NZ517623A - Diagnostic assays of secreted biological fluids for detection of infection and inflammatory conditions - Google Patents

Abstract

A method for detecting an inflammatory response in a breast of a lactating mammal comprises measuring the presence or amount of a Serum Amyloid A (SAA) protein or mRNA encoding the protein in a sample of milk obtained from the breast. The amount of the SAA protein or mRNA present in the sample being positively correlated with the inflammatory response. Preferably the inflammatory response is mastitis. Also described are methods to assess the ability of a treatment or agent to increase or decrease inflammation in breast tissue, to evaluate quality of a milk sample and to determine the presence or amount of colostrums in a milk sample

Description

New Zealand Paient Spedficaiion for Paient Number 517623 DIAGNOSTIC ASSAYS OF SECRETED BIOLOGICAL FLUIDS FOR DETECTION OF INFECTION AND INFLAMMATORY CONDITIONS FIELD OF THE INVENTION The present invention relates generally to the field of diagnostics. In particular, the invention provides a method for diagnosing infection and other 5 inflammatory conditions by measuring the presence of Serum Amyloid A in a secreted biological fluid. The invention further provides a test kit for use in the method.

BACKGROUND OF THE INVENTION Several scientific or patent publications are referenced in this patent application to describe the state of the art to which the invention pertains. Each of these publications is incorporated by reference 15 herein, in its entirety.

Mammals respond to tissue injury, trauma or infection by executing a complex series of biological reactions in an effort to prevent further tissue damage, to initiate repair of damaged tissue, and to isolate and 20 destroy infective organisms. This process is referred to as the inflammatory response, the early and intermediate stages of which are referred to as the acute phase response.

The acute phase response involves a wide 25 variety of mediators, including cytokines, interleukins and tumor necrosis factor. It also involves a radical alteration in the biosynthetic profile of the liver.

Under normal circumstances, the liver synthesizes a range of plasma proteins at steady state concentrations. Some 3 0 of these proteins, the "acute phase" proteins are induced WO 01/14580 PCT/US99/19418 in the inflammatory response to a level many times greater than levels found under normal conditions. Acute phase proteins are reviewed by Steel & Whitehead (Immunology Today 15: 81-87, 1994).

One of the massively induced acute phase proteins is Serum Amyloid A (SAA). SAA actually comprises a family of polymorphic proteins encoded by many genes in a number of mammalian species. SAAs are small apolipoproteins that accumulate and associate 10 rapidly with high-density lipoprotein 3 (HDL3) during the acute phase of the inflammatory response. Most SAAs are induced in response to inflammation; however, certain SAAs (e.g., human SAA4) appear to be constitutively expressed or minimally induced in the inflammatory 15 response.

SAAs are regulated transcriptionally and post transcriptionally, though transcriptional regulation appears to predominate. SAA mRNA levels have been observed to increase up to 1,000 fold in the hours 20 following an inflammatory stimulus. Likewise, plasma concentrations of SAA protein have been shown to increase as much as 1,000 fold, to levels approaching 1 mg/ml, for short periods following an inflammatory stimulus.

The massive increase in SAA plasma levels in 25 response to both infective and non-infective inflammatory stimuli has led to its use as a diagnostic marker of inflammation. Among the most effective assays are immunoassays utilizing antibodies raised in a species that does not produce detectable amounts of SAA. For 30 instance, McDonald et al. (J. Immunol. Meth. 144: 149- 155, 1991) describe an antibody sandwich assay using two purified rat monoclonal antibodies raised against human SAA. Immunoassays utilizing these antibodies were demonstrated to be reliable and sensitive, and do not 35 require denaturation of the specimen prior to assay (McDonald et al., 1991, supra) . Similarly, Satoh et al.

WO 01/14580 PCT/US99/19418 (Am J. Vet. Res. 56: 1286-1291, 1995) describe an ELISA assay for measuring SAA levels in horse serum using rabbit anti-horse SAA antibodies. Though effective, these and similar immunoassays are invasive in that they 5 require a blood sample. Moreover, they may not be appropriate or effective for early detection of localized inflammation, which is common in connection with a variety of infectious and non-infectious tissue trauma.

One excellent example of a non-systemic 10 inflammatory-related disease of great economic importance to the dairy industry is mastitis. Mastitis is generally regarded as an inflammation of the mammary gland. The disease can affect any mammal, but is most economically significant in dairy heifers and cows. Mastitis usually 15 results from colonization of the mammary gland by pathogenic bacteria. However, physical injuries or local mechanical or chemical stresses in the udder can also trigger a local inflammation cascade without the involvement of any primary bacterial infection (sometimes 20 referred to as sterile mastitis).

