WO1994021280A1 - Use of bactericidal/permeability increasing protein and lipopolysaccharide binding protein levels and ratios thereof in diagnosis - Google Patents

Abstract

Methods are provided for diagnosing and predicting the outcome of an inflammatory disorder in a subject, which comprises obtaining a bodily fluid sample from the patient, quantitatively determining the amount of BPI in the sample, comparing the amount of BPI Present in the sample with levels of BPI in samples obtained from normal subjects and with levels of BPI in samples obtained from individuals suffering from an inflammatory disorder in order to diagnose and predict the outcome of an inflammatory disorder in the subject. In addition, methods are provided for diagnosing and predicting the outcome of an inflammatory disorder in a subject, which comprises obtaining a bodily fluid sample from the subject, quantitatively determining the amount of BPI and LBP in the sample, determining the ratio of BPI to LBP in the sample, and then comparing the resulting ratio of BPI to LBP with BPI:LBP ratios obtained from normal subjects and from individuals suffering from an inflammatory disorder in order to diagnose and predict the outcome of the inflammatory disease in the subject.

Description

USE OF BACTERICIDAL/PERMEABILITY INCREASING PROTEIN AND LIPOPOLYSACCHARIDE BINDING PROTEIN LEVELS AND RATIOS THEREOF IN DIAGNOSIS

Field of the Invention This invention relates to the field of diagnosing and predicting the outcome of inflammatory diseases. More specifically, the invention concerns methods for diagnosing and predicting the outcome of inflammatory diseases by quantitatively determining the amount of bactericidal/permeability increasing protein (BPI) present in a sample or by determining the ratio of BPI to lipopolysaccharide binding protein (LBP) in a sample.

Background Art

Bactericidal/Permeability Increaginq Protein (BPI)

BPI is a neutrophil granule protein first discovered in 1975 [Weiss et al . (1975) J. Clin . Invest . 55:33]. BPI, a 57 kD protein, was obtained in highly purified form from human neutrophils in 1978 and was shown to increase membrane permeability and have bactericidal activity against gram-negative bacteria when assayed in phosphate buffered saline in vitro [Weiss et al . (1978) J. Biol . Chem . 253(8) :2664- 2672]. Weiss et al. [J. Biol . Chem . 254(21) :11010- 11014 (1979) ] further showed that BPI increases phospholipase A2 activity, suggesting a proinflammatory activity for BPI in addition to its in vitro bactericidal activity. Rabbit BPI was purified in 1979 [Elsbach et al . (1979) J. Biol . Chem . 254(21) :11000-11009] and shown to have bactericidal and permeability increasing properties identical to those of BPI from humans, providing a further source of material for study. Both BPI from rabbit and human were shown to be effective against a variety of gram-negative bacteria in vitro, including Kl-encapsulated E. coli [Weiss et al . (1982) Infect . Immun . 38(3) :1149-1153] . A role for lipopolysaccharide (LPS) , herein used synonymously with "endotoxin," in the in vitro bactericidal action of BPI was proposed in 1984 by Weiss et al . [J. Immunol . 132(6) :3109-3115 (1984)]. These investigators demonstrated that BPI binds to the outer membrane of gram-negative bacteria, causes the extracellular release of LPS, and selectively stimulates LPS biosynthesis.

In 1984, a protein with properties similar to those of BPI was isolated from human neutrophils and designated cationic antimicrobial protein 57

(CAP57) [Shafer et al . (1984) Infect . Immun . 45:29]. This protein is identical to BPI as determined by the N-terminal amino acid sequence, amino acid composition, molecular weight and source [Spitznagel et al . (1990) Blood 76:825-834]. Another group, Hovde and Gray, reported a bactericidal glycoprotein with virtually identical properties to BPI in 1986 [Hovde and Gray (1986) Infect . Immun . 54(1) :142-148] . In 1985, Ooi et al . reported that BPI retained its in vitro bactericidal activity after cleavage with putative neutrophil proteases, suggesting that fragments of the molecule retain activity [Ooi and Elsbach (1985) Clin . Res . 33(2):567A]. This group reported that all of the in vitro bactericidal and permeability increasing activities of BPI are present in the N-terminal 25 kD fragment of the protein [Ooi et al . (1987) J. Biol . Chem . 262:14891].

Evidence that BPI binds to a structure associated with endotoxin on the outer membrane of bacteria is as follows: (1) increased sensitivity of rough strains of E. coli relative to smooth strains to the permeability increasing activities of BPI [Weiss et al . (1986) Infect . Immun . 51:594]; (2) the Prm A mutation which results in altered endotoxin structure causes decreased binding of both polymyxin B (PMB) and BPI [Farley et al . (1987) Infect . Immun . 56:1536-1539; Farley et al . (1988) Infect . Immun . 58:1589-1592]; (3) PMB competes with BPI for binding to S. typhimurium [Farley et al . (1988), supra] ; and (4) BPI shares amino acid sequence homology and immunocrossreactivity to another endotoxin-binding protein termed lipopolysaccharide binding protein (LBP) [Tobias et al . (1988) J. Biol . Chem . 263(27) :13479-13481] .

A cDNA encoding BPI was obtained and sequenced by Gray et al . [Clin . Res . 36:620A (1988); Gray et al . (1989) J. Biol . Chem . 264(16) :9505-9506] . Gray et al . suggested that BPI is a membrane protein which can be cleaved and released in soluble form as a 25 kD fragment.

BPI binding to gram-negative bacteria was originally reported to disrupt LPS structure, alter microbial permeability to small hydrophobic molecules and cause cell death [Weiss et al . (1978), supra]. More recently, these same author, have demonstrated that such effects occur only in the absence of serum albumin. BPI has no bactericidal activity when added to bacteria cultured in the presence of serum albumin, thus suggesting that BPI does not kill bacteria in vivo where albumin is ubiquitous [Mannion et al . (1990) J. Clin . Invest . 85:853-860; Mannion et al . (1990) J. Clin . Invest . 86:631-641]. Thus it has been previously understood in the art that the beneficial effects of BPI are limited to in vitro bactericidal effects.

