exacerbation in the subject.

32. A method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising performing the method according to any one of claims 1 to 31 at different time points on a subject and comparing the results obtained.

5

33. The method of claim 32 wherein a time point against which the comparison is made is the time of diagnosis of the inflammatory condition or infection and another time point is after said diagnosis.

10 34. The method according to claim 32 or 33 wherein the comparison indicates that the subject has not improved or recovered from the inflammatory condition or infection or has deteriorated, as determined by there being no change in the amount of the MUC5B apoprotein, oligosaccharide or modified glycosylation of a mucin at the different time points.

15

35. A method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising detecting in a biological sample from said subject an enhanced or reduced level of a MUC5B apoprotein relative to the level of said MUC5B apoprotein in the subject at

20 diagnosis of the inflammatory condition or infection, said MUC5B apoprotein comprising an amino acid sequence selected from the group consisting of: (i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and (ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

25 NO: 1.

36. The method according to claim 35 wherein the MUC5B apoprotein consists of the amino acid sequence set forth in SEQ ID NO: 1.

30 37. The method according to claims 35 or 36 wherein the MUC5B apoprotein is detected by a process comprising contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of SEQ ID NO: 1 and wherein the presence of the complex indicates the presence of the MUC5B apoprotein.

38. The method of claim 37 wherein the MUC5B apoprotein was absent or present at a reduced level in the sample at diagnosis of the inflammation or infection in the subject.

39. A method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising detecting in a biological sample from said subject an enhanced or reduced level of a modified MUC5B apoprotein relative to the level of said modified MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said modified MUC5B apoprotein has a sequence that does not comprise an amino acid sequence selected from the group consisting of:

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and (ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

NO: 1.

40. The method according to claim 39 wherein the modified MUC5B apoprotein has a sequence an amino acid sequence consisting of residues from about 2345 to about 4922 of SEQ ID NO: 1.

41. The method of claim 39 or 40 wherein the modified MUC5B apoprotein is detected by a process comprising:

SEQ ID NO: 1 ; (ii) contacting a reference sample from the subject obtained at diagnosis with the antibody for a time and under conditions sufficient to form an antigen- 5 antibody complex and detecting the complex formed; and

10 42. The method according to any one of claims 35 to 41 wherein the biological sample comprises sputum or saliva.

43. The method according to any one of claims 35 to 42 wherein the relative proportion of the modified MUC5B apoprotein and native MUC5B apoprotein is

15 determined.

44. A method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the

20 level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex2NeuAc2; HexNac2Hex₂Fucι NeuAci ; HexNac₃Hexι; HexNac₃Hex₃Fuc₂NeuAcι; HexNac₃Hex₃FucιNeuAc₂Sulfι;

25 HexNac₃Hex3Fuc2NeuAc2; HexNacsHex∑Fuc∑; HexNac₃Hex₂Fuc₂Sulfι; HexNac₂Hex₂FucιNeuAcιSulfι; HexNac₂Hex₂FucιNeuAc₂; and HexNac₃Hex₃FucιNeuAcι and wherein a similar or elevated level of the ion relative to the level at diagnosis indicates that the subject has not recovered and a reduced level of the ion relative to the level at diagnosis indicates that the

30 subject has recovered.

45. The method of claim 44 wherein the oligosaccharide comprises a composition selected from the group consisting of:

HexNac₂Hex₂NeuAc₂; HexNac₂Hex Fucι NeuAci ; HexNac₃Hex₃Fucι NeuAc₂Sulf| ; HexNac₃Hex₃Fuc₂NeuAc₂; HexNac₃Hex₂Fuc₂Sulfι; and

5 HexNac₂Hex₂FucιNeuAcι.

46. The method according to claim 45 wherein wherein oligosaccharide comprises the composition HexNac Hex2FucιNeuAc2.

10 47. The method according to any one of claims 44 to 46 wherein the level of the oligosaccharide is determined by reference to an internal control oligosaccharide comprising a composition selected from the group consisting of HexNac₂Hex₂NeuAc₁; HexNac₂Hex₂FucιNeuAcι; and HexNac₂Hex₂NeuAc₂.

15 48. The method according to claim 47 wherein the internal control oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex2NeuAcι; and HexNac₂Hex₂NeuAc2.

