EA045401B1 - METHODS FOR QUANTITATIVE DETERMINATION OF INTER-ALPHA INHIBITORY PROTEINS - Google Patents

Description

State of the art

IAIPs (inter-alpha inhibitory proteins) are a family of naturally occurring, immunomodulatory plasma proteins that circulate in high concentrations in the blood of all mammals. IAIPs are mainly produced in the liver, enter the blood, and their subunit (bikunin) is excreted in the urine. IAIPs play an important role in modulating inflammation. They have broad protective effects against severe inflammation caused by infection, trauma, and injury, and importantly, the protective effects of IAIPs are independent of pathogenic microbial agents or triggers. Members of this family consist of heavy and light polypeptide subunits that are covalently linked by glycosaminoglycan. IAIPs can be found in vivo as inter-alpha inhibitor (IaI), a 250 kDa molecule consisting of two heavy chains (H1 and H2) and one light chain (L), and pre-alpha inhibitor (PaI), a molecule the size 125 kDa, consisting of one heavy (H3) and one light chain (L).

When the body generates inflammatory signals, such as those caused during injury or infection, IAIPs enter the tissue and directly reach the site of inflammation. IAIP heavy chains enhance the anti-inflammatory response by binding to proteins that are part of the inflammatory cascade. In addition, when the heavy chains are cleaved, a light chain with its associated GAG (called bikunin due to its two Kunitz-type domains) is released and the serine protease inhibitory activity of this light chain is activated. Bikunin inhibits the activity of serine proteases such as trypsin, elastase, plasmin, cathepsin G and furin. IAIPs exert their anti-inflammatory effects through multiple mechanisms. They have been shown to bind proteins that increase inflammation, such as complement proteins and extracellular histones (damage signals), thereby attenuating inflammatory processes. Through heavy chains, IAIPs can bind extracellular matrix (ECM) proteins and have been shown to promote lung epithelial repair after injury in both in vitro and in vivo models. IAIP has been shown to reduce inflammatory cytokines such as TNF-α and IL-6 in several in vivo models. Bikunin-deficient (and therefore IAIP-deficient) mice have been shown to have decreased inflammatory markers of cell adhesion, VCAM-1 and ICAM-1.

In healthy individuals, the amount of IAIP circulating in the blood is relatively high (in the range of 400-800 mg/l). However, IAIP levels decrease rapidly during systemic inflammation/sepsis in neonates and adults (Waeck YW, et al. J Pediatr. 2003; 143:11-15; Lim YP, et al. J Infect Dis. 2003; 188:919 -926 and Opal SM, et al. Crit Care Med. 2007;35:387-392), and decreased IAIP levels have been shown to strongly correlate with disease progression. As diseases progress to more serious and life-threatening stages, IAIP levels drop rapidly, suggesting that IAIP has clinical value as a prognostic and therapeutic marker, helping clinicians monitor disease progression and make informed treatment decisions for such diseases. , as severe inflammatory diseases such as severe pneumonia, sepsis and associated organ damage, NEC (necrotizing enterocolitis), wound healing, burn, cancer, stroke, Alzheimer's disease, epilepsy and others.

A standardized IAIP competitive immunoassay was used to measure IAIP in blood samples obtained from patients with systemic inflammation following bacterial and viral infections. The IAIP competitive immunoassay provides a measurement of IAIP that detects only the light chain, so this assay detects both intact IAIP and cleaved bikunin. The competitive assay does not detect other key parts of IAIP that are very important for its anti-inflammatory and tissue repair properties; namely heavy chains and glycosaminoglycan. Thus, the IAIP competitive immunoassay has limitations in assessing active IAIP in a patient sample. Given the importance of IAIP as a critical component of the body's innate immune defenses and its potential use as a prognostic biomarker, there is a need for improved methods for quantifying IAIP. An ideal assay would measure both light chain and heavy chain subunits to capture the entire molecule.

The essence of the invention

The present invention provides methods for quantifying IAIP in a sample from a subject by direct detection with agents that bind to and detect IAIP (eg, agents that bind to intact IAIP, IAIP heavy chain, or glycosaminoglycan (GAG) from IAIP). Quantitative methods may be used to evaluate, diagnose, treat, or monitor subjects or to assess the severity of a disease or the effectiveness of a treatment in a subject. The invention also provides kits that can be used to quantify IAIP in accordance with the methods described herein.

A first aspect provides a method for quantifying inter-alpha inhibitory protein (IAIP) in a sample from a subject by: a) contacting the sample with a binding agent to produce an IAIP-binding agent complex, wherein the binding agent is bound to a support; b) contacting the IAIP-binding agent complex with the detection agent; and c) determining the amount of detection agent bound to the IAIP-binding agent complex to quantify the IAIP in the sample.

In some embodiments, the IAIP is intact IAIP.

In some embodiments, the binding agent is an IAIP ligand that binds to IAIP. In some embodiments, the binding agent is an antibody that specifically binds to IAIP.

In some embodiments, the detection agent is or contains an IAIP ligand. In some embodiments, the detection agent further comprises an antibody that binds to an IAIP ligand detecting agent (eg, an IAIP ligand). In some embodiments, the detection agent is an antibody that specifically binds to IAIP. For example, in specific embodiments, the binding agent is an antibody that specifically binds to IAIP (e.g., mAb 69.26 or mAb 69.31), and the detection agent is or contains an IAIP ligand that binds to IAIP (e.g., heparin, hyaluronic acid, endotoxin (LPS) or histone). In other embodiments, the binding agent is an IAIP ligand that binds to IAIP (eg, heparin, hyaluronic acid, LPS, or histone), and the detection agent is an antibody that specifically binds to IAIP (eg, mAb 69.26 or mAb 69.31).

In some embodiments, IAIP is present in an IAIP-IAIP ligand complex.

In some embodiments, the binding agent is an IAIP ligand that binds to IAIP. In some embodiments, the IAIP ligand of the IAIP-IAIP ligand complex is different from the binding agent. In some embodiments, the binding agent is an antibody that binds to the IAIP ligand of the IAIP-IAIP ligand complex. In some embodiments, the binding agent is an antibody that specifically binds to the IAIP of the IAIP-IAIP ligand complex.

In some embodiments, the detection agent contains an IAIP ligand that binds to the IAIP. In some embodiments, the detection agent further comprises an antibody that binds to the IAIP ligand detection agent. In some embodiments, the IAIP ligand of the IAIP-IAIP ligand complex is different from the IAIP ligand detecting agent. In some embodiments, the detection agent is an antibody that binds to the IAIP ligand of the IAIP-IAIP ligand complex. In some embodiments, the detection agent is an antibody that specifically binds to the IAIP complex of the IAIP ligand IAIP.

In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is mAb 69.26 or mAb 69.31.

In some embodiments, the IAIP ligand is selected from the group consisting of endotoxin (LPS), heparin, histone, hyaluronic acid, vitronectin, fibronectin, laminin, tenascin C, aggrecan, von Willebrand factor, pentraxin-3 (PTX3), TNF-stimulated gene -6 (TSG-6), factor IX, complement component, factor XIIIa and tissue transglutaminase. In some embodiments, the complement component is C1q, C2, C3, C4, C5, C6, C8, properdin, or factor D.

In some embodiments, the detection agent comprises a label. In some embodiments, the label is biotin, an enzyme, an enzyme substrate, a radiolabel, a luminescent compound, colloidal gold, a particle, or a fluorescent dye.

In some embodiments, the support is a plate, particle, nanoparticle, resin, membrane, biochip, container, test strip, or bead.

In some embodiments, the method further includes a washing step between steps a) and b).

In some embodiments, the method further includes a washing step between steps b) and c).

In some embodiments, the method further includes a blocking step before step a) or step b).

In some embodiments, the contacting in steps a) and/or b) is carried out at a pH of from about 7.0 to about 3.5. In some embodiments, the pH is from about 5.0 to about 3.5. In some embodiments, the pH is 4.0.

In some embodiments, the sample is a liquid. In some embodiments, the fluid is blood, plasma, serum, urine, cerebrospinal fluid, synovial fluid, amniotic fluid, interstitial fluid, follicular fluid, peritoneal fluid, bronchoalveolar lavage fluid, breast milk, sputum, lymph, bile, or tissue homogenate.

In some embodiments, the subject is a human subject. In some embodiments, the subject has been identified as having or being at risk

- 2 045401 development of an inflammatory disease, or pathological condition, or infection. In some embodiments, the subject has not been identified as having or at risk of developing an inflammatory disease or condition or infection. In some embodiments, the method is performed before, after, or simultaneously with diagnosing a subject as having or at risk of an inflammatory disease or condition or infection. In some embodiments, the method is performed substantially simultaneously with treating the subject for an inflammatory disease or condition or infection. In some embodiments, the method is performed before treating the subject for an inflammatory disease or condition or infection. In some embodiments, the method is performed after the subject has been treated for an inflammatory disease or condition or infection.

In some embodiments, the inflammatory disease or condition is selected from the group consisting of sepsis, septic shock, sterile sepsis, trauma, injury, stroke, acute inflammatory disease, SIRS, acute lung injury, ARDS, pneumonia, necrotizing enterocolitis, acute pancreatitis, disease kidney, acute kidney injury, liver injury, acute circulatory failure, preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral arterial disease, type 1 or type 2 diabetes, atherosclerosis of the cardiovascular system, intermittent claudication, critical ischemia limbs, myocardial infarction, carotid occlusion, umbilical cord occlusion, low birth weight, prematurity, surgically induced inflammation, abscess-induced inflammation, multiple sclerosis, pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia, bacterial infection, wounds, burns, lacerations, contusions, bone fractures, surgical procedures, tissue ischemia, rheumatoid arthritis, meningitis, inflammatory bowel disease, chronic obstructive pulmonary disease, rhinitis, premature birth or infectious disease.

In some embodiments, the infection is caused by gram-negative bacteria, such as Neisseria species, including Neisseria gonorrhoeae and Neisseria meningitidis, Branhamella species, including Branhamella catarrhalis, Escherichia species, including Escherichia coli, Enterobacter species, Proteus species, including Proteus mirabilis, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas mallei, and Pseudomonas pseudomallei, Klebsiella spp., including Klebsiella pneumoniae, Salmonella spp., Shigella spp., Serratia spp., Acinetobacter spp.; Haemophilus spp., including Haemophilus influenzae and Haemophilus ducreyi, Brucella spp., Yersinia spp., including Yersinia pestis and Yersinia enterocolitica, Francisella spp., including Francisella tularensis, Pasturella spp., including Pasteurella multocida, Vibrio cholerae, Flavobacterium spp., meningosepticum, Campylobacter spp., including Campylobacter jejuni, Bacteroides spp. (oral, pharyngeal), including Bacteroides fragilis, Fusobacterium spp., including Fusobacterium nucleatum, Calymmatobacterium granulomatis, Streptobacillus spp., including Streptobacillus moniliformis, and Legionella spp., including Legionella pneumophila.

In some embodiments, the subject is a newborn, child, adolescent, or adult.

In some embodiments, the method is performed one or more times per year. In some embodiments, the method is performed one or more times per month. In some embodiments, the method is performed one or more times per week. In some embodiments, the method is performed one or more times per day. In some embodiments, the method is performed one or more times per hour.

In some embodiments, the method is performed at least once, at least twice, at least three times, at least five times, or at least ten times.

In some embodiments, the method further comprises administering to the subject a formulation comprising an IAIP or therapeutic agent. In some embodiments, the subject has an IAIP concentration of 200 μg/ml or lower. In some embodiments, the sample from the subject has an elevated level of the IAIP-IAIP ligand complex relative to the reference sample. In some embodiments, the subject has or is at risk of developing an inflammatory disease or condition or infection.

In some embodiments, the method includes administering the IAIP and a therapeutic agent to a subject.

In some embodiments, the therapeutic agent is selected from the group consisting of an antibiotic agent, an antiviral agent, an antifungal agent, an antiparasitic agent, an anti-inflammatory agent, an anticancer agent, an anticoagulant, an immunomodulatory agent, a bronchodilator, a complement inhibitor, a vasopressor, a sedative, or a ventilator.

In some embodiments, the subject has been sick for at least one day. In some embodiments, the subject has been sick for at least one week. In some embodiments, the subject has been sick for at least one month. In some embodiments, the subject has been ill for at least one year.

In some embodiments, the method is designed to: a) assess the health status of a subject; b) monitoring the health status of the subject; c) diagnosing the subject as having or at risk of an inflammatory disease or condition or infection; d) assessing the effectiveness of treatment administered to the subject; or e) assessing the severity of the disease in the subject.

In some embodiments, the method further includes comparing the amount of IAIP and/or IAIP-IAIP ligand complex found in the sample with the amount of IAIP and/or IAIP ligand IAIP complex found in the sample from a normal subject, or a threshold value. In some embodiments, an amount of IAIP in a sample that is lower than an amount of IAIP in a sample from a normal subject or relative to a threshold value indicates that the subject has or is at risk of developing an inflammatory disease or condition or infection. In some embodiments, an amount of IAIP-IAIP ligand complex in a sample that is higher than the amount of IAIP-IAIP ligand complex in a sample from a normal subject or relative to a threshold value indicates that the subject has or is at risk of developing an inflammatory disease or condition , or infections. In some embodiments, the amount of IAIP in a sample obtained from a normal subject, or threshold value, is >250 μg/ml. In some embodiments, the amount of IAIP in a sample from a normal subject is from about 260 to about 540 μg/ml.

In some embodiments, determining that a subject has an IAIP concentration of 250 μg/mL or less indicates that the subject has or is at high risk of developing an inflammatory disease or condition or infection, or is diagnosed with an increased risk of disease and/or mortality.

In some embodiments, the subject has an IAIP concentration of 200 to 300 μg/ml. In other embodiments, the method is performed at least once a year, at least twice a year, at least once a month, at least once a week, at least once a day, or at least once an hour.

In some embodiments, the subject has a pre-existing inflammatory disease or condition or infection.

In some embodiments, the method is performed prior to treatment and/or one or more times during the course of treatment. In some embodiments, the method is performed after treatment has begun and/or after treatment has completed. In some embodiments, treatment is considered effective if the concentration of IAIP increases in the subject relative to the result of a preliminary measurement of IAIP in the subject and/or if the concentration of the IAIP-IAIP ligand complex in the subject decreases relative to the result of the preliminary measurement of the IAIP-IAIP ligand complex in the subject. In some embodiments, treatment is considered ineffective if the concentration of IAIP decreases or remains constant in the subject relative to the result of the preliminary measurement of IAIP in the subject and/or if the concentration of the IAIP-IAIP ligand complex increases or remains constant in the subject relative to the result of the preliminary measurement of the IAIP-IAIP ligand complex in subject. In some embodiments, the method further includes modifying or changing the treatment.

A second aspect provides a method of treating a subject (e.g., a human such as a newborn, child, adolescent, or adult) who has or is at risk of developing an inflammatory disease or infection in which the subject has been determined to be in need of treatment in accordance with the method of the first aspect by administering to the subject a therapeutically effective amount of IAIP and/or a therapeutic agent selected from the group consisting of an antibiotic agent, an antiviral agent, an antifungal agent, an antiparasitic agent, an anti-inflammatory agent, an anticancer agent, an anticoagulant, an immunomodulatory agent, a bronchodilator, a complement inhibitor, a vasopressor , sedation or artificial ventilation.

In some embodiments, the inflammatory disease or condition is selected from the group consisting of sepsis, septic shock, sterile sepsis, trauma, injury, stroke, acute inflammatory disease, SIRS, acute lung injury, ARDS, pneumonia, necrotizing enterocolitis, acute pancreatitis, disease kidney, acute kidney injury, liver injury, acute circulatory failure, surgery-induced inflammation, abscess-induced inflammation, multiple sclerosis, preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral arterial disease, type 1 or type 2 diabetes, atherosclerosis cardiovascular system, intermittent claudication, critical limb ischemia, myocardial infarction, carotid occlusion, umbilical cord occlusion, low birth weight, prematurity, pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia, bacterial infection, wounds, burns, lacerations, bruises, bone fractures, surgical procedures, tissue ischemia, rheumatoid arthritis, meningitis, inflammatory bowel disease, chronic obstructive pulmonary disease, rhinitis, premature birth or infectious disease.

In some embodiments, the infection is caused by gram-negative bacteria, such as Neisseria species, including Neisseria gonorrhoeae and Neisseria meningitidis, Branhamella species, including Branhamella catarrhalis, Escherichia species, including Escherichia coli, Enterobacter species, Proteus species, including Proteus mirabilis, Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonas mallei, and Pseudomonas pseudomallei, Klebsiella spp., including Klebsiella pneumoniae, Salmonella spp., Shigella spp., Serratia spp., Acinetobacter spp.; Haemophilus spp., including Haemophilus influenzae and Haemophilus ducreyi, Brucella spp., Yersinia spp., including Yersinia pestis and Yersinia enterocolitica, Francisella spp., including Francisella tularensis, Pasturella spp., including Pasteurella multocida, Vibrio cholerae, Flavobacterium spp., meningosepticum, Campylobacter spp., including Campylobacter jejuni, Bacteroides spp. (oral, pharyngeal), including Bacteroides fragilis, Fusobacterium spp., including Fusobacterium nucleatum, Calymmatobacterium granulomatis, Streptobacillus spp., including Streptobacillus moniliformis, and Legionella spp., including Legionella pneumophila.

In a third aspect, a kit is provided for the quantitation of IAIP or the IAIP ligand IAIP complex in a sample, the kit comprising an IAIP binding agent and an IAIP detecting agent, and optionally one or more of the following: a wash buffer, a blocking agent, a label detection substrate and instructions for quantifying the level of IAIP or the IAIP-IAIP ligand complex in a sample. One or more kit components may be provided in a container, such as a tube or vial, and/or in a form ready for use (eg, for application to a kit support (eg, a plate or test strip).

In some embodiments, the binding agent is immobilized on the support.

In some embodiments, the detection agent is labeled.

In some embodiments, the IAIP binding agent is an IAIP-specific antibody or IAIP ligand.

In some embodiments, the kit further comprises an IAIP ligand binding agent. In some embodiments, the IAIP ligand binding agent is an antibody that binds to the IAIP ligand.

In some embodiments, the IAIP detecting agent is an IAIP-specific antibody or IAIP ligand.

In some embodiments, the kit further comprises an IAIP ligand detecting agent. In some embodiments, the IAIP ligand detecting agent is an antibody that specifically binds to the IAIP ligand.