While Gray et al . [J^". Biol . Chem . 264(16) :9505-9509 (1989)] proposed that BPI is a membrane protein which must be cleaved to the 25 kD fragment to be released from the neutrophil granule membrane in water soluble form, it has subsequently been determined that BPI is active and fully water soluble in its native, full-length form [Marra et al . (1990), J. Immunol . 144:662-666]. Further, no additional reports have confirmed the existence of the proposed 25 kD fragment in vivo.

While BPI is a neutrophil granular protein, it is readily solubilized without detergent from neutrophil membranes and is stable and functionally active in aqueous solution [Marra et al . (1992), J^". Immunol . 148:532-537]. Moreover, BPI is now known to be a protein which binds LPS and inhibits the immunostimulatory and toxic activities of LPS in vitro and in vivo.

Turning now to the subject of bacterial endotoxin, the management of gram-negative sepsis continues to be one of the major challenges in clinical medicine in that the frequency of its occurrence continues to increase while the treatment options available remain quite limited [Parrillo, J.E. (1993) N. Engl . J. Med. 328:1471-1477; Bone, R.C. (1991) Ann . Intern . Med. 115:457-460]. Bacterial endotoxin is known to be the principal mediator of the pathophysiology of septic shock caused by gram- negative bacteria [Morrison, D.C. and R.J. Ulevitch (1978) Am . J. Pathol . 93:525]. The presence of high levels of endotoxin in the systemic circulation may portend an unfavorable prognosis in septic patients [Brandtzaeg et al . (1989) J. Infect . Dis . 159:195-204; Casey et al . (1993) Ann . Intern . Med. 199:771-778]. These findings have generated considerable interest in the development of therapeutic measures to inhibit or neutralize endotoxin and thereby avoid the detrimental consequences from this toxic microbial product. The host response to the presence of endotoxin is complex and varies substantially among mammalian species and probably among individual patients [Riveau et al . (1987) J. Clin . Microbiol . 25:889-892]. Endotoxin may be metabolized [Mumford,

R.S. and CA. Hall (1986) Science 234:204-205], taken up by high density lipoproteins [Harris et al . (1993) J. Clin . Invest . 91:1028-1034; Flegel et al . (1989) Infect . Immun . 57:2237-2245] or cleared from the systemic circulation by immune or non-immune-mediated mechanisms [Hampton et al . (1991) Nature 352:342-344; Wortel et al . (1991) J. Infect . Dis . 166:1367-1374]. Many of the patho-physiologic effects of endotoxin appear to be mediated by the activation of mononuclear cells with the subsequent generation of the proinflammatory cytokines, TNF-alpha and interleukin- 1. Macrophage responsiveness to endotoxin varies over time and is dependent upon several factors [Morrison, D.C. and J.L. Ryan (1987) Ann . Rev. Med. 38:417-432]. Endotoxin-signaling pathways within the macrophage are complex and not entirely understood. However, it has become increasingly apparent that endotoxin signaling is initiated with its attachment to the CD14 antigen on the macrophage cell surface [Wright et al . (1990) Science 249:1431-1433]. Endogenous proteins which facilitate the delivery of endotoxin to the macrophage cell surface, such as LPS-binding protein (LBP) [Schumann et al . (1990) Science 249:1429-1431] or the septin pathway [Wright et al . (1992) J. Exp. Med.

176:719-72 ] stimulate the monocyte/macrophage cell line to produce proinflammatory cytokines and activate a cascade of host-derived inflammatory mediators which jointly participate in the genesis of septic shock [Parrillo, J.E. (1993), supra; Bone, R.C. (1991), supra .

BPI and the hepatically synthesized serum protein LBP are two structurally similare endotoxin- binding proteins which ultimately determine the host response to endotoxin which is released into the systemic circulation during gram-negative bacterial infection. LBP and BPI share up to 44% amino acid sequence homology and both contain a high affinity- binding domain for the lipid A component of bacterial endotoxin Schumann et al . (1990), supra ; Gray et al . (1989) , supra] . Despite these structural similarities, BPI and LBP function as molecular antagonists in relationship to their ability to deliver endotoxin to the macrophage. LBP binds to endotoxin and this complex then binds readily to the CD14 antigen on the macrophage cell surface. This LBP-endotoxin - CD14 complex participates in triggering the intracellular message for activation of macrophages and the subsequent release of proinflammatory cytokines [Schumann et al . (1990), supra; Tobias et al . (1988), supra] . In contrast, BPI interaction with endotoxin blocks endotoxin delivery to the CD14 antigen [Marra et al . (1990) J. Immunol . 144:662-666; Marra et al . (1992) J. Immunol .

148:537-537]. BPI binding to endotoxin has been shown to attenuate cytokine release by mononuclear cells and to inhibit endotoxin mediated activation of neutrophils [Marra et al. (1990), supra; Marra et al . (1992), supra ; Weiss et al . (1992) J. Clin . Invest . 90:1122-1130; Heumann et al . (1993) J. Infect . Dis . 176:1351-1357]. Competitive inhibition of LBP activity by BPI has been demonstrated both in vitro and in vivo in experimental models of endotoxic shock [Marra et al . (1992), supra; Opal et al . (1990) Clin . Res . 30:451A]. The physiologic role of BPI in modulating endotoxin interactions with LBP in vivo has been questioned owing to the large molar excess of LBP in the systemic circulation when compared to BPI [Heumann et al . (1993), supra]. Moreover, the solubility and extracellular availability of this neutrophil granule-specific protein during active clinical infection is unknown [Marra et al . (1992), supra] .

Diagnostic Methods

Methods of determining TNF, IL-6, IL-1, IL- 8, and endotoxin levels in patients have been used to diagnose inflammatory disorders and to determine the likelihood of recovery in patients with such disorders [see, e . g. , Van DeVenter et al . (1988) Lancet March 18; Waage et al . (1989) J. Exp. Med. 170(6) :1859] . However, TNF, IL-6, IL-1, IL-8, and endotoxin levels are not very sensitive indicators of inflammatory disorders or likelihood of recovery therefrom in such patients.

Evidence of BPI release (in both free and cell surface-bound form) from isolated human neutrophils in vitro has been reported [Marra et al. (1992) J. Immunol . 148:532-537]. However, the release of free BPI in these experiments was only observed using non-physiological stimuli, i.e., stimulation with fMLP (formyl-met-leu-phe) with cytochalasin B. Cells stimulated with LPS, TNF or fMLP alone displayed an increase in cell surface BPI, but not in free BPI. Despite these findings, there have been no reports of elevated BPI levels in vivo following endotoxin challenge. Furthermore, no diagnostic test for BPI has been reported.

Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference in order to more fully describe the art to which this invention pertains.

Disclosure of the Invention

The invention provides methods for diagnosing and predicting the outcome of inflammatory disorders in individual subjects. Specifically, the instant invention provides a method for diagnosing and predicting the outcome of an inflammatory disorder in a subject, which comprises obtaining a bodily fluid sample from the patient, quantitatively determining the amount of BPI in the sample, comparing the amount of BPI present in the sample with levels of BPI in samples obtained from normal subjects and with levels of BPI in samples obtained from individuals known to be suffering from an inflammatory disorder in order to diagnose and predict the outcome of an inflammatory disorder in the subject.

Inflammatory disorders within the present invention include, but are not limited to, systemic inflammatory response syndrome (SIRS) , adult respiratory distress syndrome (ARDS) , microbial infections including viral or bacterial sepsis, peritonitis, meningitis, hemorrh gic shock following gut ischemia with bacterial translocation. In a preferred embodiment, the etiological agent causing the microbial infection is a gram-negative bacterium. Samples within the present invention include, but are not limited to, any bodily fluid sample, such as urine, plasma, whole blood, CSF, pus, sputum, inflammatory exudate, bronchoalveolar lavage fluid, joint fluid, peritoneal fluid, bursa fluid, pleural fluid, pericolonic fluid, periappendiceal fluid, ascitic fluid, pericardial fluid and abscess drainage.

The amount of BPI measured by methods within the present invention includes free BPI and neutrophil surface-bound BPI.

The instant invention additionally provides for a method for diagnosing and predicting the outcome of an inflammatory disorder in a subject, which comprises obtaining a bodily fluid sample from the subject, quantitatively determining the amount of BPI and LBP in the sample, determining the ratio of BPI to LBP in the sample, and then comparing the resulting ratio of BPI to LBP with BPI:LBP ratios obtained from normal subjects and from individuals suffering from an inflammatory disorder in order to diagnose and predict the outcome of the inflammatory disease in the subject.

Brief Description of the Drawings

Figure 1: Time course of BPI(A) and TNF(B) release in whole blood samples incubated in vitro with varying concentrations of bacterial LPS.

Figure 2: Circulating BPI levels in vivo in endotoxin treated volunteers.

Figure 3: Polymorphonuclear leukocyte surface BPI measured in endotoxin-treated human volunteers.

Figure 4: Surface BPI on peripheral blood PMN leukocytes in patients with gram-negative sepsis.

Figure 5: Circulating free BPI in patients with gram-negative sepsis as determined by ELISA methods described infra (A) , and neutrophil counts as determined by standard flow cytometry methods (B) . Figure 6: BPI and LBP concentrations in biological fluids based upon the presence of white blood cells.

Figure 7: circulating BPI levels in normal subjects and in patients with sepsis. Figure 8: Circulating LBP levels in normal subjects and in patients with sepsis.

Modes of Carrying Out the Invention

One aspect of this invention is based on the discovery that BPI levels are elevated in vivo in response to endotoxin challenge and as a result of inflammatory disease (e.g., systemic inflammatory response syndrome (SIRS)) . The elevated levels of BPI include free BPI and cell surface-bound BPI. These findings demonstrate that the level of BPI in a subject is a marker of endotoxin challenge, and that the level of BPI in a subject is useful as a diagnostic marker of an inflammatory disorder. These findings further demonstrate that the level of BPI in a subject can be used to determine the prognosis of the subject.

It was unexpected that free BPI would circulate at all, and that the level of circulating free BPI would correlate with any disease condition or endotoxin challenge. First because BPI release by neutrophils previously was shown to occure in vitro only under nonphysiologic conditions and, secondly, because the circulating half-lives of recombinant BPI in rodents, rabbits and non-human primates (see Table 1) are very short and, therefore, may not have been expected to be elevated consistently in subjects with inflammatory disorders.

TABLE 1

CIRCULATING HALF-LIVES OF BPI IN DIFFERENT MAMMALIAN SPECIES

minutes minutes

Mouse 3.1 41.9

Rabbit 0.49 6.2

Baboon 0.23 18.2

The increased levels of BPI in vivo indicate that neutrophils are activated in the circulation. The level of cell-associated BPI indicates the endotoxin-neutralizing capacity of these cells in response to circulating endotoxin/sepsis. Measuring BPI levels is thus advantageous over measuring levels of any other neutrophil granule protein (e .g. , elastase) since BPI is the only known endotoxin- neutralizing component of human neutrophil granules. For example, it has been discovered that low levels of circulating free BPI in an endotoxemic patient indicate immunodepletion associated with a greater risk of mortality or morbidity. Furthermore, because BPI release in whole blood in vitro is triggered by lower concentrations of bacterial endotoxin than that required to trigger the release of cytokines such as TNF, methods of diagnosing and predicting the outcome of inflammatory disorders by measuring BPI levels would be expected to be more sensitive than other previously described methods.

Endotoxin is known to activate the systemic inflammatory response, multi-organ failure, and hemodynamic changes which may occur as a consequence of systemic gram-negative bacterial infection. The endogenous serum protein LBP is known to bind to the lipid A component of bacterial endotoxin and facilitate its delivery to the CD14 antigen on the macrophage. This triggers the release of pro-inflammatory cytokines and the subsequent activation of a cascade of host mediators which result in septic shock. The neutrophil granular protein bactericidal/permeability-increasing protein (BPI) competes with LBP for endotoxin-binding and may function as a molecular antagonist of LBP-endotoxin interactions in vivo.One aspect of the instant invention relates to the discovery that BPI is present in high concentrations in abscess fluids during actual clinical infection and is present in quantities far in excess of those measured for LBP. These findings support a physiologic role for BPI as a regulatory protein which functions to attenuate or prevent the systemic effects of endotoxin in invasive gram- negative infection.