49. The method according to claim 48, wherein the internal control 20 oligosaccharide comprises HexNac₂Hex₂NeuAc2.

50. The method according to any one of claims 44 to 49 determining the ratio of the level of an oligosaccharide comprising the composition HexNac₂Hex₂FucιNeuAc₂ to the level of an oligosaccharide comprising the

25 composition HexNac₂Hex₂NeuAc, wherein a similar or higher ratio for the test sample relative to the ratio for the subject at diagnosis indicates that the subject has not recovered and a reduced ratio indicates that the subject has recovered.

51. A method for determining the progression of an inflammatory condition or 30 infection of the respiratory tract in a subject, said method comprising obtaining a

MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac3Hex₂FucιSulf₁; HexNac₃Hex₂Fuc₂Sulf₂; HexNac₃Hex₃FuciSulf2; HexNac₃Hex2Fuc2Sulf₂; HexNac₄Hex₃FucιSulf₂; HexNac₂HexιNeuAcι; HexNac₄Hex₃Fuc2Sulf₂; HexNac₄Hex₃Fuc₃Sulf₂; HexNac4Hex4Fuc₂Sulf2; HexNac₃Hex₂Sulf₁;

HexNac₄Hex₄Fuc₃Sulf₂; HexNac₂Hex₂NeuAcιSulfι; HexNac₃Hex₂FucιSulfι; and HexNac₂Hex₂FucιNeuAcιSulfι and wherein a reduced or similar level of the ion relative to the level at diagnosis indicates that the subject has not recovered and an elevated or enhanced level of the ion relative to the level at diagnosis indicates that the subject has recovered.

52. The method of claim 51 wherein the oligosaccharide comprises a composition selected from the group consisting of HexNac3Hex₂Sulf-ι; and HexNac2Hex₂Fucι NeuAci Sulfi .

53. The method of claim 51 or 52 wherein the level of the oligosaccharide is determined by reference to an internal control comprising a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; HexNac₂Hex₂Fuc-|NeuAci; and HexNac₂Hex₂NeuAc₂.

54. The method according to claim 53 wherein the internal control oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; and HexNac₂Hex₂NeuAc₂.

55. The method according to claim 54, , wherein the internal control oligosaccharide comprises HexNac₂Hex₂NeuAc₂.

56. A method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of:

57. The method according to claim 56 wherein the mucin is MUC5B.

58. The method of claim 56 or 57 wherein a decrease in sulfation of the mucin and an increase in fucosylation of the mucin are determined.

59. The method of claim 56 or 57 wherein a decrease in sulfation of the mucin is determined.

60. The method of claim 56 or 57 wherein a decrease in sulfation of the mucin and a decrease in sialylation of the mucin are determined.

61. The method of claim 56 or 57 wherein a decrease in sulfation of the mucin and a decrease in sialylation of the mucin and an increase in fucosylation of the mucin are determined.

62. The method of claim 56 or 57 wherein a decrease in the ratio sulfate: fucose in the mucin is determined.

63. The method of claim 56 or 57 wherein an increase in the ratio fucose: sulfate in the mucin is determined.

64. A method for determining the progression of an inflammatory condition or infection of the respiratory tract in a subject, said method comprising obtaining a

MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of:

65. The method according to claim 64 wherein the mucin is MUC5B.

66. The method of claim 64 or 65 wherein a decrease in sulfation of the mucin and a decrease in fucosylation of the mucin are determined.

67. The method of claim 64 or 65 wherein a decrease in sulfation of the mucin is determined.

68. The method of claim 64 or 65 wherein a decrease in sulfation of the mucin and an increase in sialylation of the mucin are determined.

69. The method of claim 64 or 65 wherein a decrease in sulfation of the mucin and an increase in sialylation of the mucin and a decrease in fucosylation of the mucin are determined.

70. The method of claim 64 or 65 wherein a decrease in the ratio sulfate: sialic acid in the mucin is determined.

71. The method of claim 64 or 65 wherein an increase in the ratio sialic acid: sulfate in the mucin is determined.

72. The method according to any one of claims 56 to 71 wherein the glycosylation is determined by a process comprising performing mass spectrometry on the MUC5B-containing fraction.

73. The method according to any one of claims 56 to 71 wherein the glycosylation is determined by a process comprising contacting an antibody or lectin with the MUC5B-containing fraction for a time and under conditions sufficient for the antibody or lectin to bind to a sugar residue in the mucin.