In some embodiments, the IAIP-specific antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is mAb 69.26 or mAb 69.31.

In some embodiments, the support is a plate, resin, container, membrane, biochip, particle, nanoparticle, test strip, or bead.

In some embodiments, the label is an enzyme, enzyme substrate, biotin, particle, fluorescent dye, luminescent compound, or radioactive label.

In some embodiments, the IAIP ligand is selected from the group consisting of endotoxin (LPS), heparin, histone, hyaluronic acid, laminin, tenascin C, aggrecan, vitronectin, fibronectin, von Willebrand factor, pentraxin-3 (PTX3), TNF-stimulated gene -6 (TSG6), factor IX, complement component, factor XIIIa and tissue transglutaminase.

Definitions.

As used herein, the term about refers to a value that is no more than 10% greater or less than the value described. For example, the term about 5 nM means the range from 4.5 nM to 5.5 nM.

As used herein, the term administration refers to providing or providing a therapeutic agent (eg, IAIP) to a subject by any effective means. Exemplary routes of administration are described herein below.

As used herein, the term antibody (Ab) refers to an immunoglobulin molecule that specifically binds or is immunologically capable of reacting with a particular antigen, and contains at least a heavy chain variable domain, and typically contains at least the heavy chain and light chain variable domains of an immunoglobulin . Antibodies and antigen binding fragments, variants or derivatives thereof include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, heteroconjugate antibodies (e.g. bi-, tri- and tetraspecific antibodies, diabodies, tri-bodies and tetrabodies ), single domain antibodies (sdAb), epitope binding fragments, e.g. Fab, Fab' and F(ab') ₂ , Fd, Fv, single chain Fv (scFv), rlgG, single chain antibodies, disulfide-linked Fv (sdFv), fragments, containing either the V _L or VH domain, fragments produced by the Fab expression library, and anti-idiotypic (anti-Id) antibodies. The antibody molecules of the invention can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1,

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IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In addition, unless otherwise specified, the term monoclonal antibody (mAb) includes both intact molecules and antibody fragments (such as, for example, Fab and F(ab') ₂ fragments) that are capable of specifically binding to the target protein. The Fab and F(ab') ₂ fragments lack the Fc fragment of the intact antibody, are cleared more quickly from the animal's circulation, and may have less nonspecific tissue binding than the intact antibody.

As used herein, the term antigen-binding fragment refers to one or more antibody fragments that retain the ability to specifically bind a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibody fragments may be a Fab, F(ab') ₂ , scFv, SMIP, diabody, tribody, affibody, nanoantibody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term antigen-binding antibody fragment include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the _VL , VH, _CL , and CH1 domains; (ii) F(ab') ₂ fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the V _L and VH domains of one arm of the antibody, (v) a dAb including the V _L and VH domains; (vi) dA b fragment (Ward et al., Nature 341:544-546, 1989), which consists of VH domains; (vii) dAt, which consists of a VH or _VL domain; (viii) a dedicated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains of the Fv fragment, _VL and _VH , are encoded by separate genes, they can be joined recombinantly by a synthetic linker that allows them to create a single protein chain in which the pair of VL and VH regions form monovalent molecules ( known as single-chain Fv (scFv); see, for example, Bird et al., Science 242:423426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be prepared using conventional techniques known to those skilled in the art, and the fragments can be screened for functionality in the same manner as for intact antibodies.

Antigen binding fragments can be produced using recombinant DNA techniques, enzymatic or chemical digestion of intact immunoglobulins, or, in some embodiments, using chemical peptide synthesis methods known in the art.

As used herein, the term inter-alpha inhibitory proteins or IAIPs refers to large multicomponent glycoproteins in a family of structurally related immunomodulatory proteins. IAIPs have been shown to play an important role in the inhibition of a number of proteases, including neutrophil elastase, plasmin, trypsin, chymotrypsin, granzyme K, proprotein convertase, furin, cathepsin G and acrosin. IAIP has a wide range of anti-inflammatory mechanisms, in addition to serine protease inhibitory activity, such as binding and inactivating complement, extracellular histones and coagulation factors, reducing pro-inflammatory cytokines such as TNF-alpha and IL-6, reducing adhesion factors such as VCAM and ICAM, and a decrease in the amount of NFkB. IAIPs also play an important role in promoting tissue protection and repair, where heavy chains are transferred to matrix proteins to promote cell migration and proliferation. In human plasma, IAIPs are found at relatively high concentrations (400-800 mg/L). Unlike other inhibitor molecules, this family of inhibitors typically includes a combination of polypeptide chains (light and heavy chains) covalently linked by a chondroitin sulfate chain. IAIP heavy chains (H1, H2 and H3) are also called hyaluronic acid (HA) binding proteins. The main forms of IAIP found in human plasma are inter-alpha inhibitor (IaI), which contains two heavy chains (H1 and H2) and one light chain (L), and pre-alpha inhibitor (PaI), which contains one heavy chain (H1 and H2). H3) and one light chain (L). Another IAIP is a light chain (also called bikunin (bi-kunitz inhibitor) with two Kunitz domains) linked to glycosaminoglycan, which is known to broadly inhibit plasma and tissue serine proteases. Another IAIP is an H4 heavy chain-linked molecule that circulates in the blood without binding to bikunin. Another IAIP is a molecule associated with the H5 heavy chain. IaI and PaI present in the plasma fraction have apparent molecular weights ranging from about 60 kDa to about 280 kDa.

As used herein, the term IAIP ligand refers to a molecule or fragment thereof that binds to IAIP in vivo or in vitro (e.g., endotoxin (LPS), heparin, histone, hyaluronic acid, extracellular matrix proteins (e.g., vitronectin, fibronectin, tenascin C, laminin, aggrecan), von Willebrand factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), coagulation factors (eg, factor IX and factor XIIIa), complement component, divalent cations, such like Ca ²⁺ and tissue transglutaminase). IAIP ligands also include molecules predicted to bind to IAIP based on domain structure (e.g., proteins with domains

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RGDs that bind to the von Willebrand factor A domain of heavy chains IAIP). IAIP ligands for use in the methods described herein include ligands that bind to the IAIP heavy chain, the IAIP complex, or the IAIP GAG (eg, ligands that do not bind exclusively to bikunin).

As used herein, an IAIP-specific antibody or an antibody that specifically binds to an IAIP refers to any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule, such as at least one heavy or light complementarity determining region (CDR). a ligand-binding chain or portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof that is capable of binding IAIP and that does not specifically bind to any other protein. An antibody that specifically binds to IAIP will bind to IAIP and provide a signal that is at least twice the background signal or noise and typically more than 10 to 100 times the background signal.

As used herein, the term monoclonal antibody refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic or phage clone, and not to the method by which it is produced.

As used herein, the term pharmaceutical composition or pharmaceutical preparation is a composition or preparation having pharmacological activity or other direct effect in mitigating, treating or preventing a disease, and/or a finished dosage form or preparation. The composition is, for example, indicated for human use (for example, according to regulatory requirements for drugs or biological products, such as those published by the FDA (U.S. Food and Drug Administration) and/or EMA (European Food and Drug Administration). the field of medicines)).

As used herein, the term pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic reaction, or other complications. commensurate with a reasonable benefit/risk ratio.

As used herein, the expression reducing the likelihood of development refers to prophylactic treatment of a patient (e.g., a human) who is susceptible or otherwise at risk of a particular disease, syndrome, or condition (e.g., the conditions described herein, such as an inflammatory disease or infection) , or is at risk of increasing the extent or severity of a current disease, syndrome, or condition, such as a patient with community-acquired pneumonia (CAP) who is at risk of progression to severe community-acquired pneumonia (sCAP).

As used herein, the term reference refers to a parameter (eg, protein level, concentration, nucleic acid expression level, and gene copy number) of a sample or standard that is used for comparison with a parameter in the test sample. For example, the reference sample may be obtained from a healthy person (eg, a person who does not have an inflammatory disease or infection). The reference level may be the level (or average thereof) of expression or concentration of an analyte (eg, protein (eg, IAIP), nucleic acid, carbohydrate, etc.) determined from one or more reference samples. For example, the reference may be an average level of an analyte (eg, IAIP) (eg, mean level or median level) among a plurality of individuals (eg, healthy individuals or individuals who do not have an inflammatory disease or infection). In other cases, the reference level may be a predetermined threshold level, such as based on an analyte level or concentration as otherwise determined, such as through empirical assays.

As used herein, the term sample refers to a sample (e.g., blood, a blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, bronchopulmonary lavage, cerebrospinal fluid, tissue (e.g., tissue biopsy or tissue homogenate), pancreatic juice , synovial fluid and cells) isolated from a subject (eg, a mammal such as a human).

As used herein, the expression specifically binds refers to a binding reaction that determines the presence of an analyte (eg, a protein such as IAIP) in a heterogeneous population of proteins and other biological molecules. The analyte may be an antigen that is recognized, for example, by an antibody or an antigen-binding fragment thereof. Specific binding between the analyte being measured and the binding agent (eg, antibody, or antigen-binding fragment, or ligand) has a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen (eg, IAIP) binds to the antigen with a KD of up to 100 nM (eg, between 1 nM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or its

- 7 045401 epitope (eg, IAIP), exhibits a K _D of greater than 100 nM (eg, greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope thereof. Various immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein or carbohydrate. For example, enzyme-linked immunosorbent assays are commonly used to screen for antibodies that are specifically immunoreactive with a protein or carbohydrate. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

As used herein, the term sepsis refers to a systemic response to infection (referred to herein as infectious sepsis) or a non-infectious process associated with acute tissue injury and innate immune activation (referred to interchangeably herein as sterile inflammation or sterile sepsis), which may cause tissue damage, organ failure and death. Infectious sepsis can result from an infection caused by bacteria, viruses, fungi, or other microorganisms such as parasites (eg, protozoan parasites). Sterile sepsis may occur after hemorrhagic shock, polytrauma, pancreatitis, transplant rejection, autoimmune disease, inorganic compounds, crystals, chemicals, or ischemia/reperfusion, and is not associated with the presence of a known infection.

As used herein, the term subject refers to a mammal, including, but not limited to, a human or non-human mammal such as a primate, bovine, horse, pig, sheep, cat or dog. The subject may be a patient.

As used herein, the term treatment refers to the reduction or improvement of a disorder and/or symptoms associated with it. It should be kept in mind that, although not impossible, treatment of a disorder or condition does not require that the disorder or symptoms associated with it be completely eliminated.

Brief description of graphic materials

In fig. 1 is a diagram depicting the structures of circulating (eg, in the blood) IAIPs (IaI and PaI) and free light chains (LC, bikunin), for example, excreted in urine. The heavy and light chains of IAIP are linked only by glycosaminoglycan (GAG).

In fig. 2A is a diagram depicting the IAIP competitive ELISA. In this assay, purified IAIP is immobilized on a support, such as a multiwell plate, and then a biological sample and a labeled antibody targeting IAIP (eg, mAb 69.26) are added to the purified IAIP.

In fig. 2B is a graph showing the standard curve of data obtained using the IAIP competitive ELISA. This assay provides an indirect measurement of IAIP based on competitive antibody binding between the IAIP in the sample and immobilized, purified IAIP. A lower signal indicates a higher amount of IAIP in the sample.

In fig. 3A is a diagram depicting a sandwich ELISA using labeled IAIP ligands (eg, biotinylated heparin or LPS) and illustrative standard curves generated using the said ELISA. In this assay embodiment, an antibody specific for IAIP (eg, mAb 69.26) is immobilized on a support, such as a multiwell plate, and the biological sample is then added to the support containing the immobilized antibody. If IAIP is present in the sample, it will bind to the antibody and then be detected by adding a labeled IAIP ligand.

In fig. 3B and 3C are graphs showing sandwich ELISA standard curves for heparin-IAIP and endotoxin (LPS)-1A1P, respectively. This assay provides a direct measurement of IAIP concentration, with increased signal indicating higher amounts of IAIP in the sample.

In fig. 4 is a graph showing IAIP concentration measurements obtained using a competitive ELISA as shown in FIG. 2A. IAIP was measured in plasma samples from patients with severe community acquired pneumonia (sCAP) on consecutive days during hospitalization and compared with IAIP levels in healthy control subjects. Competitive ELISA revealed a mean IAIP concentration of 250 μg/mL in sCAP subjects and 330 μg/mL in healthy controls and found significant differences between IAIP levels in sCAP subjects and healthy controls at 0, 1, and 3. th day.

In fig. 5 is a graph showing IAIP concentration measurements obtained using the ligand-IAIP sandwich ELISA as shown in FIG. 3A. The results shown in Fig. 5 were prepared using heparin as the IAIP ligand. The same samples assessed using the competitive ELISA in FIG. 4 were also measured using this assay. Heparin-IAIP ELISA revealed mean IAIP concentrations between 125 and 150 μg/mL in sCAP subjects and 422 μg/mL in healthy controls and found differences between IAIP levels in sCAP subjects and healthy controls that were more statistically significant than the differences observed at

- 8 045401 using competitive ELISA at all points in time.

In fig. 6 is a graph showing IAIP concentration measurements obtained using the ligand-IAIP sandwich ELISA as shown in FIG. 3A. The results shown in Fig. 6 were prepared using LPS as the IAIP ligand. The same samples assessed using the competitive ELISA in FIG. 4 and heparin-IAIP ELISA in FIG. 5 were also measured using this assay. The LPS-IAIP ELISA revealed mean IAIP concentrations between 118 and 145 μg/ml in sCAP subjects and 338 μg/ml in healthy controls and found differences between IAIP levels in sCAP subjects and healthy controls that were more statistically significant than the differences observed using the competitive ELISA at all time points. The LPSIAIP ELISA performed comparable to the heparin-IAIP ELISA in terms of increased sensitivity and a more measurable difference between IAIP levels in subjects with sCAP and healthy controls.

In fig. Figure 7 shows a series of blots demonstrating the binding of heparin and IAIP-specific antibody (mAb 69.26) to IAIP. As shown in the middle blot, biotinylated heparin bound to purified IAIP (250 kDa IaI and 125 kDa PaI) but did not bind to the IAIP light chain, bikunin, or the negative control human serum albumin. In contrast, mAb 69.26 bound to both purified IAIP and bikunin. These data suggest that heparin binds to the heavy chain of IAIP, which may lead to more accurate measurement of circulating intact IAIP in the heparin-IAIP ELISA.

In fig. 8A-8C are a series of graphs showing IAIP concentration measurements obtained using a competitive ELISA as shown in FIG. 2A and sandwich ELISA of IAIP ligand as shown in FIG. 3A. IAIP was measured in plasma samples from patients with severe pneumonia, severe sepsis, and healthy control subjects. Below each graph shows the mean ± SEM of the IAIP concentration and the number of patient samples tested (in parentheses). As shown in FIG. 8A, competitive ELISA revealed a mean IAIP concentration of 246 μg/ml in patients with severe pneumonia, 250 μg/ml in patients with severe sepsis, and 330 μg/ml in healthy controls, and found significant differences between IAIP levels in subjects with severe pneumonia and healthy controls, but not between subjects with severe sepsis and healthy subjects. As shown in FIG. 8B, LPS-IAIP ELISA revealed a mean IAIP concentration of 141 μg/ml in patients with severe pneumonia, 150 μg/ml in patients with severe sepsis, and 338 μg/ml in healthy controls, and found significant differences between IAIP levels in subjects with severe pneumonia or severe sepsis and healthy controls. As shown in FIG. 8C, a competitive heparin-IAIP ELISA revealed a mean IAIP concentration of 145 μg/mL in patients with severe pneumonia, 193 μg/mL in patients with severe sepsis, and 422 μg/mL in healthy controls, and found significant differences between IAIP levels among subjects with severe pneumonia and healthy controls, but not between subjects with severe sepsis and healthy controls. The LPS-IAIP ELISA performed comparable to the heparin-IAIP ELISA in terms of increased sensitivity and providing more statistically significant results compared to the competitive ELISA.

In fig. 9 is a histogram showing the binding of biotinylated LPS to immobilized plasma-derived IAIP, bovine serum albumin (BSA), and IgG IAIP-specific mAb 69.26. LPS showed significant binding to IAIP and virtually no binding to mAb 69.26, which served as a negative control. Notably, LPS did not exhibit binding to the IAIP light chain, bikunin, indicating that the IAIP heavy chain may contribute to binding to LPS.

In fig. 10 is a bar graph showing the binding of biotinylated IAIP to LPS, BSA, IgG IAIP-specific mAb 69.26, and nonfat dry milk. As expected, IAIP bound to mAb 69.26 and LPS and showed minimal binding to BSA and nonfat dry milk. These data confirm the results of Fig. 9.

In fig. 11 is a histogram showing the effect of pH on the binding of biotinylated IAIP to LPS, BSA and IgG IAIP-specific mAb 69.26. These data demonstrate that IAIP bound most strongly to LPS at pH 5, with reduced binding at pH 6 and pH 7, and weak or no binding at pH 8 or higher or pH 4 or lower. In contrast, IAIP bound to IgG mAb 69.26 at pH 5-pH 9, but did not bind at pH 4 or lower. As in previous experiments, IAIP did not bind to BSA.

In fig. 12 is a bar graph showing the effect of salt concentration (NaCl) on the binding of biotinylated IAIP to LPS, BSA and IgG IAIP-specific mAb 69.26. These data demonstrate that salt had no effect on the binding of IAIP to either LPS or mAb 69.26, indicating strong and specific binding. Binding to BSA was not observed at any of the salt concentrations tested.

In fig. 13 is a histogram showing the effect of the concentration of nonionic detergent NP-40 on the binding of biotinylated IAIP to LPS, BSA and IgG IAIP-specific mAb 69.26. IAIP binding to LPS was enhanced by the addition of 0.05% NP-40 and was still observed in

- 9 045401 presence of 1% NP-40, indicating a strong binding interaction. Binding to BSA was not observed at any of the tested concentrations of NP-40.

In fig. 14 is a histogram showing the effect of the concentration of the nonionic detergent Tween-20 on the binding of biotinylated IAIP to LPS, BSA and IgG IAIP-specific mAb 69.26. Binding of IAIP to LPS was enhanced by the addition of 0.05% Tween-20 and was still observed in the presence of 1% Tween-20, indicating a strong binding interaction. Binding to BSA was not observed at any of the Tween-20 concentrations tested.