Mastitis can be expressed at clinical or subclinical levels, and may be localized to only a portion of the udder. Subclinical or localized mastitis is economically damaging because it often remains 25 undetected and untreated, yet results in decreased milk production. Accordingly, it is important in the early diagnosis of mastitis to be able to detect infection before clinical symptoms arise, and to be able to localize the infection to specific regions of the udder. 3 0 Current clinical laboratory methods used for the diagnosis of mastitis include estimation of somatic cell counts (SCC), various electrolyte levels, and soluble proteins, such as lactate dehydrogenase (LDH) and N-acetyl-p-D glucosamidase (NAG), in milk samples, all of 35 which reflect a breakdown in the blood-milk barrier due to the inflammatory response caused by the infection.

WO 01/14580 PCT/US99/19418 Certain of these parameters, e.g., SCC and electrolyte estimates, are unable to differentiate infected from uninfected regions of the udder (Zank & Slatterer, J. Vet. Med. 45.: 41-51, 1998), while others, e.g., LDH and 5 NAG detection, may not be sufficiently sensitive for very early diagnosis. Accordingly, new indicators that are highly predictive of the onset of mastitis infection at an early stage are needed.

SUMMARY OF THE INVENTION According to one aspect of the invention, a rapid and convenient method for detecting an inflammatory response in a breast of a lactating mammal is provided. The method comprises measuring the presence or amount of 15 a Serum Amyloid A (SAA) protein or mRNA encoding the protein, in a sample of milk obtained from the breast, the amount of the SAA protein or mRNA present in the sample being positively correlated with the inflammatory response. In a preferred embodiment, the inflammatory 20 response reflects mastitis in the animal, caused either by infection with a pathogenic organism or by a non-infective stress or trauma to the breast tissue.

The SAA detected and quantified in the milk preferably comprises one or more inflammation-responsive 25 isoforms of SAA. Specifically, the SAA comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-15.

In one embodiment, the method comprises measuring the amount of SAA protein in the milk sample. 30 Preferably, the amount of SAA protein is measured using an immunological assay with antibodies immunologically specific for one or more isoforms of SAA. Most preferably, the immunological assay is an ELISA assay.

Another embodiment comprises measuring the WO 01/14580 PCT/US99/19418 amount of SAA mRNA in the milk sample. Preferably, the amount of SAA mRNA is measured using a hybridization assay with nucleic acid molecules complementary to the SAA mRNA.

The methods of the invention may be adapted for a variety of purposes. In one embodiment, a method is provided for evaluating a therapeutic agent or treatment for its ability to decrease or increase the inflammatory response in breast tissue. In this embodiment, the 10 therapeutic agent or treatment is applied to a test subject. A milk sample is taken from the test subject and from a control subject that did not receive the agent or treatment. SAA levels in the test sample and the control sample are compared. An increase in SAA amount 15 in the test sample as compared with the control sample indicates that the therapeutic agent has a detrimental side effect of increasing inflammation in the breast tissue. A decrease in SAA in the test sample as compared with the control sample indicates that the therapeutic 20 agent or treatment decreases inflammation in the breast tissue.

In another embodiment, the methods of the invention are employed to monitor milk quality. Samples of milk batches are assayed for the presence and amount 2 5 of SAA, and compared to a sample accepted as a known standard for a pre-determined milk quality. The comparative amount of SAA in the test sample versus the standard is used to assign a milk quality rating to the batch of milk. 3 0 In another embodiment, the methods of the invention are used to determine the presence and amount of colostrum in a milk sample. This embodiment makes use of the inventors' discovery that SAA levels are elevated in colostrum, but not in normal milk. Test samples of WO 01/14580 PCT/US99/19418 milk batches suspected of containing colostrum are assayed for SAA (preferably a colostrum-specific SAA) and compared to a sample known to be colostrum-free. The comparative amount of SAA in the test sample versus the 5 control sample is determinative of whether the test sample is tainted with colostrum.

According to another aspect of the invention diagnostic kit for screening milk samples to detect inflammation in breasts of lactating mammals are 10 provided. In one embodiment, a test kit comprises a container containing one or more antibodies immunologically specific for one or more SAA isoforms, and instructions for performing immunological assays of milk samples for SAA, using the antibodies. In another 15 embodiment, the test kit comprises a container containing one or more nucleic acid molecules that specifically hybridizes to mRNA encoding or more SAA isoforms, and instructions for performing hybridization assays of milk samples for SAA, using the nucleic acids. These kits 2 0 may further comprise at least one additional reagent for performing the immunologic_or hybridization assays.