74. The method according to any one of claims 56 to 71 wherein the glycosylation consists of sulfation and wherein the sulfation is determined by a process comprising staining the MUC5B-containing fraction with sulfuric acid Alcian Blue (sAB).

75. The method of claim 74 further comprising staining the MUC5B-containing fraction with Periodic Acid-Schiff s reagent (PAS).

76. The method of claim 74 or 75 further comprising staining the MUC5B- containing fraction with acetic acid Alcian Blue (aAB).

77. The method according to any one of claims 35 to 76 wherein the subject suffers from cystic fibrosis (CF).

78. The method according to claim 77 wherein the inflammatory condition or infection consists of an acute pulmonary exacerbation in the subject.

79. A method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting in a biological sample from said subject an amount of a modified MUC5B apoprotein following treatment and comparing the amount of the modified MUC5B apoprotein to the amount of said modified MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said modified MUC5B apoprotein has a sequence that does not comprise an amino acid sequence selected from the group consisting of:

(i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

80. The method according to claim 79 wherein the modified MUC5B apoprotein has an amino acid sequence consisting of residues from about 2345 to about 4922 of SEQ ID NO: 1 and wherein an amount of the modified MUC5B apoprotein following treatment that is not reduced compared to the amount at diagnosis indicates that the subject has not responded to treatment.

81. The method of claim 79 or 80 wherein the modified MUC5B apoprotein is detected by a process comprising: (i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of

SEQ ID NO: 1 ;

82. The method according to any one of claims 79 to 81 wherein the biological sample comprises sputum or saliva.

83. A method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting in a biological sample from said subject an amount of a MUC5B apoprotein following treatment and comparing the amount of the MUC5B apoprotein to the amount of said MUC5B apoprotein in the subject at diagnosis of the inflammatory condition or infection, wherein said MUC5B apoprotein comprises an amino acid sequence selected from the group consisting of: (i) a sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 ; and

(ii) a sequence contained within residues from about 4923 to 5703 of SEQ ID

84. The method of claim 83 wherein the MUC5B apoprotein consists of the amino acid set forth in SEQ ID NO: 1 and wherein an amount of the MUC5B apoprotein following treatment that is enhanced following treatment compared to the amount at diagnosis indicates that the subject has responded to treatment.

85. The method of claim 83 or 84 wherein the MUC5B apoprotein is detected by a process comprising:

(i) contacting the biological sample from the subject with an antibody for a time and under conditions sufficient to form an antigen-antibody complex and detecting the complex formed, wherein the antibody binds specifically to an amino acid sequence contained within residues 1 to about 2344 of SEQ ID NO: 1 or contained within residues from about 4923 to 5703 of SEQ ID NO: 1 ;

(iii) comparing the amount of complex formed at (i) and (ii).

86. The method according to any one of claims 83 to 85 wherein the biological sample comprises sputum or saliva.

87. A method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂FucιNeuAcι; HexNac3Hexι; HexNac₃Hex₃Fuc₂NeuAc-ι; HexNac₃Hex₃Fuc₁NeuAc₂Su.fi;

HexNac₃Hex₃Fuc₂NeuAc₂; HexNac3Hex₂Fuc2; HexNac3Hex2Fuc2Su.fi; HexNac₂Hex₂Fuc1NeuAc₁Su.f1; HexNac₂Hex2FucιNeuAc2; and HexNac₃Hex₃FucιNeuAcι and wherein a similar or elevated level of the ion relative to the level at diagnosis indicates that the subject has not responded to treatment and a reduced level of the ion relative to the level at diagnosis indicates that the subject has responded to treatment.

88. The method of claim 87 wherein the oligosaccharide comprises a composition selected from the group consisting of:

HexNac₂Hex₂NeuAc₂; HexNac₂Hex₂Fucι NeuAci ; HexNac3Hex3Fucι NeuAc₂Sulfι ; HexNac3Hex₃Fuc₂NeuAc2; HexNac₃Hex2Fuc₂Sulfι; and HexNac₂Hex₂FucιNeuAcιSulfι.

89. The method according to claim 88 wherein wherein oligosaccharide comprises the composition HexNac2Hex₂FucιNeuAc₂.

90. The method according to any one of claims 87 to 89 wherein the elevated level of the oligosaccharide is determined by reference to an internal control oligosaccharide comprising a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; HexNac₂Hex2FucιNeuAcι; and HexNac2Hex2NeuAc₂.