In fig. 15A-15B are histograms depicting blood levels of IAIP (Fig. 15A) and CRP (C-reactive protein) (Fig. 15B) in infants with NEC (n=14) and SBI (n=13) , as well as in healthy controls matched for sex, weight and gestational age (n=26) at initial presentation. Decreased IAIP levels were found in infants with established NEC (mean ± SD: 139 ± 21 μg/ml), while levels in healthy controls (276 ± 110 μg/ml) or infants with SPC (319 ± 72 μg /ml) were significantly higher (p <0.05 and p <0.005). In contrast, there was no statistically significant difference between IAIP levels in infants with SPC and controls (p > 0.4). When examining CRP levels, no significant differences were found between SPC, NEC and the control group (p > 0.05).

In fig. 16A-16B are graphs depicting ROC curves of IAIP and CRP in infants with NEC. The predictive value of IAIP level is higher with sensitivity 100%, specificity 88.2%, PPV 41% and NPV 100%, (Fig. 16A) compared to CRP (Fig. 16B) with sensitivity 100%, specificity 64.7%, PPV 18% and NPV 98%.

In fig. Figure 17 shows a graph showing the results of longitudinal studies of plasma IAIP levels in infants with NEC (n=8) and SPC (n=9). Blood was collected sequentially at the time before and after the onset of the disease. No significant differences in IAIP levels were found in both groups of infants with SPC and NEC before the onset of the disease (p>0.6), but IAIP levels in infants with NEC were significantly lower compared with levels in children with SPC (p<0 ,04) up to 10 days after the onset of the disease.

In fig. 18 shows a diagram of the IAIP rapid test based on immunochromatographic analysis.

In fig. 19 is a graph depicting the standard curve of the IAIP rapid test using the ESEQuant reader. The values for each point were plotted as the mean + SD from a total of 13 independent analyses. The standard rapid test curve is suitable for plasma samples with IAIP levels ranging from 17.5 to 1100 μg/mL.

In fig. 20A-20C are a series of graphs depicting correlations between results obtained using the IAIP rapid test using the ESEQuant reader, Detekt reader, iCalQ reader, and results obtained using an established competitive ELISA format.

In fig. 21A-21B are a series of graphs depicting the quantification of IAIP using a sandwich ELISA in which hyaluronic acid is immobilized on a 96-well IAIP capture plate and IAIP is detected using biotin-conjugated mAb 69.26 (an anti-human IAIP monoclonal antibody). The assay can be used to quantify IAIP in serial dilutions of both human plasma (Figure 21A) and purified IAIP (Figure 21B) using 50 ng, 100 ng or 200 ng hyaluronic acid per well.

Detailed Description of the Invention

Methods are described for measuring the concentration of IAIP in a sample (e.g., a sample from a subject, such as a blood sample) using a reagent that can be used to measure the amount of IAIP in the sample (e.g., a reagent that directly binds to IAIP in the sample, such an IAIP ligand, or a reagent that binds to an IAIP ligand that is bound to IAIP). The reagent can be measured using a detectable label. These methods can be used to identify subjects who have or are at risk of developing an inflammatory disease or condition or infection, to determine the health status or severity of a disease in a subject, and to monitor treatment of a condition in a subject (e.g., an inflammatory disease or condition , or infection) to which a therapeutic agent (eg, an IAIP or an agent for treating an inflammatory disease or condition or infection) is administered.

Assays for quantification of IAIP.

Methods for quantifying IAIP in sample(s) collected from a subject are presented. Previous methods for detecting IAIP relied on competitive antibody-based ELISAs, which provide an indirect measurement of the concentration of IAIP in a sample. This method detects both intact IAIP complexes (eg, IAIPs containing one or more heavy chains (H1-H5) and a light chain, bikunin) and bikunin alone. In contrast, the methods described herein use a reagent (eg, an IAIP ligand) that detects IAIP in a sample directly by binding to the IAIP heavy chain, the heavy and light chain of the intact IAIP complex, or the IAIP GAG complex, but does not bind bikunin alone.

- 10 045401

Without wishing to be bound by theory, the IAIP ligand-based assays described herein demonstrate greater sensitivity and reliability than competitive ELISA due to the detection of IAIP heavy chain or intact IAIP complexes. These methods provide an improved readout of the amount of functional IAIP in a sample relative to antibody-based assays that detect cleaved or disrupted IAIP light chain.

The methods described herein involve the use of an IAIP ligand to detect IAIP, such as IAIP, captured on a support (e.g., a solid support, such as a plate, resin, particle, container, membrane, or bead) using, for example, an IAIP-specific agent such as an antibody, using an IAIP ligand to capture IAIP for subsequent detection with an IAIP-specific antibody, and using an IAIP ligand to capture IAIP for subsequent detection with a second, different IAIP ligand.

A first method that can be used to detect intact IAIP in a sample involves an IAIP binding agent (eg, an IAIP-specific antibody or IAIP ligand) that is attached to a support and used to capture the IAIP in the sample. An IAIP detecting agent (eg, an IAIP ligand or an IAIP-specific antibody) is then added which binds to the IAIP that has formed a complex with the IAIP binding agent. The concentration of IAIP can then be quantified, for example, by detecting the presence of a label that is either attached directly to the IAIP detecting agent or attached to a reagent that binds to the IAIP detecting agent.

A second method that can be used to determine the amount of IAIP in a sample involves detecting IAIP-IAIP ligand complexes that have formed in vivo and are present in the sample. This method also begins with the use of a binding agent that is attached to the substrate. The binding agent can bind to either IAIP or an IAIP ligand. A detection agent is then added that binds to another component of the IAIP-IAIP ligand complex (eg, if the binding agent is directed to IAIP, the detection agent is directed to the IAIP ligand and, conversely, if the binding agent is directed to the IAIP ligand, the detection agent is directed to IAIP). The IAIP concentration can then be measured, for example, by detecting the presence of a label that is either directly or indirectly attached to the detection agent.

The reagents used in the methods and the process steps are discussed in detail below.

IAIP ligands.

IAIP ligands for use in the methods described herein include ligands that bind to one or more IAIP heavy chains, IAIP heavy chain(s) and light chain, bikunin (eg, intact IAIP), or IAIP GAGs (eg , GAG in intact IAIP). Intact IAIP is a complex containing at least one IAIP heavy chain (H1, H2, H3, H4 and/or H5) and an IAIP light chain (bikunin). Any ligand that binds to the IAIP heavy chain or intact IAIP can be used in the methods described herein, including, for example, endotoxin (lipopolysaccharide, LPS), heparin, histone, hyaluronic acid, vitronectin, fibronectin, tenascin C, laminin, aggrecan , von Willebrand factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), coagulation proteins (eg, factor IX and factor XIIIa), complement proteins (eg, C1q, C2, C3, C4, C5, C6, C8, properdin and factor D), divalent cations (eg Ca ²⁺ ) and tissue transglutaminase. IAIP ligands can be labeled according to standard techniques known in the art (eg, using one or more detectable labels described below).

Antibodies to IAIP.

Antibodies that specifically bind to IAIP can be used as binding agents or detection agents in the methods described herein. Antibodies that specifically bind to IAIP are antibodies or antigen-binding fragments thereof that do not specifically bind to any proteins other than IAIP (eg, interaction of an IAIP-specific antibody with non-IAIP proteins produces a signal similar to background). An antibody that specifically binds to IAIP may bind to the IAIP heavy chain, the IAIP light chain, or both, or the IAIP GAG. Preferably, the antibody binds to the heavy chain of IAIP or to intact IAIP containing heavy and light chains. An anti-human IAIP antibody or an anti-IAIP antibody from another mammal (eg, a non-human primate, cow, pig, sheep, goat, cat, dog, rat, mouse, rabbit, guinea pig, or any other non-human mammal) may be obtained , which expresses IAIP). An antibody that specifically binds to IAIP may bind only human IAIP or may be capable of binding IAIP from humans and other mammals. The antibody can be obtained by immunizing an animal commonly used to obtain antibodies to IAIP (eg, rabbit, guinea pig, rat, mouse, sheep, donkey, goat, hamster and chicken). The IAIP-specific antibody may be polyclonal (eg, pAb R16, pAb P20, pAb P21), monoclonal (eg, mAb 69.26 or mAb 69.31), chimeric, or recombinant.

Tags.

- 11 045401

Labels for determining IAIP concentration in the methods described herein may be attached or conjugated to IAIP ligands, IAIP-specific antibodies, or other reagents described herein. Labels useful for detecting IAIP ligands, IAIP ligand-specific antibodies, and/or IAIP-specific antibodies include biotin, enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, acetylcholinesterase, and catalase), enzyme substrates, radioactive tracers (eg, radioisotopes), luminescent substances, particles (eg, colloidal gold (eg, gold nanoparticles), magnetic particles, or latex particles) and fluorescent dyes. The label can then be assessed directly (e.g., by fluorescent dye imaging, radioactivity detection, or particle detection), by enzyme-conjugated avidin or streptavidin (e.g., avidin or streptavidin conjugated to ALP or HRP for biotin detection), and/or through the use of a substrate, and can be visualized using known methods and devices, including, for example, a spectrophotometer, fluorometer, luminometer or liquid scintillation counter. Substrates can be colorimetric (eg, PNPP for ALP detection; or ABTS, OPD, or TMB for HRP detection), chemiluminescent, or fluorescent. Substrates also include liquid scintillators for detecting radioactivity. Standard detection methods known in the art can be used to detect the labels described herein.

Samples.

The methods described herein can be performed using a sample from a subject (eg, a human). Suitable samples include liquid samples. For example, IAIP can be measured in a blood or plasma sample from a subject (eg, a subject with an inflammatory disease or condition such as sepsis, or a subject at risk of developing an inflammatory disease or condition such as sepsis). The methods described herein can also be performed using a sample of other body fluids, such as urine, cerebrospinal fluid, synovial fluid, amniotic fluid, interstitial fluid, follicular fluid, peritoneal fluid, bronchoalveolar lavage fluid, breast milk, sputum, lymph, and bile. . Tissue samples (eg, biopsies) from a subject can be homogenized in an appropriate buffer to create a fluid for quantitating IAIP using these methods.

The volume of sample used in the assay will vary depending on the type of assay being performed (e.g., ELISA, immunochromatographic assay, or other assay) and the support (e.g., plate, membrane, test strip, or other support). The sample volume used may be from about 1 μL to about 500 μL (e.g., from about 1 μL to about 150 μL, e.g., from 1 μL to about 30 μL for an assay such as an immunochromatographic assay, or from about 50 μL to about 200 µl for analysis such as ELISA). The sample may be diluted before use in the assay with a buffer that will not interfere with binding to the binding or detection agent (e.g., water, PBS, or assay buffer) and may be diluted 1:2, 1:3, 1:4 , 1:5, 1:10, 1:15, 1:20, 1:10o or more.

Method for detecting intact IAIP.

A first quantitative IAIP assay involves contacting a sample containing IAIP with an IAIP binding agent (eg, an antibody that specifically binds to IAIP or an IAIP ligand) to form an IAIP-binding agent complex. The IAIP binding agent may be attached to a support (eg, a solid support). Suitable supports include plates (eg, multiwell plates), particles (eg, magnetic particles, nanoparticles, magnetic nanoparticles), biochips, resins, membranes (eg, nitrocellulose membranes, PVDF membranes), containers (eg, tubes), test strips (eg cellulose, glass fiber, nitrocellulose) and granules (eg protein A or protein G granules, magnetic beads, glass beads, plastic beads). The support may preferably be washed one or more times (eg, using a buffer such as TBS, TBS-T, PBS or PBS-T) to remove material that does not bind to the IAIP binding agent.

The IAIP-binding agent complex is then contacted with an IAIP detecting agent (eg, an IAIP ligand or an antibody that specifically binds to IAIP). The IAIP detection agent can be conjugated to a label (eg, one or more of the labels described above), which can then be detected using known detection methods. Alternatively, the IAIP detecting agent can be immediately detected without the use of a label. After adding the IAIP detecting agent, an additional washing step (eg, one or more) can be performed to remove unbound detecting agent.

The IAIP can then be measured based on the signal from the conjugated label or attached detection agent (eg, enzyme activity or fluorescence) using standard methods known in the art. If an enzyme is used as a tag, a substrate can be added to produce a signal (such as a color change) and can be read

- 12 045401 a device suitable for signal detection, such as a spectrophotometer. A signal (such as absorbance or fluorescence) can be applied to a standard of known concentration

IAIP to establish a standard curve or can be compared to a known reference concentration. The unknown concentration in samples can be calculated and determined based on an established standard curve or reference concentration value.

Before adding the sample to the IAIP binding agent, a blocking step can be performed to prevent or reduce nonspecific binding. Blocking agents for use in the methods described herein include, for example, milk, BSA, casein, gelatin (eg, fish gelatin) and whey (eg, goat whey, donkey whey, horse whey, fetal bovine whey).

The process steps can be performed at a pH of 7.0 to 3.5 (eg pH 5.0 to 3.5, eg pH 4.0). For example, the binding step and/or detection step can be performed at a pH of 7.0 to 3.5 (eg pH 5.0 to 3.5, eg pH 4.0). The IAIP binding agent may be prepared in a buffer of pH 7.0 to 3.5 (e.g. pH 5.0 to pH 3.5, e.g. pH 4.0) and/or the IAIP detecting agent ( eg, IAIP ligand) can be prepared in a buffer with a pH of 7.0 to 3.5 (eg, pH 5.0 to pH 3.5, eg, pH 4.0). To improve IAIP detection, a low pH buffer (eg pH 5.0 or lower, eg pH 4.0) can be used.

Additionally, to stabilize IAIP during analysis, divalent cations (e.g., Ca2+, Mg2+, Mn2+, Fe2+, etc.) can be added to the sample before or during contact with the IAIP binding agent and/or added to the buffer containing the detection agent. agent, before or during contact with the binding agent-IAIP complex. Divalent cations can be obtained at a concentration of 1 μM to 1 M (eg, 100 μM to 100 mM, 1 mM to 10 mM).

An IAIP-specific antibody binding agent and an IAIP ligand detecting agent.

In one example of a method described herein, the IAIP binding agent is an antibody that specifically binds to IAIP (e.g., mAb 69.26 or mAb 69.31), and the detection agent is a labeled IAIP ligand (e.g., heparin, hyaluronic acid, or LPS). Alternatively, the ligand may be unlabeled and detected using a labeled antibody that is specific for the IAIP ligand, or the unlabeled ligand may be detected using an unlabeled antibody that is specific for the IAIP ligand, which is then detected using a labeled secondary antibody (e.g., labeled a secondary antibody that binds to an antibody that is specific for the IAIP ligand but does not bind to the IAIP antibody, (e.g., anti-IAIP antibody and anti-IAIP ligand antibody are generated in different host species).If the ligand is not labeled and is detected using a labeled ligand-specific antibody or ligand-specific antibody and labeled secondary antibody, additional washing steps can be performed before and/or after incubation with each antibody.

An IAIP ligand binding agent and an IAIP-specific antibody detecting agent.

In another example of a method described herein, the IAIP binding agent is an IAIP ligand (eg, heparin, hyaluronic acid, or LPS) and the detection agent is an antibody that is specific for IAIP (eg, mAb 69.26 or mAb 69.31). In this example, an antibody that is specific for IAIP can be conjugated directly to a label, or the antibody can be detected using a labeled secondary antibody that binds to the IAIP-specific antibody. If a labeled secondary antibody is used, a wash step can be performed after incubation with the secondary antibody to minimize nonspecific signal prior to IAIP measurement.

An IAIP ligand binding agent and an IAIP ligand detecting agent.

In a third example of a method described herein, the binding agent is an IAIP ligand (eg, hyaluronic acid), and the detection agent is a labeled IAIP ligand that binds to another region of the IAIP (eg, heparin, hyaluronic acid, or LPS). This method can be performed with any two IAIP ligands that do not bind to the same IAIP region (e.g., two IAIP ligands that do not compete for binding with IAIP or for which binding of one of the IAIP ligands does not sterically interfere with the binding of a second IAIP ligand with IAIP). The ligand that acts as the IAIP detection agent may be unlabeled and detected using a labeled antibody that is specific for the IAIP ligand that acts as the detection agent, or it may be detected using a labeled secondary antibody that binds to the unlabeled antibody. which is specific for the IAIP ligand, which acts as a detection agent. If labeled antibodies are used to detect an IAIP ligand that acts as a detection agent, additional washing steps may be performed before and/or after incubation with each antibody.

An IAIP-specific antibody binding agent and an IAIP-specific antibody detecting agent.

In a fourth example of the method described herein, the binding agent is an IAIP-specific antibody, and the detection agent is an IAIP-13045401-labeled specific antibody. In this example, at least one of the IAIP-specific antibodies is capable of binding to either intact IAIP or IAIP that contains at least one heavy chain (e.g., the antibody does not bind to bikunin if at least one IAIP heavy chain, e.g. does not bind to cleaved or degraded IAIP, which lacks bikunin). In addition, the two IAIP-specific antibodies used in the assay bind different epitopes of IAIP (eg, the antibodies do not compete for binding to IAIP). If two IAIP-specific antibodies are generated using different host species, then the IAIP-specific antibody used as a detection agent can be unlabeled and detected using a labeled secondary antibody. If a labeled secondary antibody is used, additional washing steps may be performed before and/or after incubation with the labeled secondary antibody.

Quantitative determination of IAIP using rapid immunochromatographic assay (ICA).

In a fifth example, the methods described herein can be performed using an immunochromatographic assay-based test. In this example, a small volume of sample (e.g., 1-30 μL, e.g., 15 μL) can be applied to a test strip (e.g., cellulose, fiberglass, or nitrocellulose) undiluted or diluted (e.g., diluted 1:2, 1:5, 1:10, 1:20, 1:100 or more, for example with a buffer (eg PBS) or water) and then add the buffer to push the sample through the strip. The strip contains an IAIP binding agent (eg, an IAIP-specific antibody or IAIP ligand) to bind to the IAIP in the sample. IAIP can be detected using an IAIP detecting agent (eg, a labeled IAIP-specific antibody or a labeled IAIP ligand), which can be added to the strip before, after, or simultaneously with the addition of the sample. The test strip can be read using an appropriate reader to quantify the tag attached to the IAIP detection agent (e.g., a portable desktop test strip reader, a handheld PDA-based reader, or a smartphone/tablet-based reader, among others). The selection of the combination of binding and detection agent can be made based on the parameters described herein. This assay can be performed quickly (e.g., in 15 minutes or less, e.g., 15, 10, or 7 minutes or less) and provides a quantitative and rapid measurement of IAIP in a small sample volume.

Detection of natural IAIP-ligand IAIP complexes.