Other features and advantages of the present invention will be better understood by reference to the 25 drawings, detailed descriptions and examples that follow.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions Various terms relating to the methods and kits 3 0 of the present invention are used hereinabove and also throughout the specification and claims.

With respect to antibodies, the term "immunologically specific" refers to antibodies that bind to one or more epitopes of a protein of interest, but WO 01/14580 PCT/US99/19418 which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

With respect to oligonucleotides or other 5 single-stranded nucleic acid molecules, the term "specifically hybridizing" refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally 10 used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule, to the substantial exclusion of 15 hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.

II. Description Serum amyloid A (SAA) is an acute phase protein which is produced in the liver and occurs at elevated 20 levels in the serum of mammals in response to inflammation related to tissue injury or infection. The present invention arises from the surprising discovery that elevated levels of SAA also occur in the milk of lactating females, in response to inflammatory conditions 25 of the breast. In particular, cows with mastitis display elevated SAA levels that correlate with the severity of the infection, and that are confined to the area of the udder affected with the mastitis associated inflammation. This is believed to be the first observation of increased 30 production of SAA in a secreted biological fluid, in response to inflammation of the tissue in which that fluid is produced.

Without being limited by any particular explanation, the SAA found in milk from inflamed breast 3 5 tissue may result in part from a breakdown in the blood- WO 01/14580 PCT/US99/19418 • - 8 - milk barrier, allowing cellular material and plasma enzymes to leak into the milk. However, experimental results obtained by the inventors show that serum SAA levels in cows is independent of colostrum and whey SAA 5 levels. In samples of colostrum, whey and serum taken from test cows, serum SAA was found to be in the range of 10 yug/ml, while in colostrum, SAA was elevated to levels in the range of 4 70 /^g/ml (average of 5 cows) . These results indicate that the source of the SAA in milk 10 arises from an independent source in the mammary tissue, e.g., ductal epithelial cells.

The measurement of SAA levels in milk for early detection of inflammation related to mastitis is exemplified herein. However, it will be apparent to 15 persons of skill in the art that the principles applied to SAA detection in milk to diagnose mastitis-related inflammation likewise can be applied to detection of inflammatory conditions caused by other agents, such as mechanical injury, granulomatous disease, fibrocystic 20 disease and cancer.

Furthermore, if SAA is detected in milk as an indicator of an inflammation in the tissue from which it was secreted, it is likewise expected to be found in other biological fluids secreted from inflamed tissue. 25 For instance, elevated SAA levels are expected to occur in urine from an infected kidney or bladder. Elevated SAA levels may also occur in saliva, sputum, sweat or tears, arising from infected oral, lung, skin or lachrymose tissues, respectively. Application of the 30 methods described herein for early detection of inflammatory conditions of those tissues are considered within the scope of the present invention.

As one example, SAA content in sputum may be used to identify a variety of lung inflammatory 35 conditions in a patient (human or animal), including fungal and bacterial infections of the lung, as in WO 01/14580 PCT/US99/19418 _ 9 _ hypersensitive pneumonitis (e.g., Farmer's lung), viral and bacterial pneumonia, bacterial colonization and growth in Cystic Fibrosis flare (primarily caused by Pseudomonas), and cancer. Such application is considered 5 particularly useful in equine veterinary applications, where unspecified or sub-clinical lung inflammations can undermine the general health of valuable horses, such as racehorses (see, e.g., Equine Veterinary Journal 21:106-109, 1989; Journal Veterinary Medical Science 55: 10 1011-1116, 1993) .

As another example, SAA content in urine may be used to identify a variety of kidney or bladder inflammatory conditions in a patient (human or animal), including interstitial cystitis (bladder or kidney 15 involved) and kidney transplant rejection.

Referring now to the exemplary embodiment of the invention, milk is analyzed for the presence and amount of SAA as a diagnostic test for inflammation, usually associated with mastitis. The assays of the 20 invention comprise providing a test sample of milk from tissue suspected of being inflamed. The SAA level in the sample is measured, and preferably compared to a milk sample taken from known normal tissue. The amount of increase in SAA in the test sample as compared to the 25 control sample is directly proportional to the level of inflammation present in the tissue from which the sample was taken.