91. The method according to claim 90 wherein the internal control oligosaccharide comprises a composition selected from the group consisting of HexNac2Hex₂NeuAcι; and HexNac₂Hex₂NeuAc2.

92. The method according to claim 91 , wherein the internal control oligosaccharide comprises HexNac₂Hex2NeuAc2.

93. The method according to any one of claims 87 to 92 comprising determining the ratio of the level of an oligosaccharide comprising the composition HexNac₂Hex₂FucιNeuAc₂ to the level of an oligosaccharide comprising the composition HexNac₂Hex₂NeuAc, wherein a similar or higher ratio for the test sample relative to the ratio for the subject at diagnosis indicates that the subject has not responded to treatment and a reduced ratio indicates that the subject has responded to treatment.

94. A method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining the level of an oligosaccharide relative to the level of the oligosaccharide at diagnosis of the inflammatory condition or infection, wherein said oligosaccharide comprises a composition selected from the group consisting of HexNac₃Hex₂FucιSulfι; HexNac3Hex₂Fuc2Sulf2; HexNac3Hex₃FucιSulf2; HexNac₃Hex2Fuc2Sulf₂; HexNac₄Hex₃FuciSulf2; HexNac₂HexιNeuAcι; HexNac₄Hex₃Fuc₂Sulf₂; HexNac₄Hex₃Fuc₃Sulf₂;

HexNac Hex₄Fuc₂Sulf₂; HexNac₃Hex₂Sulfι; HexNac4Hex4Fuc₃Sulf₂; HexNac₂Hex₂NeuAcιSulfι; HexNac₃Hex₂FucιSulfι; and HexNac₂Hex₂FucιNeuAcιSulfι. and wherein a reduced or similar level of the oligosaccharide relative to the level at diagnosis indicates that the subject has not responded to treatment and an elevated or enhanced level of the oligosaccharide relative to the level at diagnosis indicates that the subject has responded to treatment.

95. The method of claim 94 wherein the oligosaccharide comprises a composition selected from the group consisting of HexNac3Hex₂Sulfι; and HexNac₂Hex₂FucιNeuAcιSulfι.

96. The method according to claim 94 or 95 wherein the elevated level of the oligosaccharide is determined by reference to an internal control oligosaccharide comprising a composition selected from the group consisting of HexNac₂Hex NeuAcι; HexNac₂Hex₂FucιNeuAc₁; and HexNac₂Hex NeuAc2.

97. The method according to claim 96 wherein the internal control oligosaccharide comprises a composition selected from the group consisting of HexNac₂Hex₂NeuAcι; and HexNac₂Hex NeuAc2.

98. The method according to claim 97, wherein the internal control oligosaccharide comprises HexNac₂Hex₂NeuAc₂.

99. A method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of: (i) a decrease in sulfation of the mucin coupled with an increase in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin;

(iv) a decrease in sulfation of the mucin coupled with a decrease in sialylation of the mucin and an increase in fucosylation of the mucin; (v) a decrease in the ratio sulfate: fucose in the mucin; and (vi) an increase in the ratio fucose: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has responded to treatment.

100. The method according to claim 99 wherein the mucin is MUC5B.

101. The method of claim 99 or 100 wherein a decrease in sulfation of the mucin and an increase in fucosylation of the mucin are determined.

102. The method of claim 99 or 100 wherein a decrease in sulfation of the mucin is determined.

103. The method of claim 99 or 100 wherein a decrease in sulfation of the mucin and a decrease in sialylation of the mucin are determined.

104. The method of claim 99 or 100 wherein a decrease in sulfation of the mucin and a decrease in sialylation of the mucin and an increase in fucosylation of the mucin are determined.

105. The method of claim 99 or 100 wherein a decrease in the ratio sulfate: fucose in the mucin is determined.

106. The method of claim 99 or 100 wherein an increase in the ratio fucose: sulfate in the mucin is determined.

107. A method for determining the response of a subject having an inflammatory condition or infection of the respiratory tract to treatment with a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising obtaining a MUC5B-containing fraction of sputum from said subject and determining in the fraction a modified glycosylation of a mucin selected from the group consisting of MUC2, MUC5B and MUC5AC relative to the glycosylation of the mucin at diagnosis of the inflammatory condition or infection, wherein the modified glycosylation is selected from the group consisting of:

(i) a decrease in sulfation of the mucin coupled with a decrease in fucosylation of the mucin; (iii) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin; (iv) a decrease in sulfation of the mucin coupled with an increase in sialylation of the mucin and a decrease in fucosylation of the mucin; (v) a decrease in the ratio sulfate: sialic acid in the mucin; and (vi) an increase in the ratio sialic acid: sulfate in the mucin; and wherein said modified glycosylation indicates that the subject has not responded to treatment.