Also provided are methods for detecting natural IAIP-IAIP ligand complexes (eg, IAIP-IAIP ligand complexes that are formed in vivo in a subject (eg, a human subject) and are present in a sample from the subject). The method involves contacting a sample (eg, a sample of a fluid such as plasma, serum, blood, bronchoalveolar fluid, cerebrospinal fluid, sputum, urine, or other body fluids) with a binding agent, such as a binding agent, that is attached to a support (eg , hard substrate). The binding agent may be an IAIP binding agent (e.g., an antibody that specifically binds to an IAIP, e.g., mAb 69.26 or mAb 69.31, or an IAIP ligand), or the binding agent may be an antibody that specifically binds to an IAIP ligand of interest (e.g. , an IAIP ligand that is predicted to form an IAIP-IAIP ligand complex in vivo). Suitable supports include plates (eg, multiwell plates), particles (eg, magnetic particles, nanoparticles, magnetic nanoparticles), biochips, resins, containers (eg, tubes), membranes (eg, nitrocellulose membranes, PVDF membranes), test strips (eg cellulose, glass fiber or nitrocellulose) and granules (eg protein A or protein G beads, magnetic beads, glass beads, plastic beads). The support may preferably be washed one or more times (eg, using a buffer such as TBS, TBS-T, PBS or PBS-T) to remove material that does not bind to the binding agent.

If the binding agent used in the assay is an IAIP binding agent (e.g., an antibody that specifically binds to IAIP, such as mAb 69.26 or mAb 69.31, or an IAIP ligand), the IAIP-IAIP ligand complex can be detected using a detection agent, which is an antibody directed against an IAIP ligand of interest by contacting the antibody with an IAIP-binding agent complex. If the binding agent used in the assay is an antibody that specifically binds to the IAIP ligand of interest, the IAIP-IAIP ligand complex can be detected using a detection agent that specifically binds to IAIP (e.g., an antibody that specifically binds to IAIP for example, mAb 69.26 or mAb 69.31 or another IAIP ligand) by contacting the detection agent with the IAIP-binding agent complex.

A washing step (eg, one or more) may be performed after incubation with the detection agent. In addition, before adding the sample to the IAIP or IAIP ligand binding agent, a blocking step can be performed to prevent or reduce nonspecific binding. Blocking agents for use in the methods described herein include,

- 14 045401 for example milk, BSA, casein, gelatin (eg fish gelatin) and whey (eg goat whey, donkey whey, horse whey, fetal bovine whey).

If the detection agent is an antibody (e.g., an IAIP-specific antibody or an IAIP ligand-specific antibody), the detection agent may be directly conjugated to a label (e.g., the label described above), or the detection agent may be visualized by adding a labeled a secondary antibody that does not bind to any other antibody used in the assay (eg, a binding agent, if it is an antibody).

If the detection agent is a ligand (eg, an IAIP ligand, such as an IAIP ligand different from that detected in the assay), the detection agent may be directly conjugated to a label (eg, the label described above) or detected using a labeled ligand. specific antibody, which is then detected. Alternatively, the detection agent can be detected using an unlabeled ligand-specific antibody and a labeled secondary antibody that does not bind to any other antibody used in the assay (eg, a binding agent if it is an antibody). If the detection agent is unlabeled and additional reagents (eg, labeled primary or secondary antibodies) are used, one or more washing steps may be performed after incubation with additional reagents to minimize nonspecific signal. The tracer can be used to measure the concentration of the IAIP-IAIP ligand complex using the same substrates and imaging methods mentioned above. The signal from a sample can be compared to the signal measured in sample(s) with a known concentration of IAIP-IAIP ligand complex (eg, to establish a standard curve). The unknown concentration in samples can be calculated from an established standard curve or from a known reference concentration value.

If the identity of the IAIP ligand in the IAIP-IAIP ligand complex is unknown, the method can be performed using an antibody that specifically binds to IAIP (eg, an antibody that binds to intact IAIP and/or IAIP heavy chain) as the binding agent. The detection step can then be performed by adding labeled secondary antibodies specific for the various IAIP ligands, washing after incubation with the labeled secondary antibodies, and detecting the signal from the label to determine the identity of the IAIP ligand in the IAIP-IAIP ligand complex. Labeled antibodies can be added and scored individually to identify the IAIP ligand, or they can be added simultaneously if different tags (eg, different fluorescent dyes) are attached or conjugated to each antibody. Once the IAIP ligand is identified, the amount of IAIP-IAIP ligand complex in the sample can be quantified as described herein.

Quantification of IAIP Captured in Assays IAIP or the IAIP-ligand complex IAIP can be quantified by performing the detection methods described herein using the sample of interest in comparison to samples containing known amounts of IAIP or the IAIP-ligand complex IAIP that are used to obtain a standard curve. A sample from a subject can be measured simultaneously with known amounts of IAIP or IAIP-IAIP ligand complex such that the concentration of IAIP or IAIP-IAIP ligand complex in the sample can be determined. The concentration in a sample from a subject can be compared to the average concentration of IAIP or the IAIP-I AIP ligand complex measured using the same assay in a control population, such as healthy controls, to determine whether the IAIP concentration in the sample falls within the normal range, or diseased controls to determine whether the concentration of IAIP in the sample is within the range for the pathological condition.

The concentration of IAIP or IAIP-IAIP ligand complex in a sample from a subject can also be compared with the concentration of IAIP or IAIP-IAIP ligand complex in healthy controls by measuring IAIP or IAIP-IAIP ligand complex in both the test sample and the control sample at the same time time using the methods described in this document. Control samples include samples obtained from the same starting material (eg, both the test sample and the control sample are obtained from the same body fluid or the same tissue type). In addition to being obtained from the same starting material, the test sample and control sample may also be collected from subjects of the same age and/or the same sex to minimize possible differences between subjects. If IAIP concentrations are directly compared between a subject and a healthy control, a decrease in a subject's IAIP concentration of 25% or more compared to a healthy control would indicate that the subject has or is at risk of developing an inflammatory disease or condition or infection.

Alternatively, the concentration of IAIP or IAIP-IAIP ligand complex in a sample from a subject can be compared to a predetermined threshold value for the concentration of IAIP or IAIP-IAIP ligand complex under known conditions (eg, a healthy state or a disease state). The threshold value may be the average concentration of IAIP or the IAIP ligand IAIP complex determined from a population of healthy subjects or diseased subjects.

Using the methods described herein, it was found that healthy controls

- 15 045401 subjects have 400 ± 140 μg/mL IAIP in plasma, although higher concentrations are also observed in healthy subjects. In patients with severe inflammatory disease, mean IAIP concentrations were found to be below about 200 μg/mL. An IAIP concentration of about 250 μg/ml can be used as a cutoff value for classifying subjects as having or at risk of developing a disease or condition (eg, an inflammatory disease or condition, or infection). This classification can then be used to recommend subjects for treatment or further diagnostic testing. Subjects with moderate or low IAIP levels (eg, 300 to 200 mcg/mL) may benefit from repeated testing over time (eg, once a week, twice a month, once a month, once every two months, three once a year or twice a year) to determine whether IAIP levels are constant or changing (eg, increasing or decreasing), since these levels may indicate the risk of developing an inflammatory disease, or a pathological condition, or an infection, the presence of an inflammatory disease, or pathological condition, or infection, or they may represent a normal baseline for the subject.

Methods for determining a pathological condition or disease risk using methods for determining IAIP and the IAIP-IAIP ligand complex.

The methods for detecting IAIP and IAIP-IAIP Ligand complexes described herein can be used to measure IAIP and/or IAIP-IAIP Ligand complexes in various subjects, such as a subject having or suspected of having a disease or condition (eg, an inflammatory disease, or pathological condition, or infection (for example, bacterial infection)). The level of IAIP and the IAIP-IAIP ligand complex in such a subject may be determined using any of the above assays, for example, for the purpose of diagnosing the presence of a disease or condition in the subject or the risk of the subject developing or will develop a disease or condition, or monitoring the subject in regarding the development or elimination of a disease or pathological condition.

Inflammatory diseases or pathological conditions that can, for example, be diagnosed or monitored using assay methods include, for example, acute inflammatory disease, sepsis, septic shock, systemic inflammatory response syndrome (SIRS), trauma and/or injury (e.g., wounds, burns, lacerations, contusions, bone fractures, surgical procedures), stroke (eg, ischemic stroke, hemorrhagic stroke), acute lung injury, acute respiratory distress syndrome (ARDS), pneumonia (eg, severe pneumonia, severe or non-severe community-acquired pneumonia , nursing home acquired pneumonia), necrotizing enterocolitis, acute pancreatitis, kidney disease (eg, acute kidney injury, liver injury, acute circulatory failure), preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral arterial disease, diabetes 1- type 2 or type 2, cardiovascular atherosclerosis, intermittent claudication, critical limb ischemia, myocardial infarction, carotid artery occlusion, umbilical cord occlusion, low birth weight, prematurity, surgically induced inflammation, abscess-related inflammation, pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia (eg, neonatal hypoxic-ischemic brain injury or hypoxic-ischemic encephalopathy), tissue ischemia (eg, ischemia of skeletal muscle, smooth muscle, cardiac muscle, brain, skin mesenchyme, connective tissue, gastrointestinal tract) -intestinal tract or bones), rheumatoid arthritis, meningitis, multiple sclerosis, inflammatory bowel disease (eg, Crohn's disease), chronic obstructive pulmonary disease, rhinitis, premature birth, or an infectious disease (eg, influenza or a viral infection such as Dengue fever or West Nile fever).

Infections that can, for example, be diagnosed or monitored using the assay methods include, for example, infections caused by gram-negative bacteria such as Neisseria species, including Neisseria gonorrhoeae and Neisseria meningitidis, Branhamella species, including Branhamella catarrhalis, Escherichia species, including Escherichia coli , Enterobacter spp., Proteus spp., including Proteus mirabilis, Pseudomonas spp., including Pseudomonas aeruginosa, Pseudomonas mallei, and Pseudomonas pseudomallei, Klebsiella spp., including Klebsiella pneumoniae, Salmonella spp., Shigella spp., Serratia spp., Acinetobacter spp.; Haemophilus spp., including Haemophilus influenzae and Haemophilus ducreyi, Brucella spp., Yersinia spp., including Yersinia pestis and Yersinia enterocolitica, Francisella spp., including Francisella tularensis, Pasturella spp., including Pasteurella multocida, Vibrio cholerae, Flavobacterium spp., meningosepticum, Campylobacter spp., including Campylobacter jejuni, Bacteroides spp. (oral, pharyngeal), including Bacteroides fragilis, Fusobacterium spp., including Fusobacterium nucleatum, Calymmatobacterium granulomatis, Streptobacillus spp., including Streptobacillus moniliformis, and Legionella spp., including Legionella pneumophila.

The assays described herein can be used to measure IAIP levels in a subject at risk of developing an inflammatory disease or condition or infection. Risk factors include immunosuppression, immunodeficiency (eg, immunocompromised subject), advanced age, burns (eg, thermal burns), trauma, surgery, foreign bodies, cancer, prematurity (eg, newborn born prematurely), obesity and metabolic syndrome.

The methods described herein may be performed as part of a routine physical examination or as a general health assessment.

Diagnosis of a pathological condition or risk of disease.

The assays described herein can be used along with traditional diagnostic methods to determine whether a subject has or is at risk of developing an inflammatory disease or condition or infection. The IAIP measurement obtained from the assays can also be used to determine whether a patient is a candidate for treatment with IAIP or anti-inflammatory or anti-infective therapy or to predict response to IAIP administration (eg, a patient with low IAIP levels may be treated with IAIP and/or may benefit respond to IAIP treatment). IAIP measurement may be accompanied by administration of IAIP, anti-inflammatory, or anti-infective therapy to the subject if deemed appropriate (eg, if IAIP levels are determined to be low, e.g., at least 25% lower than what is considered a normal IAIP level in a healthy subject, or if IAIP levels are below 200 µg/ml).

Measurement of IAIP and/or the IAIP-IAIP ligand complex in a sample from a subject can be used to determine whether the subject is at risk for developing an inflammatory disease or condition or infection. The method includes measuring the level of IAIP and/or the IAIP-IAIP ligand complex using one of the methods described above and comparing the level to a reference value (eg, a reference sample from a healthy patient or an average value obtained from measurements of a population of clinically healthy patients). A reduced IAIP level in a subject compared to a healthy control (e.g., a level that is 25%, 30%, 40%, 50% lower or more in a subject compared to a control) or an IAIP concentration of about 250 μg/mL or lower means, that the subject has or is at risk of developing an inflammatory disease or condition or infection. An increased level of the IAIP-IAIP ligand complex in a subject compared to healthy controls (e.g., a level that is 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% higher or more in a subject by compared to controls) indicates that the subject has or is at risk of developing an inflammatory disease or infection.

The identity of the ligand bound to IAIP in the natural IAIP-IAIP ligand complex may also provide insight into the type of inflammatory disease or infection that the subject has or is at risk of developing. For example, detection of elevated levels of the IAIP-LPS complex in a sample from a subject may indicate that the subject has or is at risk of developing an infection (eg, a bacterial infection), and detection of elevated levels of the IAIP-histone complex in a sample from a subject may indicate that that the subject has or is at risk of developing an acute systemic inflammatory disease (eg, sepsis or stroke). Such measurements can be used to diagnose patients with certain inflammatory diseases or infections, or to recommend types of therapies or courses of treatment.

The severity of the disease.

The methods described herein can also be used to assess the severity of a disease. Subjects with IAIP concentrations below about 200 μg/ml, measured using the assays described herein, may be classified as having, or at high risk of developing severe inflammation or infection, or at greater risk of morbidity and/or mortality. Alternatively, subjects with elevated levels of the IAIP-IAIP ligand complex (e.g., subjects with a level of the IAIP-IAIP ligand complex, such as the IAIP-LPS complex, that is 10%, 20%, 30%, 40%, 50% higher or more compared to healthy controls or a known reference concentration) measured using the assays described herein may be classified as having or having a high risk of developing severe inflammation or infection, or having a greater risk of morbidity and/or mortality. After assessing the severity of the disease, an appropriate course of treatment can be recommended. Subjects who have an IAIP concentration that indicates severe inflammation may be selected for more frequent or more aggressive treatment than subjects whose IAIP concentration indicates a moderate or low risk of having an inflammatory disease or condition. The assays described herein can be used to measure intact IAIP, and thus can be used to identify life-threatening conditions and assess the need for an appropriate therapeutic response.

Monitoring.

A subject who has previously had or who is at risk of developing an inflammatory disease or condition or infection (eg, a subject with a genetic predisposition, a subject who has been exposed to others with the disease or infection, or a subject who has any of the risk factors described above) can be controlled using the methods described. Control may also be an appropriate approach for subjects with moderate to low IAIP levels (eg, 300 to 200 μg/mL) and/or subjects with slightly elevated IAIP-IAIP ligand complex levels (eg, subjects with IAIP-IAIP ligand complex levels such as the IAIP-LPS complex, which is 1%, 5% or 10% higher compared to healthy controls or a known reference concentration), especially if subjects do not have obvious symptoms of inflammation or infection. Measurements of IAIP and/or the IAIP-IAIP ligand complex can be taken at regular intervals (e.g., once a year, biannually, once every three months, once a month, twice a month, or once a week), to determine whether the levels of IAIP and/or the IAIP-IAIP ligand complex are constant or variable. An increase in IAIP levels and/or a decrease in the level of the IAIP-IAIP ligand complex may indicate improvement and may lead to discontinuation of monitoring and/or treatment. A decrease in the level of IAIP and/or an increase in the level of the IAIP ligand complex IAIP may indicate relapse in patients in recovery, or the development or worsening of an inflammatory disease or infection and may lead to diagnostic testing (eg, with greater frequency) and the onset of, or increase in, or change in treatment.

Treatment effectiveness.

The methods described herein can also be used to evaluate the effectiveness of treatment in a subject being treated (eg, with an antibiotic, anti-inflammatory agent, anti-infective agent, or IAIP) for an inflammatory disease or condition or infection. IAIP levels may be measured before or after initiation of treatment and then measured on an ongoing basis during treatment (eg, once daily, once weekly, twice weekly, once monthly, twice monthly, once per three months or twice a year). An increase in IAIP levels (eg, an increase of 1%, 5%, 10%, 20%, 30% or more from the previous measurement) over the course of treatment will indicate improvement and demonstrate the effectiveness of treatment, while constant or decreasing IAIP levels (eg, one or more measurements that show no change or demonstrate a decrease of 1%, 5%, 10%, 20%, 30% or more from the previous measurement) will indicate no improvement and suggest that the course treatment must be modified or changed (eg, increased in dose or frequency, or both, changed to a different therapeutic agent, or modified to include additional therapeutic agents).

Alternatively or in addition to IAIP measurement, detection of the IAIP-IAIP ligand complex (eg, IAIP-LPS) can be used to assess the effectiveness of a therapy to treat or reduce the risk of an inflammatory disease or infection. The method includes measuring the level of the IAIP-IAIP ligand complex as a biomarker in a subject undergoing therapy before or after initiation of treatment, and then measuring it on an ongoing basis during treatment (e.g., once daily, once weekly, weekly, once every month, once every two months, once every three months or twice a year). The level of the IAIP-IAIP ligand complex can be compared to a control value (eg, a reference sample from a healthy patient or an average value obtained from measurements of a population of control subjects (eg, healthy patients)) or to a preliminary measurement obtained from the subject. A decrease in the level of the IAIP-IAIP ligand complex to normal levels or a decrease at later time points during treatment (eg, a decrease of 1%, 5%, 10%, 20%, 30% or more relative to the pre-measurement) will indicate that therapy is effective. An increase in the level of the IAIP-IAIP ligand complex (eg, an increase of 1%, 5%, 10%, 20%, 30% or more from the previous measurement or normal level) indicates that the treatment is ineffective and requires modification (eg , higher dose, more frequent administration, or both, or a different therapeutic agent or combination therapy).

Methods of treatment.

The invention also provides methods for treating, preventing, or reducing the risk of developing an inflammatory disease or condition or infection (e.g., severe infection) in a subject (e.g., a human) who has been determined to be in need of care according to the diagnostic tests described herein. methods (eg, a subject with low levels of IAIP and/or increased levels of the IAIP-IAIP ligand complex compared to a reference or compared to previous measurements). The subject may be treated with a standard therapeutic agent appropriate for the disease or condition and/or IAIP. The subject may be a newborn, a child, an adolescent, or an adult.