Another aspect of the instant invention is based on the discovery that the ratio of BPI to LBP differs significantly within body fluids and this ratio is dependent upon the presence of clinical infection and correlates with the quantity of neutrophils within extravascular fluids. In samples from closed space infections, BPI was discovered to be present in greater concentration than LBP (BPI/LBP ratio 200.9± 180.9). Approximately equal amounts of BPI and LBP were discovered in the abdominal fluid of patients with peritonitis (BPI/LBP ratio - 0.304± 0.06); whereas the BPI/LBP ratio was low in non-infected body fluids (0.053± 0.022) and approached levels of BPI and LBP found in normal human plasma. BPI levels were positively correlated with the quantity of neutrophils within clinical samples

(r= +0.812; p<0.0001). These are the first data which show that sufficient quantities of BPI are available within abscess cavities to effectively compete with LBP for endotoxin. BPI concentrations in these sites may be adequate to dampen the local inflammatory response to endotoxin release and may prevent sytemic release of endotoxin which can occur as a result of localized gram-negative infections. The net effect of these interactions is that BPI predominates in localized abscesses whereas LBP predominates in the plasma and in noninfected body fluids. This illustrates one component of the complex and dynamic interaction between an infecting gram-negative invasive bacteria and the host [Cross et al . (1993) J. Jjifect. Dis . 167:112-118]. A common host defense mechanism is to limit the spread of an infecting microorganism into the localized region thereby preventing systemic dissemination. The human neutrophil protein, BPI, may serve an essential function during localized infection with gram-negative bacteria by binding readily to endotoxin and limiting its ability to activate the macrophage and other CD14- bearing cells such as the neutrophil.

As used herein, "diagnosing" means determining the presence of an inflammatory disorder in a subject and, additionally, determining the type, presence or degree of severity of an inflammatory disorder in a subject. As used herein, a "bodily fluid sample" means any fluid sample obtained from the body including, but not limited to, urine, plasma, whole blood, CSF, pus, sputum, inflammatory exudate, bronchoalveolar lavage fluid, joint fluid, peritoneal fluid, bursa fluid, pleural fluid, pericolonic fluid, periappendiceal fluid, ascitic fluid, pericardial fluid and abscess drainage. Obtaining a bodily fluid sample from a subject is accomplished using methods well known to those skilled in the art.

A quantitative determination of the amount of BPI or LBP in the sample can be determined using immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioim unoassay (RIA) methods which employ anti-BPI antibodies.

The term "antibody" in its various grammatical forms is used herein as a collective noun that refers to a population of immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.

The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab, F(ab,)2 and F(v) . Fab and F(ab,)2 portions of antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well known [see for example, U.S. Patent No. 4,342,566], Fab, antibody portions are also well known and are produced from F(ab')2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred, and is utilized as illustrative herein.

An antibody as used herein also includes chimeric antibodies, single-chain antibodies, humanized antibodies and antibodies produced recombinantly or by any animal species.

An antibody as used herein includes antibodies that specifically bind to free BPI as well as antibodies that specifically bind to a

BPI/endotoxin complex. Such antibodies can be used to distinguish free BPI from BPI complexed to LPS/endotoxin.

A polyclonal antibody within the present invention is characterized as being capable of immunoreacting with BPI.

The phrase "monoclonal antibody" in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e .g. , a bispecific monoclonal antibody.

As used herein, the term "immunoreactivity: in its various grammatical forms refers to the concentration of antigen required to achieve a 50% inhibition of the immunoreaction between a given amount of the antibody and a given amount of BPI. That is, immunoreactivity is the concentration of antigen required to achieve a B/Bo value of 0.5, where Bo is the maximum amount of antibody bound in the absence of competing antigen and B is the amount of antibody bound in the presence of competing antigen, and both Bo and B have been adjusted for background [Rodbard (1974) Clin . Chem . 20:1255-1270]. Methods for determining the affinity of a monoclonal antibody for an antigen are well known in the art [Muller (1980) J. Immunol . Meth . 34:345352; Sokal et al . Biometry (W. H. Freeman & Co., 1981)]. A subject monoclonal antibody, typically containing whole antibody molecules can be prepared using the polypeptide-induced hybridoma technology described by Niman et al . (1983) Proc. Natl . Sci . U.S.A. 80:4949-4953, which description is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a polypeptide of this invention.

It is preferred that the myeloma cell line be from the same species as the lymphocytes. Typically, a mouse of the strain 129 G1X+ is the preferred mammal. Suitable mouse myelomas for use in the present invention include the hypoxanthineaminopterin-thymidine-sensitive (HAT) cell lines P3X63-Ag8.653, and Sp2/0-Agl4 that are available from the American Type Culture Collection, Rockville, MD., under the designations CRL 1580 and CRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 1500. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody of this invention can be identified by radioimmunoassay.

A monoclonal antibody of the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate polypeptide specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody- containing medium is then collected. The antibody molecules can then be further isolated by well known techniques.

Media useful for the preparation of these compositions are both well known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al . (1959) Virol . 8:396, supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

An "immunoassay" within the present invention means an analytical method relying on the use of an antibody as the specific reagent used to quantitatively measure the amount of an analyte, in this case BPI and LBP, in a sample. The antibody may be labeled with a fluorescent or chemiluminescent labeling agent or an indicating means such as an enzyme, capable of signaling the formation of a complex containing BPI or LBP and an antibody molecule within the present invention.

The word "complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.

As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well known in the art and constitute a part of this invention only insofar as they are utilized with otherwise novel methods.

The labeling means can be a fluorescent labeling agent that chemically binds to antibodies without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyanate (FITC) , 5-dimethylamine- lnaphthalenesulfonyl chloride (DANSC) tetramethylrhoda ine isothiocyanate (TRITC) , lissamine, rhoda ine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in ANTIBODY AS A TOOL

(Marchalonis et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982)), which is incorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such as alkaline phosphatase, horseradish peroxidase (HRP) , glucose oxidase, luciferase or the like. In such cases where the principal indicating group is an enzyme such as alkaline phosphatase, HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for alkaline phosphatase include p-nitrophenyl disodium phosphate, for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine, and for luciferase include luciferin and the like. An additional reagent useful with glucose oxidase is 2,2,-azino-di-(3-ethyl- benzthiazoline-G-Sulfonic acid (ABTS) . Radioactive elements are also useful labeling agents. An exemplary radio labeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is ¹²⁵I.