SAA exists in several isoforms, most of which are increased in production in an inflammatory response. 3 0 In accordance with the present invention, any one inflammation-responsive isoform, or a combination of isoforms, may be detected in the assay. In a preferred embodiment applicable to testing cow's milk, bovine SAA isoforms are detected. One isoform of bovine SAA has 3 5 been reported to date. However, in accordance with the present invention, a new bovine SAA isoform has been WO 01/14580 PCT/US99/19418 identified, which is associated with colostrum and which may comprise part or all of the SAA found in milk from inflamed tissue. Accordingly, the present invention contemplates detecting one or both of these SAA isoforms 5 in assays of cow's milk. A portion of the amino acid sequence comprising the bovine colostrum-associated SAA is set forth herein as SEQ ID NO:1. A portion of the amino acid sequence of the bovine serum SAA isoform is set forth herein as SEQ ID NO:2. 10 In alternative embodiments, isoforms of SAA from different species are detected (partial amino acid sequences of these isoforms are set forth herein). These include, but are not limited to, human SAA1 (SEQ ID NO:3), human SAA3 (SEQ ID NO:4), rabbit SAA1 (SEQ ID 15 NO:5), rabbit SAA3 (SEQ ID NO:6), mouse SAA1 (SEQ ID NO:7), mouse SAA3 (SEQ ID NO:8), hamster SAA1 (SEQ ID NO: 9) , hamster SAA3 (SEQ ID N0:10), horse SAA (SEQ ID NO:11), horse colostrum-associated SAA (SEQ ID NO: 12), pig colostrum-associated SAA (SEQ ID NO: 13), mink SAA1 20 (SEQ ID NO:14) and dog SAAa (SEQ ID NO:15).

Any assay that detects and quantifies SAA in a sample is contemplated for use in the present invention. Two categories of assays are preferred. One is an immunological assay for SAA protein and the other is a 25 hybridization assay for mRNA encoding SAA protein. The latter assay is effective because SAA is transcriptionally regulated. Accordingly, the inflammation response comprises an increase in SAA mRNA production and a concomitant increase in protein 3 0 production.

Immunological assays for SAA require antibodies immunologically specific for one or more SAA isoforms. Polyclonal or monoclonal antibodies directed toward SAA may be prepared according to standard methods. 35 Monoclonal antibodies may be prepared according to WO 01/14580 PCT/US99/19418 general methods of Kohler and Milstein, following standard protocols. In a preferred embodiment, anti-SAA antibodies are raised in animals that produce little or no SAA themselves, since SAA is more immunogenic to these 5 animals. One preferred animal source of anti-SAA antibodies is the rat. In another preferred embodiment, antibodies are prepared that react with a plurality of SAA isoforms. Such antibodies may be polyclonal or monoclonal antibodies. In a particularly preferred 10 embodiment, antibodies such as those described by McDonald et al. (J. Imunol. Meth. 144: 149-155, 1991) are used. These antibodies, raised against human SAA, also react with a variety of SAA isoforms from other species, including bovine SAA.

A variety of immunological assays known in the art are available to detect and quantitate SAA in milk samples or samples of other secreted biological fluids. These include, but are not limited to, (1) immunoprecipitation followed by protein quantification; 20 (2) immunoblot analysis (e.g., dot blot, Western blot) (3) radioimmune assays, (4) nephlometry, turbidometric or immunochromatographic (lateral flow) assays, and (5) enzyme-coupled assays, including ELISA and a variety of qualitative rapid tests (e.g., dip-stick and similar 25 tests). Of these, ELISA assays are preferred for use in the invention, due to their ease and economy of use, and their portability for use in the field.

ELISA assays have been developed and used for measuring the amount of SAA in a sample of blood. These 30 same assays, or variations thereof as devised by one skilled in the art, may be used in the assays of the present invention. Such assays are described in detail in the art (e.g., McDonald et al., 1991, supra; Satoh et al . , Am. J. Vet. Res. .56: 1286-1291, 1995; Wilkins et WO 01/14580 PCT/US99/19418 al . , Clin. Chem. 40.: 1284-1290, 1994; Taktak & Lee, J. Immunol. Meth. 136: 11-16, 1991) . An exemplary assay is set forth in Example 1.

Hybridization assays to measure the presence 5 and amount of SAA-encoding mRNA in a sample require nucleic acid probes that specifically hybridize with the mRNA encoding one or more SAA isoforms. The availability of amino acid and nucleotide sequence information for a wide variety of SAAs enables the design and production of 10 such probes, according to known methods. In one embodiment, the probes may be designed to hybridize with an mRNA encoding a particular SAA isoform, by choosing segments of the mRNA that are unique to that isoform. In a preferred embodiment, the probes are designed to 15 hybridize to a portion of the SAA-encoding mRNA that is conserved across isoforms, thereby generating a probe that detects a plurality of SAA isoform mRNAs in the test sample.