Before administering IAIP or other therapeutic agent to a subject in need thereof, the concentration of IAIP can be measured in a sample from the subject in accordance with the methods described herein. Alternatively, or in addition to measuring IAIP levels, the method may include detecting the IAIP-IAIP ligand complex prior to administration of IAIP. For example, the method may include detecting the IAIP-LPS complex in a subject having or suspected of having a bacterial infection (e.g., an infection caused by gram-negative bacteria), and/or measuring the concentration of IAIP in a sample from the subject, and administering the IAIP to the subject (e.g., administering the IAIP in a subject with increased levels of the IAIP-LPS complex compared to healthy controls or controls

- 18 045401 value (for example, a level of the IAIP-LPS complex that is 1%, 5%, 10%, 20%, 30% higher or higher than the level in healthy controls) or administration of IAIP to a subject at risk of systemic inflammation or shock syndrome ( for example, a subject with IAIP levels at least 25% lower than healthy controls)).

IAIP or compositions containing IAIP can be administered to a subject in need thereof (eg, as determined using one or more of the methods described herein). Subjects that can be treated with IAIP include subjects with an infection (eg, infection caused by gram-negative bacteria) or subjects at increased risk of developing an infection (eg, subjects with one or more risk factors, including immunosuppression, immunodeficiency (eg, a subject with a weakened immunity), older age, burns (eg, thermal burns), trauma, surgery, foreign bodies, cancer, recent birth (eg, newborns), prematurity (eg, newborns born prematurely), obesity, and metabolic syndrome). Infection may occur due to endotoxins caused by the release of lipopolysaccharide (LPS) molecules during infection by gram-negative bacteria. Severe infection caused by Gram-negative bacteria can lead to severe systemic inflammation, sepsis, shock syndrome, and death. As shown herein, IAIP binds to LPS, and therefore, administration of IAIP can be used to treat subjects infected with Gram-negative bacteria to reduce or prevent LPS-induced cytotoxicity.

Infections suitable for treatment with IAIP include infections caused by gram-negative bacteria such as Neisseria species including Neisseria gonorrhoeae and Neisseria meningitidis, Branhamella species including Branhamella catarrhalis, Escherichia species including Escherichia coli, Enterobacter species, Proteus species including Proteus mirabilis, Pseudomonas spp., including Pseudomonas aeruginosa, Pseudomonas mallei, and Pseudomonas pseudomallei, Klebsiella spp., including Klebsiella pneumoniae, Salmonella spp., Shigella spp., Serratia spp., Acinetobacter spp.; Haemophilus spp., including Haemophilus influenzae and Haemophilus ducreyi, Brucella spp., Yersinia spp., including Yersinia pestis and Yersinia enterocolitica, Francisella spp., including Francisella tularensis, Pasturella spp., including Pasteurella multocida, Vibrio cholerae, Flavobacterium spp., meningosepticum, Campylobacter spp., including Campylobacter jejuni, Bacteroides spp. (oral, pharyngeal), including Bacteroides fragilis, Fusobacterium spp., including Fusobacterium nucleatum, Calymmatobacterium granulomatis, Streptobacillus spp., including Streptobacillus moniliformis, and Legionella spp., including Legionella pneumophila.

Subjects who have been determined to require treatment (e.g., using one or more of the modalities described herein) or who can be treated with IAIP, once such need has been determined, also include subjects who have or are at risk of developing inflammatory disease or condition such as acute inflammatory disease, sepsis, septic shock, systemic inflammatory response syndrome (SIRS), trauma/injury (eg, wounds, burns, lacerations, contusions, bone fractures, surgical procedures), stroke ( eg, ischemic stroke, hemorrhagic stroke), acute lung injury, acute respiratory distress syndrome (ARDS), pneumonia (eg, severe pneumonia, severe or non-severe community-acquired pneumonia, nursing home-acquired pneumonia), necrotizing enterocolitis, acute pancreatitis, diseases kidneys, including acute kidney injury, liver injury, acute circulatory failure, preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral arterial disease, type 1 or type 2 diabetes, cardiovascular atherosclerosis, intermittent claudication, critical limb ischemia, myocardial infarction, carotid artery occlusion, umbilical cord occlusion, low birth weight, prematurity, surgically induced inflammation, abscess-associated inflammation, pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia (eg, neonatal hypoxic-ischemic brain injury or hypoxic-ischemic encephalopathy), tissue ischemia (eg, ischemia of skeletal muscle, smooth muscle, cardiac muscle, brain, skin mesenchyme, connective tissue, gastrointestinal tissue, or bone), rheumatoid arthritis, meningitis, multiple sclerosis, inflammatory bowel disease (eg, Crohn's), chronic obstructive pulmonary disease, rhinitis, premature birth, or an infectious disease (such as influenza or a viral infection such as dengue fever or West Nile fever); subjects with low levels of IAIP (eg, IAIP levels of 250, 225, 200, 175, 150 μg/mL or lower) and subjects with elevated levels of disease-associated IAIP-IAIP ligand complexes (eg, IAIP-LPS or IAIP-histone ).

Introduction.

An IAIP (eg, IaI and/or PaI) or a composition containing such proteins and a pharmaceutically acceptable excipient, diluent or carrier can be administered to a subject (eg, a human) having or at risk of developing an inflammatory disease or condition or infection that determined to be in need of care according to the diagnostic modalities described herein (eg, a subject with low IAIP levels and/or elevated levels of the IAIP-IAIP ligand complex compared to reference or compared to previous measurements) by any suitable route, including, for example, parenterally, by inhalation spray, topically, nasally, buccally, sublingually, intranasally, by oral administration, inhalation, suppository, rectally, vaginally or by injection. Administration by injection includes, for example, intravenous, intraperitoneal, subcutaneous, intradermal, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intranasal, intravitreal and intracranial administration. If the patient is hospitalized, the preferred route of administration is intravenous injection.

IAIP (eg, IaI and/or PaI) or a composition containing such proteins may be administered to a subject one or more times every 1, 2, 3, 4, 5, 6, 8, 12 or 24 hours; one or more times every 1, 2, 3, 4, 5 or 6 days; or one or more times every 1, 2, 3 or 4 weeks. In other cases, IAIP (eg, IaI and/or PaI) or a composition containing such proteins is administered as a continuous infusion.

IAIP (e.g., IaI and/or PaI) for use in the compositions of the invention can be obtained, for example, from human plasma and blood by methods known in the art (see, for example, US Pat. No. 9,139,641, which is incorporated herein by reference in full).

In particular, IAIPs can be obtained in purities ranging from 80% to 100% (e.g., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) from a natural source (eg, blood) and used to prepare the composition of the invention (see, for example, US Pat. No. 7,932,365, which is incorporated herein by reference in its entirety). IAIPs for use in the compositions of the invention may also be exposed to low pH conditions (eg, a wash buffer having a pH of about 4.0 or lower, e.g., about pH 3.6 or lower) during purification (as described in US Pat. No. 9,139,641 ).

The compositions may include any suitable IAIP, for example, IaI, PaI, heavy chain, light chain, or any combination thereof. For example, the composition may contain Ial, PaI and/or bikunin. In some cases, the composition may contain IaI and PaI. The heavy chain may be H1, H2, H3, H4 or H5. The light chain may be bikunin.

The proportion or concentration of IAIP (eg, IaI and/or PaI) in the compositions may vary depending on a number of factors, including dose, chemical characteristics (eg, hydrophobicity) and route of administration. IAIPs (eg, IaI and/or PaI) may be present in the composition in physiological proportions. Physiological proportions may be, for example, proportions found in a healthy human or animal and/or the ratio of IaI and PaI that is naturally occurring in human blood plasma. Physiological proportions are typically about 60% to about 80% IaI and about 20% to about 40% PaI.

IAIPs (eg, Ia1 and/or PaI) or compositions thereof may have a half-life, for example, greater than about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7 ,5, or 10 hours. IAIPs (eg, IaI and/or PaI) or compositions thereof may have a half-life of greater than about 5 hours, or preferably greater than about 10 hours. Longer half-lives are preferred, for example, because fewer doses are required to administer to a subject over time.

Dosages.

A pharmaceutically acceptable composition of the invention for administration to a subject having or at risk of developing an inflammatory disease or condition or infection who has been determined to be in need of care according to the diagnostic methods described herein (e.g., a subject with low levels of IAIP and/or increased levels of the IAIP-IAIP ligand complex compared to a reference or compared to previous measurements) contains IAIP (eg, IaI and/or PaI) at a dose known in the art (see, for example, US Pat. No. 7,932,365 , International Patent Application Publication No. WO2009154695 and US Patent Application Publication No. 2009/0190194, each of which is incorporated herein by reference in its entirety). For example, the compositions of the invention can be administered in a dose range from about 1 mg/kg to 50 mg/kg, preferably in doses from 10 mg/kg to 30 mg/kg. The dose may be administered one or more times: every 1, 2, 3, 4, 5, 6, 8, 12 or 24 hours, every 1, 2, 3, 4, 5 or 6 days, or every 1, 2, 3 or 4 weeks or as needed. Lower or higher doses than those indicated above may be preferred. Specific dosages and treatment regimens for any particular subject will depend on a variety of factors, including the potency of the particular composition used, age, body weight, general health, sex, diet, time of administration, rate of elimination, combination of drugs, severity and course of disease ( e.g., the patient's condition and/or symptoms), the subject's predisposition to the disease, and the decision of the health care professional (e.g., physician). The IAIP can be combined with a carrier material to form a unit dosage form.

Once the patient's condition has improved, assessed by improvement of symptoms or measurement of IAIP and/or IAIP-IAIP ligand complex as described herein, as appropriate

- 20 045401 a maintenance dose of the IAIP composition or combination therapy can be prescribed. Subsequently, the dose or frequency of administration, or both, may be reduced depending on the reduction of symptoms to a level at which the improved condition is maintained. If symptoms have decreased to the desired level or the level of IAIP has been increased and/or the level of IAIP-IAIP ligand complexes has been reduced to the desired level, treatment may be discontinued. However, subjects may require long-term intermittent therapy if there is any recurrence of disease symptoms or a decrease in IAIP levels. Improvement may also be judged by the level of IAIP in a biological specimen obtained from the patient (eg, blood (eg, whole blood, plasma, or serum), bronchoalveolar lavage fluid (BALF), sputum, urine, cerebrospinal fluid (CSF), or tissue homogenate (eg, liver biopsy homogenate) The level of IAIP and/or the IAIP-IAIP complex in a biological sample can be determined using one or more of the assays described herein.

Compositions.

The invention provides methods for administering IAIP to a subject having or at risk of developing an inflammatory disease or condition or infection that has been determined to be in need of care according to the diagnostic methods described herein (for example, a subject who has low levels of IAIP and/or increased levels of the IAIP-IAIP ligand complex, such as the IAIP-LPS complex, when measured using the methods described herein). The methods include administration of an IAIP (eg, IaI and/or PaI), a composition that contains an IAIP (eg, IaI and/or PaI) and a pharmaceutically acceptable excipient, carrier or diluent, or such compositions in combination with a secondary treatment as described herein document. The compositions may be formulated as a solid or liquid. The compositions can be formulated for administration by any suitable means, including those described herein.

Injectable forms of IAIP for administration are particularly preferred. IAIPs and compositions containing them can be formulated for intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intradermal, intravitreal, intranasal, and intracranial injection or infusion methods. The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, a sterile injectable aqueous or oleaginous suspension. The suspension can be formulated in accordance with methods known in the art, using suitable dispersing or wetting agents (such as, for example, TWEEN® 80) and suspending agents. The sterile injectable preparation may be in the form of a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.

The compositions may also be formulated for oral administration in any orally acceptable dosage form, including, but not limited to, capsules, tablets, pills, emulsions and aqueous suspensions, dispersions and solutions. For the preparation of solid compositions, such as tablets, IAIP can be mixed with a pharmaceutical excipient to form a solid composition before being formulated into a dosage form containing a homogeneous mixture. This solid preformulation can then be divided into unit dosage forms of the type described above, containing, for example, from 1 mg/kg to about 50 mg/kg IAIP (eg, IaI and/or PaI). The solid preformulation may contain from 10 mg/kg to 30 mg/kg IAIP (eg, IaI and/or PaI). The tablets or pills of the present invention may be coated or otherwise mixed to form a dosage form providing the benefit of a sustained release.

Liquid forms in which the compositions may be formulated, for oral or injectable administration, include aqueous solutions, suitable flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, and also elixirs and similar pharmaceutical carriers.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients described herein and/or known in the art. The compositions can be administered by the oral or nasal route for local or systemic action. The compositions can be nebulized using inert gases.

Topical application of the compositions is useful when the desired treatment involves areas or organs that are readily accessible for topical application. For topical application to the skin, the composition must be formulated with a suitable ointment containing the active ingredients suspended or dissolved in a vehicle. Alternatively, the pharmaceutical composition may be formulated with a suitable lotion or cream containing the active composition suspended or dissolved in a carrier with suitable emulsifying agents.

- 21 045401

The pharmaceutical compositions can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the composition of this invention with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the active components. Topical transdermal patches are also included in this invention.

Compositions administered to a subject may be in the form of one or more of the pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization methods or sterile filtered. Aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration.

Other delivery systems may include sustained release, delayed release, or extended release delivery systems. Repeated administration of the compositions of the invention can be avoided using such systems, increasing convenience for the subject and the physician. Many types of controlled release delivery systems are available and well known to those of ordinary skill in the art. These include polymer-based systems such as those described in (US Patent No. 3,773,919; European Patent No. 58,481; European Patent No. 133,988; Sidman, K. R. et al., Biopolymers 22: 547-556, and Langer, R. et al., J Biomed Mater Res 15:267-277; Langer R Chem Tech 12:98-105). Other examples of sustained release compositions include semi-permeable polymer matrices in the form of molded articles, such as films or microcapsules. Delivery systems also include non-polymeric systems, which are: lipids; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; etc. Methods for preparing such compositions will be apparent to those skilled in the art (see, for example, US Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660, 3,832,253 and 3,854,480).

Methods for preparing pharmaceuticals are known in the art, for example, Niazi, Handbook of Pharmaceutical Manufacturing Formulations (Second Edition), CRC Press 2009, describes the development of formulations for liquid, sterile, compressed, semi-compressed and over-the-counter forms. Transdermal and mucosal delivery, lymphatic delivery, nanoparticles, controlled drug release systems, theranostics, protein and peptide drugs, and biologics delivery are described in Wang et al., Drug Delivery: Principles and Applications (Second Edition), Wiley 2016; formulation and delivery of peptide and protein agents are described, for example, in Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems (Third Edition), CRC Press 2015.

Combination therapies.

The methods of the invention also include the administration or co-administration of a second drug (eg, as a separate therapy or in addition to an IAIP (eg, Ia1 and/or PaI) or a composition thereof) to treat an inflammatory disease or condition (eg, sepsis, septic shock, sterile sepsis, SIRS, trauma/injury (eg, wounds, burns, lacerations, contusions, bone fractures, surgical procedures), stroke (eg, ischemic stroke, hemorrhagic stroke), acute lung injury, ARDS, pneumonia (eg, severe pneumonia, severe or non-severe community-acquired pneumonia, pneumonia acquired in nursing homes), necrotizing enterocolitis, acute pancreatitis, kidney disease, including acute kidney injury, liver injury, acute circulatory failure, preeclampsia, cancer, cancer metastasis, tumor invasion, disease peripheral artery disease, type 1 or type 2 diabetes, cardiovascular atherosclerosis, intermittent claudication, critical limb ischemic disease, myocardial infarction, carotid artery occlusion, umbilical cord occlusion, low birth weight, prematurity, surgically induced inflammation, inflammation associated with abscess, pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia, tissue ischemia, rheumatoid arthritis, meningitis, multiple sclerosis, inflammatory bowel disease (eg, Crohn's disease), chronic obstructive pulmonary disease, rhinitis, preterm labor, or infectious disease), or infection (for example, bacterial infection). For example, the second treatment may include administration of an antibiotic agent if the subject has or is at risk of developing a bacterial infection, an antiviral agent if the subject has or is at risk of developing a viral infection (eg, Dengue fever or West Nile fever), an antifungal agent if the subject has or is at risk of developing a fungal infection, an antiparasitic agent if the subject has or is at risk of developing a parasitic infection, an anti-inflammatory agent if the subject has or is at risk of developing an inflammatory disease or condition described herein, an anticancer agent if the subject has or is at risk of developing cancer or metastasis

- 22 045401 for cancer, an anticoagulant if the subject has or is at risk of developing stroke or myocardial infarction, an immunomodulatory agent if the subject has cancer or an autoimmune disease or condition (for example, inflammatory bowel disease or rheumatoid arthritis), and a bronchodilator, complement inhibitor, vasopressor, sedative, mechanical ventilation if the subject has or is at risk of developing acute lung injury, ARDS, or pneumonia.

When the method includes administering a combination of IAIP (eg, Ia1 and/or PaI), or a composition containing IAIP (eg, IaI and/or PaI) and a pharmaceutically acceptable excipient, diluent or carrier, and one or more second therapeutic agents, each agent is present at dose levels from 1 to 100%, and more preferably from about 5 to 95% of the dose typically used in a course of monotherapy. The agent(s) from the second treatment can be administered separately, as part of a multiple-dose regimen, from an IAIP (eg, IaI and/or PaI) or a composition thereof. The IAIP and the agent(s) from the second treatment may be administered simultaneously or sequentially in any order. Alternatively, the agent(s) from the second treatment may be part of a unit dosage form, for example, mixed with IAIP (eg, IaI and/or PaI) in one composition.