108. The method according to claim 107 wherein the mucin is MUC5B.

109. The method of claim 107 or 108 wherein a decrease in sulfation of the mucin and a decrease in fucosylation of the mucin are determined.

110. The method of claim 107 or 108 wherein a decrease in sulfation of the mucin is determined.

111. The method of claim 107 or 108 wherein a decrease in sulfation of the mucin and an increase in sialylation of the mucin are determined.

112. The method of claim 107 or 108 wherein a decrease in sulfation of the mucin and an increase in sialylation of the mucin and a decrease in fucosylation of the mucin are determined.

113. The method of claim 107 or 108 wherein a decrease in the ratio sulfate: sialic acid in the mucin is determined.

114. The method of claim 107 or 108 wherein an increase in the ratio sialic acid: sulfate in the mucin is determined.

115. The method according to any one of claims 99 to 114 wherein the glycosylation is determined by a process comprising performing mass spectrometry on the MUC5B-containing fraction.

116. The method according to any one of claims 99 to 114 wherein the glycosylation is determined by a process comprising contacting an antibody or lectin with the MUC5B-containing fraction for a time and under conditions sufficient for the antibody or lectin to bind to a sugar residue in the mucin.

117. The method according to any one of claims 96 to 114 wherein the glycosylation consists of sulfation and wherein the sulfation is determined by a process comprising staining the MUC5B-containing fraction with sulfuric acid Alcian Blue (sAB).

118. The method of claim 117 further comprising staining the MUC5B- containing fraction with Periodic Acid-Schiff s reagent (PAS).

119. The method of claim 117 or 118 further comprising staining the MUC5B- containing fraction with acetic acid Alcian Blue (aAB).

120. The method according to any one of claims 77 to 119 wherein the subject suffers from cystic fibrosis (CF).

121. The method according to claim 120 wherein the inflammatory condition or infection consists of an acute pulmonary exacerbation in the subject.

122. A method of treatment of a subject suffering from an inflammatory condition or infection of the respiratory tract comprising performing the method according to any one of claims 1 to 121.

123. An isolated synthetic or recombinant peptide consisting essentially of residues 1 to about 2344 of SEQ ID NO: 1 or residues from about 4922 to about 5703 of SEQ ID NO: 1.

124. An isolated synthetic or recombinant peptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID Nos: 2-17.

125. Use of the isolated synthetic or recombinant peptide of claim 123 or 124 in the preparation of a reagent for detecting a MUC5B apoprotein or a modified MUC5B apoprotein in a biological sample from a human subject.

126. The use of claim 125 wherein the subject suffers from cystic fibrosis.

127. The use of claim 125 or 126 wherein the subject suffers from an inflammation of the respiratory tract or a respiratory infection.

128. The use of claim 127 wherein the subject suffers from an acute pulmonary infection.

129. An isolated antibody that binds specifically to the peptide according to any one of claims 123 or 124.

130. A method for diagnosing cystic fibrosis (CF) or a past or present acute pulmonary exacerbation in a CF subject said method comprising determining a reduced sulfation of mucins in a MUC5B-containing fraction of sputum from the subject relative to the level of sulfation in a MUC5B-containing fraction of sputum from a non-CF subject.

131. The method according to claim 130 wherein the sulfation is determined by a process comprising performing mass spectrometry on a MUC5B-containing fraction.

132. The method according to claim 130 or 131 wherein the glycosylation is determined by a process comprising contacting an antibody or lectin with the MUC5B-containing fraction for a time and under conditions sufficient for the antibody or lectin to bind to a sulfate residue in the mucin.

133. The method according to any one of claims 130 to 132 wherein the sulfation is determined by a process comprising staining the MUC5B-containing fraction with sulfuric acid Alcian Blue (sAB).

134. The method of claim 133 further comprising staining the MUC5B- containing fraction with Periodic Acid-Schiff s reagent (PAS).

135. The method of claim 133 or 134 further comprising staining the MUC5B- containing fraction with acetic acid Alcian Blue (aAB).