As is known in the art, suitable hybridization 20 probes comprise single stranded DNA or RNA molecules. Preferably they are between 10 and 200 nucleotides in length, and most preferably between 18 and 100 nucleotides in length. The probes may be synthesized with a detectable label; alternatively, they may be 25 detectably labeled after synthesis. Probes also may be obtained from cloned DNA that encodes SAA.

Methods in which the aforementioned nucleic acids may be utilized as probes for hybridization assays include, but are not limited to: (1) dot blot 30 hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR). Preferred for use in the present invention are rapid hybridization assay, such as dot blots or portable PCR-based assays. 35 These assays are suitable for use in a clinical or field environment. Such assays are described in detail in the WO 01/14580 PCT/US99/19418 art (see, e.g., "Current Protocols in Molecular Biology", eds. Frederick M. Ausubel et al., John Wiley & Sons, 1999).

The assays of the invention are of great 5 utility and value in a variety of applications. Focusing on the exemplary embodiment of the inventions, the assays are particularly suitable for use in early and localized detection of mammary inflammation associated with mastitis. Expanding on this utility, the assays can also 10 be used to monitor the therapeutic effectiveness of treatment of mastitis. The treatment may comprise an established treatment. Alternatively, the assays can be used to screen and evaluate the effectiveness of novel therapeutic agents and treatment regimens. In this 15 embodiment, the milk of control and test animals (subjected to the new treatment or agent) are assayed for the presence and amount of SAA. A decrease in SAA in the test sample, as compared to the control sample, is indicative that the new therapeutic agent or treatment is 20 effective in controlling the inflammation.

The assays described above also may be used to determine if a therapeutic or other agent applied to udder tissue causes any detrimental inflammatory side effects. In this embodiment, as above, milk samples from 25 test animals receiving the treatment is compared with milk samples from control animals for the presence and amount of SAA. In this embodiment, an increase in SAA level in the test samples as compared to the control sample indicates that the treatment has a detrimental 30 inflammatory side effect.

The assays of the invention also may be used to monitor milk quality. A current practice in many parts of the world is to assign a quality rating to a milk batch. Thus, a batch of milk being sold by a farmer is 35 currently assessed for quality by measuring the number of somatic cells present in a sample of the milk, which is WO 01/14580 PCT/US99/19418 correlated with the prevalence of clinical and subclinical mastitis in the dairy herd. The assays of the present invention provide a rapid, convenient alternative to the somatic cell measurement to evaluate the quality 5 of a batch of milk.

As another useful embodiment, since SAA is elevated in colostrum and not in milk from normal breast tissue, the measurement of SAA in a milk sample can be used to differentiate colostrum from milk. Accordingly, 10 in instances where it is undesirable to have milk that contains colostrum (some countries have laws to this effect), the methods of the invention may be used to detect colostrum-tainted milk.

The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.

EXAMPLE 1 2 0 Immunoassay for SAA 1. Preparation of Microtiter Plate: 96-well microtiter plates were prepared by incubating each well with 100 /il of rat monoclonal 25 antibody to bovine SAA (titer = 5.0 /zg/ml) in carbonate buffer (0.015 M Na2C03, 0.035 M NaHC03 and a pH of 9.6) . This plate coating antibody is designated C-100-8.

Plates were incubated for 12 hours at 4° C then the antisera solution is aspirated and the plates washed by 30 filling and emptying all wells 4 times with PBS-Tween which consists of 0.05% v/v Tween 20 (polyoxyethylene sorbitan monolaurate) in phosphate buffered saline (PBS = 0.010 M Na2HP04.2H20, 0.003 M KH2 P04, 0.132 M NaCl, pH of 7.2). Plates were treated with StabilCoat 35 (SurModics Inc., Eden Prairie, MN) for 1 hour at 22°C, dried for 8 hours at 37° C, then sealed in foil bags.

WO 01/14580 PCT/US99/19418 2. Reference standard: A solution containing known standard concentration of bovine SAA at 37.5 ng/ml was further diluted two-fold serially 6 times in PBS-Tween. Fifty 5 microliters (5 0//1) of the standard and each of the subsequent 6 serial dilutions were used on the plate to establish a standard curve. The standard contained 37.5 ng/ml and the subsequent two-fold dilutions contained 18.8, 9.4, 4.7, 2.3, 1.20 and 0.60 ng/ml, respectively.