Agents that may be administered in combination with IAIPs (eg, IaIs and/or PaI) include didoxynucleosides, such as zidovudine (AZT), 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxycytidine (ddC ), lamivudine (3TC), stavudine (d4T), and TRIZIVIR (abacavir + zidovudine + lamivudine); nonnucleosides, such as efavirenz (DMP-266, DuPont Pharmaceuticals/Bristol Myers Squibb), nevirapine (Boehringer Ingleheim), and delaviridine (Pharmacia-Upjohn); TAT antagonists such as Ro 3-3335 and Ro 24-7429; protease inhibitors, eg furin inhibitors, indinavir (Merck), ritonavir (Abbott), saquinavir (Hoffmann LaRoche), nelfinavir (Agouron Pharmaceuticals), 141 W94 (Glaxo-Wellcome), atazanavir (Bristol Myers Squibb), amprenavir (GlaxoSmithKline), fosamprenavir (GlaxoSmithKline), tipranavir (Boehringer Ingleheim), KALETRA (lopinavir+ritonavir, Abbott), and other agents such as 9-(2hydroxyethoxymethyl)guanine (acyclovir); interferon, such as interferon alpha, interleukin II and phosphonoformate (Foscarnet); or cell entry inhibitors, for example, T20 (enfuvirtide, Roche/Trimeris) or UK-427857 (Pfizer), levamisole or thymosin, cisplatin, carboplatin, docetaxel, paclitaxel, fluorouracil, capecitabine, gemcitabine, irinotecan, topotecan, etoposide, mitomycin, gefitinib, vincristine, vinblastine, doxorubicin, cyclophosphamide, celecoxib, rofecoxib, valdecoxib, ibuprofen, naproxen, ketoprofen, dexamethasone, prednisone, prednisolone, hydrocortisone, acetaminophen, misonidazole, amifostine, tamsulosin, phenazopyridine, ondansetron, granisetron, alosetron, palonosetron, promethazine, prochlorperazine, trimethobenzamide, aprepitant, diphenoxylate with atropine, and/or loperamide; and anticoagulants, such as antithrombin III and activated protein C.

Additional exemplary agents that may be administered in combination with IAIP (eg, Ial and/or PaI) or compositions thereof are discussed below.

Antibiotics.

If the subject has or is at risk of developing a bacterial infection (eg, necrotizing enterocolitis or gram-negative bacterial infection), the second treatment may include an antibiotic that is used to treat the bacterial infection. Non-limiting examples of antibiotics include: amoxicillin, penicillin, doxycycline, clarithromycin, benzylpenicillin, azithromycin, daptomycin, linezolid, levofloxacin, moxifloxacin, gatifloxin, gentamicin, macrolides, cephalosporins, azithromycin, ciprofloxacin, cefuroxime, amoxicillin /potassium clavulanate, erythromycin, sulfamethoxazole-trimethoprim, doxycycline monohydrate, cefepime, ampicillin, cefpodoxime, ceftriaxone, cefazolin, erythromycin ethyl succinate, meropenem, piperacillin-tazobactam, amikacin, erythromycin stearate, cefepime in dextrose, doxycycline hydrochloride, ampicillin/sulbactam, ceftazidime, gemifloxacin, gentamicin sulfate, erythromycin lactobionate, imipenem/ cilastatin, cefoxitin, cefditoren pivoxil, ertapenem, doxycycline benzoyl peroxide, ampicillin/sulbactam, meropenem, cefuroxime, cefotetan, piperacillin/tazobactam, broad-spectrum fluoroquinolones (which can be used, for example, to treat pneumonia caused by atypical pathogens such as Mycoplasma pneumoniae or Chlamydophila pneumoniae), and others known in the art.

Antiviral agents.

If the subject has or is at risk of developing a viral infection (eg, dengue fever or West Nile fever), the second treatment may include an antiviral agent that is used to treat the viral infection. Non-limiting examples of antiviral agents include zanamivir, oseltamivir, permivir, ribavirin, acyclovir, ganciclovir, foscarnet, cidofovir and others known in the art.

Antifungal agents.

If the subject has or is at risk of developing a fungal infection, the second treatment may include an antifungal agent that is used to treat the fungal infection. Non-limiting examples of antifungal agents include amphotericin, caspofungin, voriconazole, raconazole, posaconazole, fluconazole, flucytosine, and others known in the art.

Antiparasitic agents.

If the subject has or is at risk of developing a parasitic infection, the second treatment may include an antiparasitic agent that is used to treat the parasitic infection (eg, infection caused by protozoan parasites. Non-limiting examples of antiparasitic agents include nitazoxanide, melarsoprol, eflornithine, metronidazole, tinidazole, miltefosine, mebendazole , pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, albendazole, praziquantel, rifampicin and others known in the art.

Anti-inflammatory agents.

If the subject has or is at risk of developing an inflammatory disease or condition described herein, the second treatment may include an anti-inflammatory agent that is used to treat or reduce the inflammation. Non-limiting examples of anti-inflammatory agents include corticosteroids, statins, steroids, non-steroidal anti-inflammatory drugs, glucocorticoids and others known in the art.

Bronchodilators.

If the subject has or is at risk of developing acute lung injury, ARDS, or pneumonia, a second treatment may include a bronchodilator, which is used to relax the bronchial muscles, allowing the airways to be larger and allow air to pass through the lungs. Non-limiting examples of bronchodilators include beta-2 agonists, xanthines, ipratropium, oxytropium, muscarinic receptor antagonists, ipratropium, oxitropium, theophylline, theobromine, caffeine, salbutamol, isoproterenol, albuterol, levalburerol, pirbuterol, metaproterenol, terbutaline, salmeterol, formoterol and others known in this field of technology.

Vasopressors.

If the subject has or is at risk for developing acute lung injury, ARDS, pneumonia, or trauma/injury (eg, wounds, burns, or surgical procedures), the second treatment may include a vasopressor, which causes vasoconstriction and/or an increase in blood pressure. Non-limiting examples of vasopressors include epinephrine, isoproterenol, phenylephrine, norepinephrine, dobutamine, ephedrine, droxidopa, and others known in the art.

Sedatives.

The second treatment may include a sedative. Non-limiting examples of sedatives include propofol, diprivan, morphine, fentanyl, midazolam, lorazepam, presed, infumorph, dexmedetomidine, alfentanil and others known in the art.

Complement inhibitors.

If the subject has or is at risk of developing acute lung injury, ARDS, or pneumonia, a second treatment may include a complement activation inhibitor. The composition may inhibit the activation of one or more complement components, such as C1, C2, C3 (for example, C3a and C3b), C4 (for example, C4b), C5 (for example, C5a and C5b), C6, C7, C8, C9, membrane attack complex, factor B, factor D, MASP-1 and MASP-2 or fragments thereof. Complement inhibitors may include protease inhibitors such as C1-INH and Rhucin/rhC11NH, soluble complement regulators such as sCR1/TP10, CAB-2/MLN-2222, therapeutic antibodies such as eculizumab/SOLIRIS®, pexelizumab, ofatumumab, inhibitors complement components such as compstatin, receptor antagonists such as PMX-53 and rhMBL.

Sets.

The invention also includes kits for use in measuring IAIP in a sample (eg, a fluid sample) of a patient (eg, a human patient, such as a newborn, child, adolescent, or adult). The kit may include one or more of the following components: support (e.g., plate (e.g., multiwell plate)), particles (e.g., magnetic particles, e.g., nanoparticles, magnetic nanoparticles), biochips, resins, containers (e.g., tubes), membranes (for example, nitrocellulose membranes, PVDF membranes), test strips (for example, cellulose, fiberglass or nitrocellulose) or beads (for example, protein A or protein G beads, magnetic beads, glass beads, plastic beads) containing an immobilized agent, IAIP binding agent (eg, IAIP-specific antibody or IAIP ligand), IAIP-detecting labeling agent (eg, IAIP ligand or IAIP-specific antibody), wash buffer, blocking agent, label detection substrate, diluting agent, and assay instructions detection. The binding agent and detection agent may be provided in containers, or the binding agent may be provided pre-attached to the support (eg, the binding agent is already attached to the plate or test strip).

The invention also includes kits for use in measuring the IAIP-IAIP ligand complex in a sample (eg, a fluid sample) of a patient. The kit may include one or more of the following components: support (e.g., plate (e.g., multiwell plate)), particles (e.g., magnetic particles, e.g., nanoparticles, magnetic nanoparticles), biochips, resins, containers (e.g., tubes), membranes (e.g. nitrocellulose membranes, PVDF membranes), test strips (e.g. cellulose, glass fiber or nitrocellulose) or beads (e.g. protein A or protein G beads, magnetic beads, glass beads, plastic beads) containing immobilized binding agent (eg, an IAIP-specific antibody, another IAIP ligand, or an antibody that specifically binds to an IAIP ligand), a labeled detection agent (eg, another IAIP ligand, an IAIP-specific antibody, or an antibody that specifically binds to an IAIP ligand), washes buffers, blocking agent, label detection substrate, dilation agent, and instructions for performing the detection assay. The binding agent and detection agent may be provided in containers, or the binding agent may be provided pre-attached to the support (eg, the binding agent may already be attached to the plate or test strip).

Examples

The following examples are presented to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood from their illustrative nature that other procedures, methodologies or techniques known to those skilled in the art may alternatively be used.

Example 1 Heparin-IAIP Assay.

Preparation of biotinylated heparin.

Heparin (heparin sodium injection (USP), Sagent Pharmaceuticals, cat. no. NDC 25021-40030) was conjugated to biotin using biotin hydrazide reagent (ApExBIO, cat. no. A87007) according to the manufacturer's instructions. Briefly, a 1000 IU heparin solution was mixed with 0.25 mg EDC cross-linking reagent (1-(3-dimethylaminopropyl)-3-3-ethylcarbodiimide hydrochloride, Alfa Aesar, cat. no. A10807) and 0.5 mM biotin hydrazide, which had previously been dissolved in DMSO in 0.1 m MES buffer pH 4.7 with gentle stirring at room temperature for 3 hours. Removal of unconjugated biotin and buffer exchange were performed by ultrafiltration on an Amicon ultracentrifugal filter device with a 5 kDa cutoff filter membrane (Millipore). After dilution in d-H2O, biotinylated heparin was ready for use in the assay.

Heparin-IAIP sandwich ELISA.

Purified mouse monoclonal antibody against human IAIP light chain (mAb 69.26) was immobilized on a 96-well microplate (Immulon 600, Greiner BioOne) at a concentration of 200 ng/well at 32°C for 2 hours. After blocking with 5% nonfat dry milk for 1 h and washing with TBS-T (TBS+0.05% Tween-20), unknown samples and known IAIP standard were diluted in TBS+0.1% Tween-20 and added to the microplate (final volume 50 µl/well).

Samples and serially diluted IAIP standard solution were incubated for 1 h at 32°C. After several washes of the TBS-T microplate, biotinylated heparin was diluted in a buffer containing 20 mM acetic acid + 25 mM NaCl, pH 4.0 (1:2500) and 50 μl per well was added. Biotinylated heparin was incubated for 30 minutes at 32°C, and then the microplate was washed at least three times using TBS-T. Finally, HRP-conjugated streptavidin (Pierce) diluted 1:5000 (50 μl/well) was added to the microplate. After washing, 50 μl of TMB substrate (Neogen Enhanced K-Blue TMB substrate) was added and the reaction was stopped by adding 50 μl of 1 M HCl, and the color change was read on a spectrophotometer (Molecular Devices) at a wavelength of 450 nm. A standard curve was generated using four-point logistic regression (SoftMax Pro software, Molecular Devices), and a seven-point curve was generated from a maximum IAIP concentration of 2.0 μg/ml to 0.03125 μg/ml with serial 2-fold dilutions as shown in FIG. 3B. The IAIP concentration of unknown samples was calculated based on the resulting standard curve.

Example 2. LPS-IAIP assay.

Preparation of biotinylated endotoxin/LPS (lipopolysaccharide):

Lipopolysaccharide (LPS/endotoxin) from Escherichia coli 055:B5 (Sigma, cat. no. L2280) was labeled using biotin hydrazide reagent (ApExBIO, cat. no. A87007) according to the manufacturer's instructions and similar to the protocol used for heparin. 10 mg LPS was reconstituted in 0.1 M MES buffer and 2.5 mM biotin hydrazide and 2.5 mg EDC (1-(3-dimethylaminopropyl)-3-3ethylcarbodiimide hydrochloride, Alfa Aesar, cat. no. A10807) stirred gently for 3 hours at room temperature. Removal of unconjugated LPS and buffer exchange were performed by ultrafiltration on an Amicon ultracentrifugal filtration device with a 5 kDa cutoff filter membrane (Millipore). After dilution in d-H2O, biotinylated LPS was ready for use in the assay.

Sandwich ELISA LPS-IAIP.

Similar to the protocol described above for heparin-IAIP, purified mouse monoclonal anti-human IAIP light chain antibody (mAb 69.26) was immobilized on a 96-well microplate (Immulon 600, Greiner BioOne) at a concentration of 50 ng/well at 32°C for 2 hours. After blocking with 5% nonfat dry milk for 1 hour and rinsing with TBS-T

- 25 045401 (TBS+0.05% Tween-20) unknown samples and known IAIP standard were diluted in TBS+0.1% Tween-20 and added to the microplate (final volume 50 µl/well). Samples and serially diluted IAIP standard solution were incubated for 1 h at 32°C. After several washes of the TBS-T microplate, biotinylated LPS was diluted in a buffer containing 20 mM acetic acid + 25 mM NaCl, pH 4.0 (1:32000) and 50 μl per well was added. Biotinylated LPS was incubated for 30 minutes at 32°C, and then the microplate was washed at least three times using TBS-T. Finally, HRP-conjugated streptavidin (Pierce) diluted 1:10,000 (50 μl/well) was added to the microplate. After washing, 50 μl of TMB substrate (Neogen Enhanced K-Blue TMB substrate) was added and the reaction was stopped by adding 50 μl of 1 M HCl, and the color change was read on a spectrophotometer (Molecular Devices) at a wavelength of 450 nm. A standard curve was generated using four-point logistic regression (SoftMax Pro software, Molecular Devices), and a seven-point curve was generated from a maximum IAIP concentration of 2.0 μg/ml to 0.03125 μg/ml with serial 2-fold dilutions as shown in FIG. 3C.

Example 3: Analysis of blood samples from patients diagnosed with severe community-acquired pneumonia (sCAP).

Serial blood samples were collected from patients with confirmed sCAP who were admitted to the intensive care unit of Rhode Island Hospital. 16 patients were included in the study, and plasma was collected on days 0 (time of ICU (intensive care unit) admission), days 1, 3, and 7. IAIP levels were determined by an established competitive ELISA (Fig. 2A) and both sandwich ELISAs using biotinylated heparin or biotinylated LPS as detection molecules (Fig. 3A). Blood samples from 95 healthy controls aged 17 to 71 years (obtained from healthy blood donors and purchased from the Rhode Island Blood Center) were included in this study to compare IAIP levels in healthy controls with levels measured in patients with sCAP. The results are shown in Fig. 4, 5 and 6.

The results obtained indicate that the level of IAIP in patients with sCAP was significantly lower compared to healthy controls at the time of hospitalization and during disease progression (up to 7 days). Thus, IAIP levels can be used to guide clinicians in assessing prognosis and making therapeutic decisions. Although the competitive ELISA demonstrated a reduction in IAIP levels in patients with sCAP, sandwich ELISA assays using biotin-conjugated heparin and/or LPS as specific binding ligands yielded results that were more statistically significant (p value = 0.0001 between levels IAIP on days 0, 1, 3, 7 compared to healthy controls). Sandwich ELISA assays using biotin-conjugated heparin and/or LPS also revealed lower concentrations of IAIP in patients with sCAP, resulting in a larger difference between IAIP levels measured in patients with sCAP and healthy controls. These data indicate that sandwich ELISA-based assays using labeled IAIP ligands have increased sensitivity and potentially higher accuracy than competitive ELISAs.

These same assays were also used to evaluate samples from patients with sepsis admitted to the intensive care unit at Rhode Island State Hospital.

These analyzes yielded similar results. While the IAIP values of normal healthy controls are similar in the competitive ELISA (mean ± SEM = 328.9 + 6.34 μg/ml) and in both the LPS and heparin-IAIP assays (337.7 ± 9. 05 and 421.8 ± 14.47 μg/ml), IAIP levels in the pneumonia and sepsis groups were significantly higher in the competitive ELISA, resulting in statistically less significant differences or no differences between the disease groups and healthy controls (Fig. 8A-8C ).

Example 4 Heparin IAIP Binding Assay.

The specific binding of heparin to IAIP was assessed using Western blotting. Highly purified IAIP (1 μg), normal human plasma (1 μl), purified bikunin (ulinastatin injection, Techpool, 2 μg), and human serum albumin (HSA, 2 μg) as a negative control were separated on a 7.5% gel SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% nonfat dry milk, the nitrocellulose membrane was incubated with biotin-conjugated heparin (1:500 in TBS) overnight at room temperature. After several washes with TBS+0.05% Tween, HRP-conjugated streptavidin (1:15000) was added and incubated for 1 hour at room temperature. After washing, a metal-reinforced DAB substrate (Pierce) was added to visualize the reactive bands.

Biotin-conjugated heparin specifically bound to purified IAIP (250 kDa IaI and 125 kDa PaI) as shown in lane 2 (Fig. 7), similar to mAb 69.26 (monoclonal antibody against human IAIP). Unlike mAb 69.26, which binds to the light chain (bikunin) (lane 3), biotin-conjugated heparin does not bind to the light chain of IAIP, suggesting that heparin binds to IAIP via the heavy chain. IAIP heavy chains appear to be associated specifically with heparin.

- 26 045401

Multiple reactive protein bands were detected by biotin-conjugated heparin in human plasma (Fig. 7, lane 1), and no binding to HSA was detected.

In addition, free bikunin was not detected in the heparin-IAIP or LPS-IAIP ELISAs, suggesting that only IAIP complexes with an intact heavy chain are measured using the heparin or LPS-IAIP ELISAs. These data indicate that sandwich ELISA using heparin and/or LPS (or other IAIP ligands) provides an assessment of the circulating IAIP complex in a subject (eg, a subject with a medical condition) that has improved accuracy and reliability compared to other known IAIP assays, such as the IAIP competition assay (Fig. 2A).

Example 5. Binding of biotinylated LPS to immobilized IAIP obtained from blood plasma.

To study the direct binding of LPS to IAIP in vitro, IAIP and other control proteins (BSA=bovine serum albumin; bikunin (IAIP light chain); and purified IgG mAb 69.26) at a concentration of 1000 μg/well were immobilized on a 96-well microplate ( Greiner Microlon 600) and blocked with nonfat dry milk (5% in TBS+0.1% Tween-20). After washing, 100 μg of biotinylated LPS (lipopolysaccharides from Escherichia coli O55:B5, purchased from Sigma, cat. no. L2880) was added to each well and incubated in TBS buffer + 150 mM CaCl2 for 1 h at room temperature. After several washes with TBS+0.1% Tween, HRP-conjugated streptavidin was added and incubated for 1 hour. Finally, the TMB substrate was added and the reaction was stopped by adding 1 M HCl solution. Color change and absorbance were measured using spectrometry at 450 nm. Significant binding of biotinylated LPS molecules to IAIP was found compared to BSA, which served as a positive control as described for LPS binding (David SA, et al., Innate Immunity 1995; 2(2):99-106), or IgG mAb 69.26 (negative control) (Fig. 9). LPS did not bind significantly to the IAIP light chain (bikunin), suggesting that the IAIP heavy chain facilitates binding to LPS. Binding may also be mediated by glycosaminoglycan chains.