The linking of labels, i.e., labeling of, polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a components in the culture medium [see, for example, Galfre et al . (1981) Meth . Enzymol . 73:3-46]. The techniques of protein conjugation or coupling through activated functional groups are particularly applicable [see, for example, Aurameas et al. Scand. J. Immunol . 8(Suppl. 7):7-23; Rodwell et al . (1984) Biotech . 3:889-894; U.S. Pat. No. 4,493,795]. ELISA and RIA methods are preferred methods for determining amounts of free BPI and LBP.

Quantitatively determining the amount of BPI in the sample is also accomplished using, for example, flow cytometry methods which employ anti-BPI antibodies. Results using such a flow cytometry method are provided in the Experimental Details section, infra . The flow cytometry method is best suited for determining the amounts of cell surface- bound BPI. As used herein, BPI means native human BPI, which may be free (i.e., not cell-associated), neutrophil surface-bound, or contained within the neutrophil. BPI may either be the intact, full-length protein or any fragment or complex thereof. in one embodiment, the amount of BPI in the sample is the amount of free BPI in the sample. In another embodiment, the amount of BPI in the sample is the amount of neutrophil surface-bound BPI in the sample. The neutrophils may be isolated from sites where the accumulation of activated neutrophils is associated with a pathological state, as the transient accumulation of non-activated neutrophils may not indicate a pathological state. As an example, neutrophil surface-bound BPI in the lung can be measured to diagnose ARDS.

As used herein with respect to diagnosing an inflammatory disorder in a patient, a control sample comprises the average BPI or LBP level present in subjects known to have a particular inflammatory disorder, or known to have no inflammatory disorder (i.e., a positive or negative control may be used, or both a positive and negative control may be used) . As used herein, "subject" means a human. As used herein, "prognosis" means a forecast as to the recovery from the inflammatory disorder or to overcoming some or all symptoms of the inflammatory disorder. This invention will be better understood by reference to the Examples which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

EXAMPLE 1 QUANTITATIVE DETERMINATION OF BPI BY ELISA

Materials

Immulon-4 96-well microtitration plates (Dynatech Laboratories, Inc., Chantilly, VA, 011-010- 3750); 12-channel 50-200 μl pipettor; P20, P200, P1000 pipettors; micropipette and multichannel pipette tips; reagent reservoirs (Costar, Cambridge, MA) ; racked 1- ml tubes (BioRad, Richmond, CA) ; and polypropylene 15- ml conical tubes.

Reagents Coating Buffer: 25 mM Sodium Borate pH 9.5.

Blocking Buffer: 5% BSA (Sigma Chemical Co., St. Louis, MO, A-7030) in PBS.

Standard and sample diluent - Wash/Sample Buffer or appropriate solution for unknowns (e . g. , if testing tissue culture supernatants, use REM + dFBS) .

Wash/Sample Buffer: 50 mM Tris (pH 7.4), 500 mM NaCl, 1 mg/ml BSA (Sigma, fraction V, low endotoxin, A-3675), 0.05% Tween 80, 1 μg/ml Polymyxin B Sulfate (GIbco/BRL Life Technologies, Grand Island, NY, 7900 U/mg) .

Substrate Buffer: 24.5 g MgCl₂ (make 500 ml) , 48 ml Diethanolamine, bring up to -400 ml with deionized, reverse osmosis H₂0 (Lab V, Technic, Seattle, WA) , adjust to pH 9.8, bring up to 500 ml with deionized, reverse osmosis H₂0 (Lab V, Technic) .

Antibodies: INV 1-2 IgG (protein A purified rabbit anti-BPI antibody, 5 mg/ml) ; and biotinylated anti-BPI antibody. Polyclonal anti-BPI antibodies may be produced, for example, by immunizing a rabbit with BPI, and recovering the anti-BPI antibodies produced thereby using methods known to those skilled in the art.

Other Reagents

BPI standard (1.0 mg/ml aliquots stored at - 70°C) ; PNPP substrate tablets (5 mg/tablet: Sigma 104- 105) ; Streptavidin-Alkaline Phosphatase (BioRad, Richmond, CA) .

BPI may be obtained, for example, according to the method described at column 11, line 62 through column 12, line 13 of U.S. Patent No. 5,089,274. Alternatively, recombinant BPI may be utilized. Recombinant BPI can be produced by the following method.

BPI cDNA was obtained by screening a HL-60 cDNA library in ΛgtlO with 24-base probes based on a published sequence [Gray et al . (1989) J. Biol . Chem . 264(16) :9505-9509]. The insert DNA from the purified clone containing the largest insert was sequenced in puc-19 and found to be full length. The full length BPI cDNA was inserted into an expression vector containing an SV40 promoter and terminator, a mouse dihydofolate reductase (DHFR) gene, a bacterial origin of replication, and an ampicillin resistance gene. An 5' EcoRI site was created in the cDNA by ligation of a synthesized oligonucleotide linker, and a naturally- existing Bglll site was used for the 3'-end. The cDNA was inserted into the expression vector using EcoRI (5') and BamHI (3') restriction sites. The final constructs were transfected into DUKX B-ll, a DHFR' Chinese Hamster Ovary (CHO) cell line, using lipofectin. The transfected gene was amplified through multiple rounds of growth in increasing concentrations of methotrexate (MTX) . The final expression clone, 3A1, was selected in the presence of 2.0 μM MTX, and was adapted to serum-free growth conditions.

Recombinant BPI was captured from conditioned media on a CM-SEPHAROSE column (Pharmacia Fine Chemicals, Piscataway, NJ) equilibrated in 50 mM Tris, pH 7.4, (Buffer A). The column was washed in Buffer A and eluted with 1M NaCl in Buffer A. The CM- SEPHAROSE eluate was diluted 5-fold in 50 mM Tris, pH 8.5, (Buffer B) and passed over Q-SEPHAROSE (Pharmacia Fine Chemicals) equilibrated in Buffer B. Recombinant BPI in the Q-SEPHAROSE flowthrough was adsorbed to CM- SEPHAROSE equilibrated in Buffer B and eluted with 1M NaCl in Buffer A. The eluate from CM-SEPHAROSE was loaded on SEPHAROSE CL-6B equilibrated in 10 mM succinate (pH 6.0), 110 mM NaCl and the column was developed in the same buffer.