Fifty /il of PBS-Tween without SAA established background readings. 3. Assay procedure (standard sandwich ELISA): a. Wells were washed three times with PBS- Tween wash buffer. After the last wash, wells were patted dry on absorbent paper. b. 50/ul of biotinylated rat monoclonal antibody were added to bovine SAA (1 /xg/ml) to each well. This antibody is designated C-100-7 and has a different epitope specificity for SAA than the plate-coating antibody. c. Serum, colostrum or milk -samples were at room temperature and were vortexed vigorously before dilutions were made. Samples were diluted 1:500. In 2 5 some instances, samples may require greater dilution to fall within the range of the assay. 50/^1 of the diluted sample or standard was added to each well. Sides of the plate were tapped to mix gently. d. The plate was covered with a dust cover, 3 0 and incubated for 1 hr at 37°C. e. After incubation, the plate was aspirated or decanted, then washed three times with PBS-Tween buffer. After the last wash, the plate was patted dry on absorbent paper. f. 100/^1 of streptavidin-peroxidase (100 ng/ml) was added to each of the wells.

WO 01/14580 PCT/US99/19418 g. The plate was covered and incubated at room temperature in the dark for 3 0 minutes. h. The wells were aspirated or decanted, then washed three times with PBS-Tween, and the plate patted dry after the last wash. i. 100/^1 of substrate consisting of 3, 3',5, 5' - tetramethyl benzidine at 10 g/ml (0.1 mg dissolved in 0.1 ml of dimethylsulfoxide and 9.9 ml of 0.1 M sodium acetate pH 6.0 added) was added. j. The plate was covered and incubated in the dark at room temperature for 3 0 minutes. k. 50/ul of a Stop Solution of H2S04 (prepared by adding 2.8 ml of concentrated H2S04 to 97.2 ml of water) was added. 1. The absorbance of each well was read at 450 nm having blanked the plate reader against a chromagen blank composed of 100^1 of TMB substrate and 50,ul Stop Solution only. m. The absorbance of the standards was plotted 20 against standard concentration on graph paper. The background absorbance for the 0 ng/ml may be subtracted from each of the data points, including the standards, unknowns and controls prior to plotting. n. The concentrations of the test samples and 25 controls was determined from the standard curve by multiplying the interpolated value by the appropriate dilution factor (e.g., colostrum or milk diluted 500 should be multiplied by 500). Samples which have a signal greater than the highest standard were further 30 diluted in diluent buffer and re-analyzed.

EXAMPLE 2 Evaluation of SAA in Colostrum and Subsequent Serial Samplings of Milk The purpose of this study was to evaluate colostrum and subsequent serial milk samplings to 17 - determine SAA content. Samples were obtained from Holstein dairy cows at the University of Nebraska -Lincoln Dairy Research Facility. Samples of colostrum were taken at calving, and subsequent milk samples were taken twice weekly for three weeks. Samples from all four udder quadrants were pooled. Results are shown in Table 2.

Table 2: SAA Levels in Colostrum and Milk Samples Cow ID 83 colostrum 83 milk 83 milk 83 milk 83 milk 83 milk 83 milk Sample Day Calving +4 + 7 + 11 + 14 + 18 +21 SAA ug/ml 184 . 8 0.2 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 932 colostrum 932 milk 932 milk 932 milk 932 milk 25 932 milk 932 milk Calving +4 + 7 + 11 + 14 + 18 + 21 566 .2 229.8 217 . 7 169 . 0 6 . 4 5 . 8 26 . 0 908 colostrum 908 milk 90 8 milk 9 08 milk 908 milk 908 milk 908 milk Calving +4 + 7 + 11 + 14 + 18 + 21 135 . 6 2 . 6 9 . 1 8.2 2 . 0 2 .1 3.6 961 colostrum Calving 364 . 6 PCTAJS99/19418 961 milk +4 0 . . 3 961 milk + 7 0 . . 5 961 milk + 11 0 . . 0 961 milk + 14 0 . . 0 961 milk + 18 0 . . 0 961 milk + 21 0 , . 0 The results obtained above for cows 83, 908 and 961, each of which was free of mastitis in all quadrants, 10 shows the low basal level of SAA present in normal milk samples after colostrum has cleared.

The results obtained for cow 932 demonstrates that SAA level can effectively detect mastitis in a quadrant specific manner. Midway through the sampling 15 period, cow 932 was observed to be symptomatic for mastitis in one quadrant. Even though all four quadrants' milk were pooled in the first three milk samplings, the samples were elevated in SAA. In the subsequent samplings, the mastitis quadrant was not 20 milked, so was not pooled with the milk from the unaffected quadrants. As a result, SAA levels decreased in those samples to approximately the levels observed in the completely asymptomatic cows.

EXAMPLE 3 SAA Levels as a Quadrant-Specific Indicator of Mastitis 3 0 The purpose of this study was to determine if SAA can detect mastitis-related inflammation, and if SAA is elevated only in the affected quadrant, or in milk from all quadrants.