Example 6. Binding of biotinylated IAIP to immobilized LPS.

In the reverse order of the previous experiment, biotinylated IAIP (4 μg/well) was added and incubated for 1 h to immobilized LPS (100 μg/well), BSA (2 μg/well), IgG mAb 69.26 (2 μg/well) as a positive control, and skim milk powder (2 μg/well) as a negative control (blank). After several washes, HRP-conjugated streptavidin was added, and then the TMB substrate was added to the wells of the microplates. Significant binding of biotinylated IAIP to immobilized LPS was observed, while significantly lower binding of IAIP to immobilized BSA or nonfat dry milk was observed. 10). IgG mAb 69.26 (a monoclonal antibody specific for human IAIP), which served as a positive control in this experiment, bound strongly to biotinylated IAIP.

Example 7: Analysis of the effect of pH on IAIP-LPS binding.

To further characterize the binding of IAIP and LPS, similar solid-phase binding experiments were performed. Biotinylated IAIP was incubated with immobilized LPS, IgG mAb 69.26 (positive control), or BSA (negative control) under different pH conditions. Acetate buffer (50 mM) was used to obtain a low pH solution (pH 3-6), and Tris-HCl buffer (50 mM) was used to obtain a neutral or higher pH solution (pH 7-9). While biotinylated IAIP bound strongly at pH 5, no binding was observed at pH 3 or 4 (Figure 11). Reduced IAIP binding was observed as pH increased above pH 5. IAIP binding to its specific mAb 69.26 peaked at pH 7, but interestingly did not change significantly as pH increased to pH 9. Similarly, at pH 3 and 4, IAIP binding with monoclonal antibody was insignificant. No binding was observed between biotinylated IAIP and BSA. The results clearly indicate that optimal binding between IAIP and LPS occurs in the pH range of 4–7, and in particular around pH 5.

Example 8 Analysis of the effect of salt concentration on IAIP-LPS binding.

The effect of salt concentration was examined by adding increasing amounts of salt.

(NaCl) into the buffer during incubation of biotinylated IAIP with immobilized LPS on a microplate. The binding of IAIP to LPS decreased with increasing salt concentration, but this decrease was not significant even at a salt concentration of 1200 mM, indicating that the binding of IAIP to LPS was relatively strong and specific, similar to the specific binding of IAIP to the monoclonal antibody (mAb 69.26) against human IAIP, which was used as a positive control in this experiment (Fig. 12).

Example 9. Analysis of the effect of nonionic detergents (NP-40 and Tween-20) on IAIP-LPS binding.

Further studies were conducted to examine the effect of increasing concentrations of the nonionic detergents nonylphenoxypolyethoxylethanol (NP-40) (Figure 13) and Tween-20 (Figure 14) on the binding of biotinylated IAIP to immobilized LPS. Increased binding was observed when

- 27 045401 adding 0.05% (w/w) NP-40 or Tween-20 to the IAIP binding reaction with LPS, compared to TBS buffer alone without added detergent. Low amounts of detergent may promote binding to LPS, suggesting that the lipid-binding domain of the LPS molecule may be involved in this interaction. Even when the amount of detergent was increased to 1%, IAIP binding was still relatively strong as most of the IAIP was still bound to LPS, indicating a strong interaction between IAIP and LPS in vitro. Similarly, biotinylated IAIP binds strongly to mAb 69.26 with high specificity and affinity, and addition of detergent up to 1% does not significantly change binding. In contrast, the negative control (BSA) did not show any binding to IAIP.

Example 10 Quantitative determination of IAIP using rapid immunochromatographic assay (ICA).

The methods described herein can be used to rapidly quantify IAIP using immunochromatographic assay (ICA). The IAIP sandwich ELISA described and used in Examples 1-3 can be adapted to a point-of-care (POC) ELISA, a rapid, reliable, quantitative, and user-friendly test that can be used to identify high-risk subjects (eg, infants) , adolescents or adults) with life-threatening severe inflammatory conditions (eg, neonatal sepsis (NS) and necrotizing enterocolitis (NEC)). ICA can a) measure a linear range from 20 to 700 μg/ml IAIP; b) demonstrate high precision (eg, <5% variability or imprecision) for IAIP levels around 150 μg/mL (eg, 100 to 200 μg/mL); c) obtain results in an hour or less (eg, 15 minutes or less, eg, less than 10, 7 or 5 minutes or less); and d) make do with a small sample volume of <150 μL of plasma or whole blood sample (eg, 150, 100, 75, 50, 25, 15, μL or less).

The test can be used to quickly identify an uncontrolled inflammatory response in subjects (eg, premature newborns, infants, adolescents, or adults). Early intervention is known to be important for improving survival of patients suffering from inflammatory disease or infection.

For example, in neonates, the ability to implement early intervention protocols is often limited by the difficulty of identifying such pathological conditions (eg, NS and NEC) from other less serious diseases. The excellent predictive value of a negative IAIP result may help clinicians make difficult decisions, such as, for example, early discontinuation of antibiotic treatment in a subject (eg, infant, adolescent, or adult) in whom current tests are unremarkable. Additionally, as therapeutic proteins, IAIP may be useful in critically ill subjects (eg, infants, adolescents, or adults), and blood levels may serve as a useful prognostic and therapeutic marker to help clinicians make informed decisions about replacement therapy with IAIP or other auxiliary therapeutic agents and monitor disease progression.

A rapid point-of-care test based on IAIP that can be used to influence therapeutic decisions such as initiation and duration of treatment (eg antibiotic treatment) using a simple, convenient and portable device with accurate quantitative results readable in over a short period of time (eg, 15 minutes or less) is not only innovative, but also clinically useful in reducing morbidity and mortality.

ICA involves adding a sample from a subject to a test strip (such as cellulose, fiberglass, or nitrocellulose), which is then pushed into the strip using a buffer. The strip contains an IAIP binding agent (eg, an IAIP-specific antibody or an IAIP ligand). A labeled detection agent (eg, IAIP-specific antibody or IAIP ligand) can be added to the strip before, after, or simultaneously with the sample and visualized using standard methods for rapid quantitation of IAIP.

a) Gold-conjugated reagents: Gold nanoparticles can be used for labeling reagents in (ICA) format due to their excellent colloidal stability. A stable formulation of hydrazide-functionalized gold nanoparticles is commercially available (Innova Bioscience). While typically antibodies are covalently attached to colloidal gold as detection reagents, heparin or LPS can be similarly conjugated through their sugar chains using InnovaCoat Gold nanoparticles. Conjugation of gold to LPS and heparin can be carried out on a small scale and tested for their stability and efficiency in ICA.

b) Optimization of capture, detection, sample reagents and ISA conditions: Based on the competitive ISA development experience described below, the composition and coating conditions of mAb 69.26 on the capture strip and other factors such as blocking agents, surfactants and carbohydrates can be selected to reducing nonspecific binding and improving flow characteristics. You can also determine the optimal dilution factor and the appropriate dilution

- 28 045401 driver, which reduces interference and nonspecific binding. A multivariate design of experiments (DoE) can be used to determine the interaction of the following parameters: 1) concentration of binding agent, 2) coating pH and buffer type (range=4-10), 3) duration of coating, and 4) type of blocking agent (e.g., bovine serum albumin, fish gelatin, etc.).

c) Test Strip Components, Case and Reader: The test strip may be housed in a plastic cassette with sample wells and test reading zones located above the appropriate areas of the strip. A variety of unified spatial strip configurations can be used. You can choose a suitable housing, for example one adapted to the Detekt or ESEQuant reader. The composition of the membrane for sample application and for the conjugate, as well as the pore size of the nitrocellulose membrane, can be selected to achieve the desired rate of capillary action, hence the reaction time of the sample with the detection reagent.

Target ICA performance can be optimized by changing, for example, formulations and processes for test line reagent rotation conditions, method of conjugation of detection molecules (e.g., LIS or heparin) and titration of detection agents, and optimal membrane and absorbent membrane material and sizes as described below:

A. Optimization of test and control lines. Membrane banding conditions can be optimized for both the test line (mAb 69.26) and the control line (LPS-binding proteins or heparin-binding proteins (e.g. coagulation factor IX) test strips. Drying time is 30 minutes at 37° C can be used to immobilize control and test lines as reliable process procedures for ICA membranes.Multivariate DoE can be used to determine the interaction of the following membrane banding parameters: 1) concentration of binding agent, 2) pH and type of buffer (range=pH 4-10), 3) salt concentration and 4) banding volume (1-3 µl/strip) and jet speed. Incorporation of membrane banding improvements from initial optimization studies. Multivariate DoE can also be used to determine the effectiveness of membrane co-coating with varying concentrations (0-1 μg/lane) of protein blockers, surfactants, carbohydrates, and other agents that may reduce nonspecific binding of gold particles or improve membrane mobility characteristics. Based on our solid-phase binding studies, LPS and heparin bind significantly more strongly to IAIP at pH below 5. Similar conditions can be used in ICA.

b. Optimization of test strip parameters. The composition of the sample application membrane and the conjugate membrane, as well as the pore size of the nitrocellulose membrane, can affect the rate of capillary action, and therefore the reaction time of the sample with the gold detection reagent, and can thus be selected to achieve the desired results. We determined that fast flow NC (nitrocellulose) (HF090, Millipore) met the design specifications for sensitivity and time to result. DoE can be used to evaluate filter types, and membrane or absorbent membrane materials for analysis. Likewise, the size and geometry of the sample application membrane and the conjugate membrane can be adjusted to achieve the desired results.

The sample application membrane can be optimized to: 1) accept undiluted sample, 2) provide the proper formulation to minimize nonspecific binding, and 3) accept whole blood samples. The prototype competitive rapid IAIP test described below used a sample dilution (1:5), which reduced the volume requirement of fresh sample applied to the test strip and contained blocking proteins and surfactants that reduce nonspecific binding problems. For components (eg, protein blockers, surfactants, etc.) and their concentrations, a sample application membrane can be designed based on the formulation (eg, dried formulation). There are immunoassay additives available that can be used to effectively and reliably reduce interference and nonspecific binding. Screening studies of potential agents can be accompanied by a multivariate DoE to determine optimal concentrations of, for example, blocking agents.

Conjugation methods for LPS and heparin can be evaluated for use with colloidal gold (nanoparticle) and colored latex particle conjugation methods.

c. Filter membrane for blood separation and plastic cassette body. In addition to the sample application membrane based on the dried formulation, an integrated blood separation filter membrane suitable for the exclusion of blood cells can be selected for all blood samples. The goal is to simplify the process and make the rapid test format user-friendly, especially for non-laboratory, point-of-care applications, such as using directly applied whole blood from a finger prick or blood collection tube. Several types of such blood filters are commercially available, such as VividTM or CytosepTM membranes (Pall) or other manufacturers (GE Lifesciences) and can be used

- 29 045401 used in IHA. The plastic test strip housing can be sized for use with a reader (such as a Detekt reader).

We have determined that the RDS-150 PRO model of the DetektTM portable reader (Detekt Biomedical, Austin, TX) can be used in ICA. Detekt's horizontal flow optical readout technology for rapid testing is currently used in commercial diagnostic devices for food and beverage safety testing, biological threat detection, environmental monitoring and animal health. The reader is a handheld device that optically scans a test strip and compares the signal intensity of the test strip and the control strip using a programmed calibration algorithm. Signal integration software can be configured to interpret the IAIP dose-response curve, and this algorithm can be resident in DetektTM modules. The Vision Suite Pro development kit can be used in conjunction with the manufacturer, Detekt Biomedical LLC in Austin, Texas, to integrate IAIP test strip interpretation with the Detekt reader. A software algorithm based on the shape of the IAIP binding curve and internal controls can be developed to perform the following functions: a) to establish the shape of the IAIP binding curve relative to the test line signal intensity; b) to establish internal controls, possibly based on a control line signal, to correct for day-to-day variability in test strip usage; and c) to establish software to incorporate kit lot-specific information into the reader (e.g., by scanning a bar code).

The performance of the developed and optimized sandwich ELISA-based rapid IAIP test (according to the parameters described above and herein) can be assessed in a preclinical setting. The ICA cassette can be tested as follows:

a) Analytical sensitivity, reporting range and precision - samples/controls of known IAIP concentrations covering the likely range of IAIP concentrations (25-800 µg/ml) can be tested in multiple replicates over several days (N=10) by several skilled workers ( N=3 minimum). Data can be analyzed for precision at all points on the IAIP binding curve, including assay cutoff precision (set at 150 μg/mL).

b) Interfering Substances - Potential interfering substances can be added to sample pools of known IAIP concentrations to determine the effect on dose recovery. Clinical Laboratory Standards Institute (CLSI) protocol EP-07A can be used as a reference. Stock solutions of substances (eg, hemoglobin, bilirubin, human IgG, etc.) that may be found in high concentrations in patient samples can be added to IAIP samples and tested.

c) User Reliability Assessment - Sample pools of known IAIP concentration can be used to test the test strip's reliability to various scenarios/errors made by end users. Parameters tested include: 1) incorrect sample volume added (+25%), 2) laboratory operating conditions (temperature 65-85°F; humidity 10-70% RH), and 3) incorrect test strip reading time (+50-100 % recommended).

d) Study of product stability during shelf life - IAIP assay kits can be stored at various temperatures (e.g., ambient and 37°C) and tested using a set of IAIP controls with a known concentration of IAIP at various time intervals after manufacture ( >1 year) to determine product stability.

e) Sensitivity and specificity in preliminary clinical samples - the product can be tested using clinical samples from subjects (eg infants with suspected and confirmed diagnosis of NS and NEC).

To evaluate the IAIP rapid test in an observational clinical trial, samples may be collected from subjects (eg, subjects having or at risk of an inflammatory disease or condition or infection) undergoing routine clinical evaluation and treatment. Collection of data from multiple centers serving a broad, heterogeneous population of subjects with the same inflammatory disease or condition or infection can be used to ensure sufficient study participation and strengthen the association between IAIP and disease or infection. A rapid bedside care device that can provide real-time IAIP levels may help avoid unnecessary interventions or prolongation of unnecessary treatments (eg, antibiotic therapy) and may reduce the length/cost of hospitalization.

ICA IAIP can be used to clinically evaluate subjects as follows:

1) Collection of blood samples: Specimens may be collected from subjects having or at risk of developing an inflammatory disease or condition at various medical centers. Clinical and demographic data can be recorded for all subjects, including age, weight, gender, and laboratory results. Serial samples from the same subjects

- 30 045401 items collected at different points in time (for example, 0, 24, 48, 72 hours and 7 days) can also be used in ICA. ICA can be used to determine whether IAIP levels correlate with disease severity and progression, whether IAIP levels predict risk or subsequent disease, and whether subjects will develop more severe disease or improve after therapy.

2) Power analysis: Preliminary data and published studies of subjects having an inflammatory disease or condition or infection can be used to estimate the sample size.

3) Blood Test: Collected clinical blood plasma samples can be transferred into test tubes, labeled (de-identified), and kept frozen until tested. IAIP levels are stable for 24 hours at room temperature, up to 14 days with routine clinical refrigerated storage, and indefinitely at 20°C. IAIP levels can be analyzed using the IAIP rapid ICA test and the IAIP sandwich ELISA for a comparative study.

To evaluate the proof of concept for a rapid ICAI test, we manufactured test strip cassettes designed to measure IAIP using a competitive ICA similar to that shown in FIG. 2A. Data obtained using this immunoassay format described below confirm that ICA based on a sandwich format (similar to that shown in Fig. 3A) can also be used. Competitive ICA is described below.

Sepsis and systemic inflammatory response syndrome in newborns Advances in critical care have led to significant improvements in the survival of subjects (eg, infants, especially very low birth weight (VLBW) (<1500 g) preterm infants). Preterm infants, however, are prone to opportunistic infections and acute life-threatening conditions: neonatal sepsis (NS) and necrotizing enterocolitis (NEC). A recent multicenter study found that up to 21% of children with VLBW experience at least one case of late-onset (>72 hours of life) blood culture-confirmed sepsis, and up to 7% of children with VLBW develop NEC. NS and NEC are associated with significant morbidity, including adverse neurodevelopmental outcomes, and have relatively high mortality rates in NS (10–30%) and NEC (16–42%). Early signs and symptoms of these neonatal diseases are nonspecific, often subtle, and can easily be mistaken for noninfectious etiologies such as exacerbations of bronchopulmonary dysplasia, apnea of prematurity, gastroesophageal reflux, or functional bowel dysfunction. Even more alarming, the clinical deterioration of both diseases can progress rapidly, leading to shock, disseminated intravascular coagulation, and death within hours of clinical onset. There are currently no rapid tests that can help a doctor determine a patient's risk of disease progression and death.

Although the exact cause of NEC is still unclear, it is widely believed that the pathogenesis of NEC occurs when infectious agents translocate across the intestinal epithelial layer, evade the innate immune defense, and cause subsequent inflammation and tissue necrosis. Both NS and NEC are associated with systemic inflammatory responses. Their clinical picture, which is nonspecific and subtle, is very similar. In addition, NS and NEC often co-occur in the same episode of illness (eg, NEC with sepsis occurs in one third of NEC cases in case-control studies). Prompt medical attention with prompt antimicrobial treatment and supportive care is the standard of care for both conditions. Thus, it is of practical and clinical importance to identify the risk of these potentially lethal conditions at the earliest possible opportunity.

A biomarker for early and accurate detection of inflammatory disease or infection (eg, NS and/or NEC) would be very useful to help clinicians make difficult decisions about the initial use and continuation or early discontinuation of antibiotic treatment in subjects (eg, infants) for whom conventional tests are uninformative. Unfortunately, there is currently no widely available biomarker that is clinically useful and effective in treating these complex diseases. Recently, procalcitonin (PCT) has been approved as a biomarker to aid in antibiotic treatment in patients with lower respiratory tract infections and sepsis, but this marker is specific only for bacterial infection and is not sensitive in detecting systemic inflammatory conditions caused by viral or other nonbacterial infections. Additionally, the use of the PCT test is still controversial in pediatric patients, especially preterm infants during the first few days of life. Thus, a sensitive biomarker that provides information on the severity of inflammatory disease would be clinically useful in the treatment of inflammatory diseases or infections (eg, NS and NEC in preterm infants). This remains a serious problem.