Procedure

Coating Plates: Note: Coat plates up to 1 month in advance. Store plates at 4°C until needed. Dilute anti-BPI IgG antibody (protein A-purified) to 5 μg/ml in 25 mM Na Borate, pH 9.5 (10 ml/plate). Add 100-μl to each well of 96-well plate (Immulon-4, Dynatech Laboratories, Inc.). Incubate overnight at 37°C. Refrigerate until use. Blocking Plate: Flick coating antibody out of plates and blot plate on paper towels. Add 200 μl 5% BSA in PBS to each well. Incubate 1-4 hours at 37°C or overnight at 4°C. Flick out blocking solution, wash 3X with ELISA buffer and blot plate on paper towels.

BPI Standards and Unknowns: Note - BPI standards and samples should be diluted only in propylene tubes. Thaw new standard aliquot (0.5 ml at 1 mg/ml) every 2 months. Make stock solution of purified BPI at 100 ng/ml by: a) diluting 149713 BPI 5 μl + 495 μl diluent (= 1:100; 10 μg/ml), then again 1:100 for a final concentration of 100 ng/ml in 1 ml (this is enough 100 ng/ml stock solution for 2 standard curves in triplicate) ; and b) making 1000 μl of each of the following standard concentrations as follows:

Final TBPI^") 100 ng/ml BPI Buffer (ul)

15 150 850

10 100 900

5 50 950

2 20 980

1 20 1980

0.5 0 1000 + 1000 of

1 ng/ml

0.25 0 1000 + 1000 of 0.5 ng/ml

0 0 1000

Add 100 μl/well standards and unknowns and incubate at 37°C for 2 hours. Wash 4X with Sample/Wash Buffer and vigorously blot on paper towels.

2nd Antibody (Conjugate) : After final wash, blot plate vigorously, add 100 μl of biotinylated anti-BPI Ab at 1:2000 (= 5 μl in 10 ml of wash/sample buffer) to each well. Incubate at 37°C for 1 hour, or overnight at 4°C. Wash 4X and blot dry.

Streptavidin Alkaline Phosphatase: After final wash, blot plate vigorously, add 100 μl 1:1000 (= 10 μl in 10 ml) Streptavidin-Alkaline Phosphatase conjugate to each well. Incubate at 37°C for 45 minutes, or overnight at 4°C. Wash 4X and blot dry.

Substrate: After final wash, blot plate vigorously, and add 100 μl substrate solution (2 x 5 mg PNPP substrate tablet/10 ml substrate buffer) .

NOTE: Prepare fresh substrate immediately before use by dissolving 2 tablets/10 ml substrate buffer.

Read plate at 405 nm after developing for 25-35 min. Keep plate in the dark between readings.

EXAMPLE 2

TIME COURSE OF BPI AND TNF RELEASE IN WHOLE BLOOD SAMPLES INCUBATED IN VITRO WITH VARYING CONCENTRATIONS OF BACTERIAL LPS Human heparinized whole blood was incubated for 4 hours at 37°C on a rocking platform with increasing concentrations of E. coli 055:B5 (0-10 ng/ml final) refined standard endotoxin (LPS)

(Whitaker Bioproducts, Walkersville, MD) . At the end of the incubation period, samples were centrifuged at

500 x g at 4°C, and plasma was collected and frozen on dry ice until assays for free BPI and TNF were performed. It was previously shown that BPI is released into the media of isolated neutrophils following stimulation with fMLP and cytochalasin B.

BPI is released by cells in whole blood in vitro following treatment with very low concentrations of

LPS. Results are shown in Figures 1A (BPI) and IB

(TNF) . EXAMPLE 3

CIRCULATING BPI LEVELS IN VIVO IN ENDOTOXIN-TREATED VOLUNTEERS

Male volunteers 19-35 years of age were _ screened for an unremarkable medical history and physically examination, and for normal leukocyte counts and normal lymphocyte subsets. Ten subjects with normal values were admitted to the Cornell Medical College Clinical Research Center on the day 0 prior to the study. Subjects fasted overnight. At 7 a.m. on the day of the study, radial arterial lines were placed in the subjects. 1.5 hours later, an arterial blood sample (10 ml) was collected (t= -0.5). 0.5 hours later, 20 IU/kg of National Reference E. 5 coli 0113 LPS (provided by Dr. H.D. Hochstein, Office of Biologies, U.S. Food and Drug Administration) was administered in an intravenous bolus in order to induce experimental endotoxemia. At various times after LPS administration as indicated, 10 ml arterial 0 blood samples were collected. Because arterial lines were removed at 8 hours, blood samples at later times were obtained by venipuncture. Free BPI was measured by ELISA.

Following experimental endotoxemia, free 5 plasma BPI increased in all normal volunteers. This was especially apparent at 6 and 12 hours after LPS administration when levels of plasma BPI were elevated 4- to 5-fold (peak 58 + 20 ng/ml at 6 hours post-LPS) . Although the overall response in free plasma BPI was statistically significant (p>0.01) by ANOVA, none of the individual time points post-LPS were significantly different from the baseline mean by Newman Keuls' post hoc analysis. This was due to apparent idiosyncratic responses of the endotoxin-challenged volunteers.

The results showed that BPI can be detected as early as 30 minutes following LPS treatment, and release is maximal at two hours. In contrast, TNF release is not detectable until 2 hours following LPS treatment, and the TNF response is not as sensitive to LPS as is the BPI release. These results demonstrate that BPI is released in vivo in response to lower LPS concentrations than those required to trigger the release of TNF.

BPI levels in whole blood samples were also measured, wherein the samples were incubated with increasing concentrations of the human monoclonal anti-endotoxin antibody HA-1A (Centocor, Malvern, PA) . Interestingly, the more HA-1A present in conjunction with LPS, the more BPI is released. This effect was not seen using a human IgM antibody alone, indicating that immune complexes are effective in stimulating BPI release from human neutrophils in vitro, and that HA- 1A does not neutralize LPS-stimulation of human neutrophils in vitro. Results are shown in Figure 2.

EXAMPLE 4

POLYMORPHONUCLEAR LEUKOCYTE SURFACE BPI MEASURED IN ENDOTOXIN-TREATED HUMAN VOLUNTEERS

BPI levels on circulating PMN from volunteers as described above were measured by flow cytometric analysis. Surface BPI levels in endotoxin- treated volunteers were elevated approximately 1.5- fold from 1 to 6 hours after LPS was given. Samples at times later than 6 hours after LPS administration were not evaluated. (*p<0.01 versus t= -0.5 hours by 1-way, repeated measures analyzed by ANOVA and Newman- Keul's Test). Results are shown in Figure 3.