Samples were obtained from the Holstein dairy 35 herd at the University of Nebraska - Lincoln Dairy Research Facility. Three cows with mastitis in the left rear (LR) quadrant of the udder were selected. Each cow

Claims (20)

WO 01/14580 PCT/US99/19418 - 19 - was judged visually for the level of inflammation present in each cow. Cow 874 displayed low-grade inflammation, while cow 59 displayed significant inflammation and cow 978 displayed high grade inflammation. 5 Milk samples were taken from all four quadrants of each cow, and SAA levels were determined by ELISA for the non-pooled samples. Results are shown in Table 3. Table 3: SAA Levels in Milk from Udder Quadrants 10 Cow ID Quadrant SAA (ug/ml) 874 RF 12 874 RR 10 874 LF 11 15 874 LR (Mastitis) 73 59 RF 1 5 9 RR 1 59 LF 7 59 LR (Mastitis) 212 20 978 RF 6 978 RR 18 978 LF 8 978 LR (Mastitis) 2368 25 The results demonstrate that SAA levels provide a quadrant-specific indicator of inflammation due to mastitis. SAA elevation is confined to the affected quadrant. Moreover, a direct correlation was observed 3 0 between the visual symptoms of inflammation and SAA levels found in the affected quadrant. 35 The present invention is not limited to the embodiments described above, but is capable of modification within the scope of the appended claims. 20 WO €1/14580 PCT/US99/19418 We claim:

1. A method for detecting an inflammatory response in a breast of a lactating mammal, which comprises measuring the presence or amount of a Serum Amyloid A (SAA) protein or mRNA encoding the protein, in a sample of milk obtained from the breast, the amount of the SAA protein or mRNA present in the sample being positively correlated with the inflammatory response.

2. The method of claim 1, wherein the mammal is the inflammatory response is mastitis.

3. The method of claim 2, wherein the mastitis is caused by infection with a microorganism.

4. The method of claim 2, wherein the inflammatory response is caused by a non-infective agent.

5. The method of claim 1, wherein the SAA comprises one or more inflammation-responsive isoforms of SAA.

6. The method of claim 5, wherein the SAA comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-15.

7. The method of claim 1, which comprises measuring the amount of SAA protein in the milk sample.

8. The method of claim 7, wherein the amount of SAA protein is measured using an immunological assay with antibodies immunologically specific for one or more isoforms of SAA.

9. The method of claim 8, wherein the immunological assay is an ELISA assay. SUBSTITUTE SHEET (RULE 26) 21

10. The method of claim 1, which comprises measuring the amount of SAA mRNA in the milk sample.

11. The method of claim 10, wherein the amount of SAA mRNA is measured using a hybridization assay with nucleic acid molecules complementary to the SAA mRNA.

12. A method to assess the ability of a treatment or agent to increase or decrease inflammation in breast tissue, which comprises: (a) obtaining a milk sample from a test subject to which the treatment or agent has been administered; (b) obtaining a milk sample from a control subject which has not been administered the treatment or agent; (c) measuring the amount of SAA in the test sample and the control sample; and (d) comparing the measured amounts of SAA, an increase in SAA in the test sample as compared to the control sample indicating the ability of the treatment or agent to increase inflammation in the breast tissue, and a decrease in SAA in the test sample as compared to the control sample indicating the ability of the treatment or agent to decrease inflammation in the breast tissue.

13. The method of claim 12, adapted for screening a anti-microbial agents for treatment of mastitis. 22

14. A method of evaluating quality of a milk sample, which comprises: (a) establishing a standard that correlates a predetermined milk quality with a pre-determined concentration of SAA contained therein; (b) measuring SAA concentration in the milk sample being tested for quality; (c) comparing the amount of SAA in the milk sample with the established standard; and (d) assigning a quality rating to the milk sample based on the comparison with the standard.

15. A method of determining the presence or amount of colostrum in a milk sample, which comprises: (a) measuring SAA concentration in a milk test sample suspected of containing colostrum; (b) comparing the amount of SAA in the test sample with an amount of SAA in a control milk sample known to contain no colostrum, an elevation in SAA in the test sample as compared with the control sample being indicative of the presence and amount of colostrum in the test sample.

16. The method of claim 15, which further comprises measuring a colostrum-specific isoform of SAA.

17. A method according to claim 1 substantially as herein described or exemplified.

18. A method according to claim 12 substantially as herein described or exemplified. INTELLECTUAL PROPERTY OFFICE OF N.7. 2 5 NOV 2003 RECEIVED 23

19. A method according to claim 14 substantially as herein described or exemplified.

20. A method according to claim 15 substantially as herein described or exemplified. WO 01/14580 PCT/US99/19418 1 SEQUENCE LISTING <110> McDonald, Thomas L. Weber, Annika <120> Diagnostic Assays of Secreted Biological Fluids for Detection of Infection and Inflammatory Conditions <130> UNMC 98018A <160> 15 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 20 <212> PRT <213> Cow <400> 1 Met Trp Xaa Thr 1 Trp Arg Ala Tyr 20 <210> 2 <211> 27 <212> PRT <213 > Cow <400> 2 Xaa Xaa Trp Met Ser Phe Phe Gly Glu Ala Tyr Glu Gly Ala Lys Asp 15 10 15 Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala 20 25 <210> 3 <211> 27 <212> PRT <213> Homo sapiens <400> 3 Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala Arg Asp 15 10 15 Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala 20 25 <210 > 4 <211> 27 <212> PRT <213> Homo sapiens Phe Leu Lys Glu Ala Gly Gin Gly Ala Lys Asp Met 5 10 15 WO 01/14580 PCT/US99/19418 2 <400> 4 Gin Gly Trp Leu 1 Met Trp Arg Ala 20 <210> 5 <211> 27 <212> PRT <213> Rabbit <400> 5 Gin Arg Trp Phe Ser 1 5 Met Trp Arg Ala Tyr 20 <210> 6 <211> 27 <212> PRT <213> Rabbit <400> 6 Arg Glu Trp Leu Thr 1 5 Met Trp Arg Ala Tyr 20 <210> 7 <211> 27 <212> PRT <213> Mus musculus <400> 7 Gly Gly Phe Phe Ser Phe Val His Glu Ala Phe Gin Gly Ala Gly Asp 15 10 15 Met Trp Arg Ala Tyr Thr Asp Met Lys Glu Ala 20 25 <210> 8 <211> 27 <212> PRT <213> Mus musculus <400> 8 Gin Arg Trp Val Gin Phe Met Lys Glu Ala Gly Gin Gly Ser Arg Asp 15 10 15 Met Trp Arg Ala Tyr Ser Asp Met Lys Lys Ala 20 25 Thr Phe Leu Lys Ala Ala Gly Gin Gly Ala Lys Asp 5 10 15 Tyr Ser Asp Met Lys Glu Ala Phe He Gly Glu Ala Thr Gin Gly Ala Trp Asp 10 15 Ser Asp Met Arg Glu Ala 25 Phe Leu Lys Glu Ala Gly Gin Gly Ala Lys Asp 10 15 Ser Asp Met Lys Glu Ala 25 <210> 9 <211> 27 <212> PRT WO 01/14580 PCT/US99/194I8 3 <213> Hamster <400> 9 Gin Arg Trp Phe Gin Phe Met Lys Glu Ala Gly Gin Gly Thr Arg Asp 15 10 15 Met Trp Arg Ala Tyr Thr Asp Met Arg Glu Ala 20 25 <210> 10 <211> 27 <212> PRT <213> Hamster <400> 10 Gin Arg Trp Phe Gin Phe Met Lys 1 5 Met Trp Arg Ala Tyr Ser Asp Met 20 Glu Ala Gly Gin Gly Ser Thr Asp 10 15 Arg Glu Ala 25 <210> 11 <211> 27 <212> PRT <213> Horse <400> 11 Xaa Xaa Leu Leu Ser Phe Leu Gly Glu Ala Ala Arg Gly Thr Trp Met 15 10 15 Asp Leu Arg Ala Thr Asn Asp Met Arg Glu Ala 20 25 <210> 12 <211> 13 <212 > PRT <213> Horse <400> 12 Arg Glu Leu Lys Thr Phe Leu Lys Glu Ala Gly Gin Gly 15 10 <210> 13 <211> 10 <212> PRT <213> Pig <400 > 13 Trp Leu Leu Thr Phe Leu Lys Glu Ala Gly 15 10 <210> 14 <211> 27 <212 > PRT <213 > Mink WO 01/14580 PCT/US99/19418 <400> 14 Gin Xaa Trp Tyr Ser Phe lie Gly Glu Ala Ala Gin Gly Ala Trp Asp 15 10 15 Met Tyr Arg Ala Tyr Ser Asp Met lie Glu Ala 20 25 <210 > 15 <211> 27 <212> PRT <213> Dog <400> 15 Gin Xaa Trp Tyr Ser Phe Val Gly 1 5 Met Leu Arg Ala Tyr Ser Asp Met 20 Glu Ala Ala Gin Gly Ala Trp Asp 10 15 Arg Glu Ala 25