EXAMPLE 5

SURFACE BPI ON PERIPHERAL BLOOD PMN LEUKOCYTES IN PATIENTS WITH GRAM-NEGATIVE SEPSIS

Septic patients also manifested increased

PMN leukocyte surface BPI. However, in contrast to experimental endotoxemia where PMN cell BPI increased about 1.5-fold, these seriously ill patients expressed

PMN leukocyte surface BPI levels that were approximately 3-fold greater than the control levels.

There were no discernible differences in PMN leukocyte surface BPI levels between patients who subsequently survived and those who did not. Patients studied after recovery manifested PMN leukocyte surface BPI levels similar to those of normal volunteers studied prior to LPS administration. One of the ICU patients 5 who initially entered the study subsequently did not meet the gram-negative infection criteria. Importantly, this patient had a surface PMN leukocyte BPI level that was no different from that of baseline normal subjects prior to LPS challenge. Results are 10 shown in Figure 4.

EXAMPLE 6

CIRCULATING FREE BPI IN PATIENTS WITH GRAM-NEGATIVE SEPSIS

15 Twenty consecutive patients with a clinical gram-negative sepsis according to the Zeigler criteria were included. A proven gram-negative focus was found in 17 to 20 patients and 6 of them also had a positive blood culture for gram-negative bacteria. At

_₀ inclusion, significantly higher levels of BPI were detected in the survivors when compared to non- survivors. These data indicate that BPI levels have prognostic value in gram-negative sepsis. Results are shown in Figures 5A (free BPI) and 5B (neutrophil

₂₅ counts) .

EXAMPLE 7 DETERMINING THE BPI/LBP RATIO IN BIOLOGICAL FLUIDS Biological fluid samples were obtained from _₀ 49 consecutive patients with an average age of 68 ± 11 years and that consisted of 23 male and 26 female patients. The samples were obtained in the operating room or invasive radiology department from these patients who were hospitalized at an acute care _₅ medical-surgical hospital in Rhode Island. The samples were identified and processed as soon as possible (within 2 hours in each case) after their collection. Specimens were transferred into sterile, endotoxin-free tubes and centrifuged at 15,000 r.p.m. for 15 minutes at 4°C. Supernatants were then filtered using a 0.45 micron filter (Gelman Sciences, Ann Arbor, Michigan) and then frozen at -70°C for subsequent analysis. The medical records and clinical laboratory records for each patient were then compared in order to correlate the clinical data with the cell count, gram stain, and culture results for each biological fluid analyzed.

BPI levels were measured by sandwich ELISA using rabbit polyclonal antibodies against human BPI and LBP raised in rabbits as primary and secondary antibodies [Marra et al . (1992) J. Immunol . 148:518]. LBP levels were measured by sandwich ELISA using rabbit polyclonal antibodies against human LBP raised in rabbits as primary and secondary antibodies. Microtitration plates were developed with polyclonal rabbit anti-LBP IgG coupled to biotin, followed by streptavidin-alkaline phosphotase conjugate (Gibco/BRL) and with p-nitrophenyl disodium phosphate as a substrate (Sigma) . Absorbances were determined at 405 nm on a V^ kinetic microtitration plate reader (Molecular Devices, Menlo Park, CA) . The sensitivity of assays for both BPI and LBP was 2.5 ng/ml.

Recombinant BPI standards were prepared as described in Example 1. Recombinant LBP for the preparation of LBP standards was produced by the following method. A cDNA clone encoding human LBP was obtained by PCR using human liver poly-A+ cDNA (Clontech, Inc. , Palo Alto, CA) as a target and primers based on the published sequence [Schumann et al . (1990) Science 249:1429]. In addition, the primers contained external restriction sites for Nhe I (5') and Xho I (3') to permit insertion into the expression plasmid. The expression plasmid used, pCEP4 (Invitrogen, Inc., San Diego, CA) had been prepared for acceptance of the insert by prior digestion with Nhe I and Xho I. Recombinant clones containing the LBP insert DNA were characterized by agarose gel electrophoresis and verified by DNA sequencing. Purified plasmid DNA from one clone was used to transfect T-antigen containing human kidney (293-EBNA) cells, using lipofection (Gibco/BRL) . Cultures were selected for recombinants in growth medium containing 200 μg/ml hygromycin and 250 μg/ml Geneticin (Gibco/BRL) . Media supernatants were tested for the presence of LBP by ELISA, using polyclonal anti-LBP IgG.

Recombinant LBP was captured from media conditioned by the 293 human kidney cell line by passing the media over a S-SEPHAROSE (Pharmacia) column equilibrated in 50 mM Tris (pH 7.4) (Buffer A). The column was washed in Buffer A and eluted with 0-1 M gradient of NaCl in Buffer A. The fractions from S- SEPHAROSE containing LBP were pooled, diluted 10-fold in 50 mM Tris (Ph 8.5) (Buffer B) and passed over a column of Q-SEPHAROSE equilibrated in Buffer B. The anion exchange column was washed in Buffer B and eluted with a 0-1 M gradient of NaCl in Buffer B. Fractions containing purified LBP were pooled. Microtitration plates were developed with polyclonal rabbit anti-BPI/LBP IgG coupled to biotin, followed by streptavidin-alkaline phosphatase conjugate (Gibco/BRL) as described above. Absorbance was determined as described above. Statistical comparisons for multiple groups were analyzed by a non-parametric Kruskal-Wallis one¬ way analysis of variance. Differences between two groups were compared by the Mann-Whitney U test. Correlations between neutrophil quantity in BPI or LBP levels were calculated by the Spearman rank correlation coefficient method. Numeric results are recorded as the mean ± standard error and a probability of less than 0.05 was considered statistically significant. The 49 samples were classified in three groups which included 17 samples from closed space abscesses, 10 samples from primary or secondary peritonitis fluids, and 22 samples from non-infected body fluids (pleural fluid - 9; joint fluid - 7; peritoneal fluid - 6) . None of the patients were bacteremic at the time of the tissue fluid analysis. The microbiology of infected fluids revealed E. coli was the most common isolated organism (9) ; followed by Bacteroides fragilis (8) ; Staphylococcus aureus (4) ; Peptostreptococci (4) ; Enterococci (3) ; other gram- negative enteric bacteria (3) ; and miscellaneous bacteria (7) . Seven abscesses contained more than one organism. The relative concentrations of BPI and LBP in body fluids varied strikingly depending upon the presence or absence of infection. BPI levels exceeded LBP levels in 16 of 17 fluids from closed space infections, while BPI and LBP levels were present in similar concentration in peritoneal fluids of patients with primary or secondary peritonitis (Table 2) . BPI/LBP ratio was <1:10 in 20 of 22 non-infected body fluids. The relative concentration of BPI and LBP and the corresponding BPI/LBP ration in each category of body fluid is summarized in Table 2. The differences between the BPI/LBP ratio were highly statistically significant between the three types of body fluids (Kruskal-Wallis "H" value with 2 degrees of freedom = 36.24; p<0.0001). BPI levels tended to be higher in abscess cavities which contained gram-negative or mixed infections (n=8) when compared to gram-positive or sterile abscess cavities (n=9) (15.77 ± 5.9 μg/ml versus 3.36 ± 2.25 mcg/ml; p=0.07 by Mann Whitney U test) . BPI levels were significantly lower in non- infected fluids (0.165 ± ug/ml) than in infected fluids (peritonitis - 2.25 ± ug/ml; or abscess - 8.36 ± ug/ml; p<0.005). LBP levels were significantly lower in abscess cavities p<0.005) than in other body fluids (Table 2) .

TABLE 2

THE LEVELS OF BPI AND LBP IN EXTRAVASCULAR FLUIDS

BPI/LBP

BODY FLUID N BPI (μg/ml) LBP (μg/ml) RATIO^*

CLOSED-SPACE 17 8.36±3.15 1.19±0.26 200.9± 180.9 INFECTIONS

PERITONITIS 10 2.25±0.53 7.70±2.38 0.304±0.06

STERILE BODY 22 0.165 ±0.07 5.02±0.91 0.053 ±0.02 FLUIDS

BPI-BACTERIAL/PERMEABILITY-INCREASING PROTEIN; LBP-LIPOPOLYSACCHARIDE BINDING PROTEIN; 'BPI/LBP RATIOS SIGNIFICANTLY DIFFERED BETWEEN THE THREE BODY FLUIDS (P<0.0001)

The levels of BPI were directly proportional to the quantity of white blood cells (WBC) within body fluids. The correlation coefficient between BPI levels and WBC levels within body fluids revealed an r value of +0.81 (p<0.0001) yet no relationship was identified between LBP levels and the quantity of white blood cells in body fluids (r=0.04; p=0.73). The relationship between the quantity of white blood cells and BPI/LBP levels is demonstrated in Figure 6. BPI levels varied from as little as 9.2 ±

3.9 ng/ml in fluids without WBC's to up to 1000-fold higher levels in clinical samples with 4+ WBC's (9.1 ± 1.4 ug/ml) .

Claims

WHAT IS CLAIMED IS:

1. A method for diagnosing and predicting the outcome of an inflammatory disorder in a subject, which comprises:

(a) obtaining a bodily fluid sample from the patient;

(b) quantitatively determining the amount of BPI in the sample; and then (c) comparing the amount of BPI present in the sample with levels of BPI in samples obtained from normal subjects and with levels of BPI in samples obtained from individuals suffering from an inflammatory disorder in order to diagnose and predict the outcome of an inflammatory disorder in the subject.

2. The method of claim 1, wherein the inflammatory disorder is systemic inflammatory response syndrome.

3. The method of claim 1, wherein the inflammatory disorder is adult respiratory distress syndrome.

4. The method of claim 1, wherein the inflammatory disorder is a microbial infection.

5. The method of claim 4, wherein the microbial infection is sepsis.

6. The method of claim 4, wherein the etiological agent causing the microbial infection is bacterial.

7. The method of claim 6, wherein the etiological agent is a gram-negative bacteria.

8. The method of claim 1, wherein the bodily fluid sample selected from the group consisting of urine, plasma, whole blood, CSF, pus, sputum, inflammatory exudate, bronchoalveolar lavage fluid, joint fluid, peritoneal fluid, bursa fluid, pleural fluid, pericolonic fluid, periappendiceal fluid, ascitic fluid, pericardial fluid and abscess drainage.

9. The method of claim 1, wherein the BPI measured is the free BPI in the sample.

10. The method of claim 1, wherein the BPI measured is the neutrophil surface-bound BPI in the sample.

11. The method of claim 1, further wherein the amount of BPI/enodtoxin in the sample is determined and compared to the amount of free BPI present in the sample.

12. A method for diagnosing and predicting the outcome of an inflammatory disorder in a subject, which comprises:

(a) obtaining a bodily fluid sample from the subject; (b) quantitatively determining the amount of

BPI and LBP in the sample;

(c) determining the ratio of BPI to LBP in the sample; and then

(d) comparing the resulting ratio of BPI to LBP with BPI:LBP ratios obtained from normal subjects and from individuals suffering from an inflammatory disorder in order to diagnose and predict the outcome of the inflammatory disease in the subject.

13. The method of claim 14, wherein the inflammatory disorder is systemic inflammatory response syndrome.

14. The method of claim 12, wherein the inflammatory disorder is adult respiratory distress syndrome.

15. The method of claim 12, wherein the inflammatory disorder is a microbial infection.

16. The method of claim 15, wherein the microbial infection is sepsis.

17. The method of claim 15, wherein the etiological agent causing the microbial infection is bacterial.

18. The method of claim 17, wherein the etiological agent is a gram-negative bacterium.

19. The method of claim 12, wherein the bodily fluid sample is selected from the group consisting of urine, plasma, whole blood, CSF, pus, sputum, inflammatory exudate, bronchoalveolar lavage fluid, joint fluid, peritoneal fluid, bursa fluid, pleural fluid, pericolonic fluid, periappendiceal fluid, ascitic fluid, pericardial fluid and abscess drainage.

20. The method of claim 12, wherein the BPI measured is the free BPI in the sample.

21. The method of claim 12, wherein the BPI measured is the neutrophil surface-bound BPI in the sample.