B. Supporting Preliminary Data (SBIR Project 1 Phase I completed R43 HD069243-01A1).

Comparative studies of the prognostic value of IAIP with other markers in identifying

- 31 045401 infants with systemic inflammation and with more localized disease (spontaneous intestinal perforation).

We confirm that IAIP is an excellent biomarker of severity and that it can differentiate NEC from the more focal inflammatory disease, spontaneous intestinal perforation (SIP). The IAIP test is superior to CRP in differentiating between NEC and SPC.

IAIP can differentiate disease severity, such as local inflammatory response (LIR) versus systemic and potentially life-threatening inflammatory responses (NEC and NS). We collected blood samples from 95 infants (64 girls and 31 boys) suspected of having neonatal sepsis (NS), necrotizing enterocolitis (NEC), and spontaneous intestinal perforation (SIP) at Women & Infants' Hospital in Providence, Rhode Island. Serial samples were also collected when available from each individual patient. Most infants were <30 weeks gestational age (range 23 to 31 weeks) with a mean birth weight of 1235 grams. From these collected samples, we obtained 8 infants with established NEC, 9 infants with SPC, and 20 infants with HC. Because we have previously used the IAIP assay to determine the positive and negative predictive value of NS, we assessed the predictive value of IAIP levels in systemic inflammatory diseases such as NEC and in more localized and focal necrotizing disease such as SPC. We also compared levels of IAIP and another inflammatory biomarker C-reactive protein (CRP) in these infants with controls matched for sex, weight, and gestational age. A competitive ELISA using a single monoclonal antibody specific for human IAIP (mAb 69.26) was used to measure blood IAIP levels, and a CRP ELISA kit was used to analyze CRP levels in a blinded manner. Significant reductions in IAIP levels were found in infants diagnosed with NEC (stage II or higher according to the modified Bell criterion) compared with healthy infants (p < 0.05) and infants with SPC (without radiographic evidence of NEC, p < 0.005). However, no significant differences were found between infants with SPC and healthy controls. In contrast, elevated levels of CRP were found in both infants with NEC and those with SPC, although this increase was not statistically significant (p>0.05) (see Figures 15A-15B). Additionally, the receiver operating characteristic (ROC) of CRP at a cutoff value of <4 yielded an area under the curve (AUC) of 0.65 (p=0.01, 95% CI; 0.540.90) with a sensitivity of 100%, specificity of 64.7% , a positive predictive value (PPV) of 18.7 and a negative predictive value (NPV) of 100; while ROC IAIP at a cutoff value of <207 mg/L yielded a robust AUC of 0.98 (p < 0.0001, 95% CI; 0.840.99). The predictive value of IAIP was superior compared to CRP with a sensitivity of 100%, specificity of 88.2%, PPV41 HNPV 100 (Figures 16A-16B).

Several serial infant samples were available from some, but not all infants, before and after the onset of NEC and SPC. We next analyzed IAIP levels using an established competitive IAIP ELISA. IAIP levels in children with NEC were significantly lower compared with the levels found in patients with SPC (Fig. 17) at diagnosis and after initiation of treatment. These were random samples that were only available from residual blood samples. These results demonstrate that IAIP levels are a useful biomarker that identifies life-threatening systemic inflammatory conditions such as NEC (in addition to NS) with high sensitivity and specificity, and IAIP levels also appear to distinguish NEC from less dangerous ones for life pathological conditions in patients with SPC. The IAIP test demonstrated excellent NPV for both NEC (100%) and HC (98%). The IAIP test can be used to guide treatment decisions (eg, antibiotic treatment), such as early termination in infants for whom conventional tests are uninformative.

Development of a prototype quantitative competitive immunochromatographic assay (ICA) for IAIP that measures IAIP levels in the blood and detects NS and NEC.

a) Selection of test format and assay design: For the IAIP rapid test format, an immunochromatographic assay based test was selected (see FIG. 18). ICAs provide many advantages when developing rapid point-of-care assays because they are designed: 1) to use small sample volumes; 2) to follow well-characterized kinetics and provide short time periods for obtaining test results; 3) to be analytically sensitive and accurate; 4) that usually contain internal controls to verify the correct operation of the device; 5) for production from raw materials that are well characterized and widely available; and 6) to generate signals that can be quantitatively analyzed using a strip reader. Thus, ICA, as a rapid IAIP test, can be used, for example, for point-of-care studies.

b) Optimization of capture, detection, and reagents for sample and ICA conditions:

Initial efforts were successful in adapting the established IAIP competitive ELISA to the competitive ICA format by optimizing capture, detection, sample reagents, and immunochromatographic assay conditions. We also established the dose-response curve and evaluated the signal timing, accuracy, and repeatability of the IAIP rapid assay format. Founded

- 32 045401 on the reader, test result data analysis was originally adapted for use with the Qiagen test strip reader. After these initial tests, we began optimizing the test strips and test conditions, and tested several portable readers that are available and suitable for our test strips.

c) Purified IAIP and anti-IAIP monoclonal antibodies (mAb 69.26): Both reagents are key components used in the competition assay format. We have developed and optimized a scalable bioprocess method for the isolation of IAIP from human plasma, resulting in high yield and high purity. Hybridoma cells were grown in CELLine scale-up culture flasks (Integra Bioscience) for large-scale in vitro antibody production.

IgG was isolated from hybridoma supernatant using protein A affinity chromatography.

d) Test strip: Several pilot batches of strips were prepared and the selected formula provided a stable platform for the quantitative measurement of IAIP in human plasma: 1) Sample application membrane: cellulose membrane with buffer and surfactant; 2) membrane for the conjugate: glass fiber membrane with anti-IAIP (mAb 69.26)-gold conjugate and rabbit IgG-gold conjugate (control line); 3) Nitrocellulose: Fast Flow Nitrocellulose (Millipore HF090) coated with IAIP (Test Line) and coated with Goat Anti-Rabbit IgG (Terminal Absorbent Membrane Control Line: Cellulose Membrane (No Formulation)).

e) Sample: A small volume of plasma (15 µl of 1:5 sample dilution) was added to the strip, followed by 115 µl of buffer to push the sample through the strip. Chase buffer was optimized: 2 mM Tris pH 8.0 + 100 mM NaCl + 0.5% Brij (polyoxyethylene ether) + 0.05% Tween 20 + 1.0% fetal calf serum. Brij surfactant was added to the buffer to facilitate rapid removal of the conjugate from the upper portions of the strip.

f) Procedural time: Strips were used with plasma samples or IAIP calibrators (at three different concentrations) and then quantified using a Qiagen reader at 10, 15, 20, 25, 30, 45 and 60 minutes after sample addition. The strip passed in approximately 7 minutes (i.e., the nitrocellulose was cleared of foreign gold) and could be read at any time after that. We found that after 15 min the signals did not change significantly. Thus, we read the strips equally 15 min after adding the samples.

g) Calibrators and controls: Human blood plasma (Rhode Island Blood Center) with a known IAIP value by ELISA was used as a calibrator and internal standard control. Various doses were created by diluting plasma with 1% FCS-containing buffer. Using a Qiagen portable test reader (ESEQuant LFR), we determined a standard curve ranging from 17.5 to 1100 μg/mL (using a 1:5 sample dilution). The daily reproducibility of the calibration curves was tested by repeating and running a set of calibrators up to 13 times at different times of day over a period of 5 days. The results are shown in Fig. 19. The mean and SD of each value were plotted. The results demonstrate that the test exhibited a dense spread over a 5-day period with an excellent coefficient of variance (CV less than 15%).

h) Test Strip Readers and Software: Since our IAIP Rapid Test is designed for quantitative analysis, we have carefully selected the reader to capture the signal generated by the test strips. There are various types of readers available for sale. The choice of reader was based on features and technical specifications such as design, ergonomics, and software reliability. Because this is an important part of the rapid test, we tested three different readers with different technologies and features for the IAIP rapid test strips: 1) a portable desktop test strip reader (ESEQuant LFR from Qiagen); 2) Detekt RDS 1500 Pro handheld PDA reader (Detekt Biomedical, Austin, TX) and 3) smartphone/tablet based reading system (iCalq-iCalq reader, Salt Lake City, UT). We found that the Detekt reader generated a better linear curve in the 5 to 700 μg/mL range than other readers tested.

Determination of intra-laboratory and intermediate precision of the optimized analysis.

The intra- and intermediate-precision characteristics of the rapid test were determined using collected blood samples (n=6) that were reused for 6 consecutive days. The results were subsequently read using three different portable and hand-held readers, and the coefficient of variation (CV) was calculated from the results obtained from each individual sample. CV for the IAIP rapid test using the ESEQuant reader ranged from 4 to 16%, with an average of 13%, while the Detekt reader demonstrated an average CV of 10% (range 5-16%) and the smartphone-based iCalQ reader demonstrated average CV 16% (range 10-23%). The Detekt reader performed better with a relatively low CV than the other two readers tested. To evaluate the within- and intermediate-precision test, we performed 8 plasma samples and analyzed IAIP levels based on optimized protocols. The signal obtained from each sample was read three times on different readers and IAIP values were calculated individually based on established standard curves. Subsequently, the CV was determined from three replicate readings and a range of 2 to 8% was established with an average CV of 5% for all three tested

- 33 045401 readers.

Evaluating the performance of a rapid assay in infant plasma.

Using samples collected from infants at the Women & Infants' Hospital, we performed an IAIP assay based on the optimized conditions for the IAIP test strips described above. The resulting signals were read sequentially using three different readers, and the results were calculated based on the corresponding standard curve of the readers. The results of the rapid tests were compared with the results obtained using an established competitive ELISA assay and the values of each individual sample were used to plot against the results of the rapid test (Fig. 20A-20C). The results showed an excellent correlation between 6 hours of competitive ELISA and the results of the rapid competitive ICA test, which were obtained within 15 minutes using the ESEQuant reader (Correlation coefficient R2 0.832, n=311) and Detekt reader (R2=0.84, n= 339). However, the smartphone-based iCalQ reader reproduced test results that were less comparable to ELISA results, especially in samples containing high levels of IAIP (>600 μg/mL).

In summary, we have successfully modified the 6-hour laboratory competitive ELISA into a rapid point-of-care IAIP test that is capable of measuring circulating IAIP in the blood (ranging from 10-800 μg/ml) accurately (correlation coefficient R2>0.8 according to compared with ELISA results) within 15 minutes with acceptable intra-laboratory and intermediate precision (less than 20% CV). We confirmed that blood IAIP level is a useful prognostic marker not only for NS but also for NEC with high sensitivity (100%) and high specificity (88%). The IAIP test is more specific in detecting NEC than the CRP test, and the IAIP level is also useful in distinguishing between patients with NEC and SPC.

Given the successful adaptation of the competition assay to the ICA format, we expect that the sandwich ELISA-based IAIP assay described herein (e.g., as shown in Fig. 3A) can also be adapted to the ICA format. Rapid quantification of IAIP can lead to early identification of subjects (eg, human subjects such as infants, children, adolescents and adults) who have or are at risk of developing an inflammatory disease or condition, or infection (eg, sepsis, NEC, bacterial infection , or other disease or pathological condition) and timely initiation of optimal treatment.

Example 11. Solid phase analysis of hyaluronic acid-IAIP.

Hyaluronic acid, sodium salt (Sigma-Aldrich), was dissolved in dH ₂ O (stock concentration 1 mg/ml) and further diluted in 20 mM NaHCO ₃ /Na ₂ Co ₃ buffer at pH 9.0. Fifty, 100 and Greiner BioOne, 200 ng of hyaluronic acid per well were immobilized in a 96-well microplate (Microlon 600) at 37°C for 120 minutes. After blocking with 5% nonfat dry milk in TBS-T (20 mM Tris-buffered saline pH 7.3 + 0.05% Tween-20 (v/v)) at 37°C for 60 minutes and 3 washes with TBS-T, a serially diluted solution containing IAIP (human blood plasma and highly purified IAIP in TBS) was added to the microplate and incubated at 37°C for 60 minutes. Human plasma was obtained by cryoprecipitation of fresh frozen plasma obtained from a local blood bank. A cryosupernatant with an IAIP concentration of 250 μg/mL was used in this experiment. The initial concentration of purified IAIP was 1 mg/ml. The microplate was then washed 3 times with TBS-T, and biotin-conjugated mAb 69.26 (anti-human IAIP monoclonal antibody) was added at a 1:1000 dilution in TBS and incubated at 37°C for 30 minutes.

After three additional washes with TBS-T, streptavidin-conjugated horseradish peroxidase (HRP) diluted to 1:5000 in TBS (Inova Bioscience) was added and incubated at 37°C for 30 minutes. After washing 3 times in TBS-T, 50 μl of Enhanced K-Blue TMB Enhanced K-Blue TMB Substrate (Neogen) was added to each well and the reaction was stopped by adding 50 μl of 1 M hydrochloric acid (HCl). The color change was read using a spectrophotometer (Molecular Devices SpectraMax Plus microplate reader) at 650 nm, and a standard curve was generated for human plasma or purified IAIP at 50, 100 and 200 ng/well hyaluronic acid immobilized (FIGS. 21A-21B ). These data indicate that IAIP can be quantified in biological mixtures by capturing the complex IAIP molecule using hyaluronic acid and detecting the captured IAIP using a monoclonal antibody specific for the IAIP light chain (eg, mAb 69.26). As shown in FIG. 21A-21B, the standard curve can be optimized and adapted using purified IAIP or human plasma with a known amount of IAIP. Therefore, this assay can be used to determine the unknown amount of IAIP in a biological sample from a subject. An alternative approach to this method can be used in which an IAIP-specific monoclonal antibody is used to capture IAIP and biotin-labeled hyaluronic acid is used as a ligand to detect bound IAIP, similar to the methods used in Examples 1 and 2.

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Claims (37)

Other embodiments of the invention All publications, patents and patent applications mentioned above in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application had been specifically and separately identified for inclusion by reference in their entirety. completeness. US Provisional Application Nos. 62/490003 and 62/614333 are specifically incorporated herein in their entirety. Various modifications and variations of the described methods, pharmaceutical compositions and kits according to the invention will be apparent to those skilled in the art without departing from the scope and spirit of the claimed invention. Although the disclosure has been described in connection with specific embodiments, it should be understood that it is capable of further modifications and that the claimed invention should not be unduly limited to such specific embodiments. CLAIM 1. A method for quantifying intact inter-alpha inhibitory protein (IAIP) in a sample obtained from a subject, comprising: a) contacting the sample with a binding agent to produce an IAIP binding agent complex, wherein the binding agent is bound to a support, and wherein the binding agent comprises an antibody that specifically binds to bikunin; b) contacting the IAIP-binding agent complex with a detecting agent, wherein the detecting agent comprises an IAIP ligand that binds to at least one IAIP heavy chain, and wherein the IAIP ligand is selected from the group consisting of endotoxin (LPS), heparin, histone , hyaluronic acid, vitronectin, fibronectin, laminin, tenascin C, aggrecan, von Willebrand factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), factor IX, complement component, factor XIIIa and tissue transglutaminase; c) determining the amount of detection agent bound to the IAIP-binding agent complex; And d) comparing the results obtained in step c) with a known reference concentration, where the sample is blood, plasma, serum, urine, cerebrospinal fluid, synovial fluid, amniotic fluid, interstitial fluid, follicular fluid, peritoneal fluid, bronchoalveolar lavage fluid, breast milk, sputum, lymph, bile or tissue homogenate; and wherein the intact IAIP comprises bikunin and at least one IAIP heavy chain selected from the group consisting of H1, H2 and H3; thereby quantifying intact IAIP in the sample. 2. The method of claim 1, wherein at least one of a), b) or c) is carried out at a pH of from about 7.0 to about 3.5. 3. The method of claim 2, wherein the pH is from about 5.0 to about 3.5. 4. The method of claim 3, wherein the pH is approximately 4.0. 5. The method of claim 1, further comprising: a) adding divalent cations to the sample, and wherein the divalent cations are Ca ²⁺ , Mg ²⁺ , Mn ²⁺ or Fe ²⁺ . 6. The method of claim 1, wherein the subject is a human subject. 7. The method according to any of the previous claims, wherein the IAIP ligand is heparin, hyaluronic acid or histone. 8. The method according to any of the previous paragraphs, wherein the IAIP ligand is heparin. 9. A method according to any of the preceding claims, wherein the detection agent further comprises a label, and wherein the label is biotin, a gold nanoparticle, a magnetic particle, a latex particle, an enzyme, an enzyme substrate, a fluorescent dye, a luminescent compound, or a radioactive label. 10. The method of claim 9, wherein the label is an enzyme, and wherein the enzyme is horseradish peroxidase. 11. The method according to claim 9, where the label includes a gold nanoparticle. 12. Method according to any one of claims 9 to 11, wherein the detection agent comprises biotin and one of streptavidin or avidin. 13. The method according to claim 9, where the enzyme is horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, acetylcholinesterase and catalase. 14. The method of claim 9, wherein the enzyme substrate is p-nitrophenyl phosphate (PNPP), 2,2'azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD ), 3,3',5,5'-tetramethylbenzidine (TMB) or 3,3'-diaminobenzidine (DAB). 15. A method according to any of the preceding claims, wherein the sample is blood, cerebrospinal fluid, bronchoalveolar lavage fluid, synovial fluid or sputum. 16. The method according to any of the previous paragraphs, where the sample is blood. - 35 045401 17. The method according to any of the previous paragraphs, where the sample is plasma or serum. 18. The method of claim 9, wherein the label is a gold nanoparticle and the IAIP ligand is heparin. 19. A method according to any of the preceding claims, wherein the support is a plate, particle, nanoparticle, resin, membrane, biochip, container, test strip or bead. 20. The method according to claim 19, where the substrate is a test strip. 21. The method of claim 20, wherein the detection agent is added to the test strip before, after or simultaneously with the sample. 22. A kit for the quantitative determination of intact IAIP in a sample, the kit comprising: a support, a binding agent containing an antibody that specifically binds to bikunin, where the binding agent is bound to the support, a detection agent containing a label, and an IAIP linand that binds to at least at least one IAIP heavy chain selected from the group consisting of H1, H2 and H3, wherein the IAIP ligand is selected from the group consisting of endotoxin (LPS), heparin, histone, hyaluronic acid, vitronectin, fibronectin, laminin, tenascin C, aggrecan, von Willebrand factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), factor IX, complement component, factor XIIIa and tissue transglutaminase; and instructions for quantifying IAIP in a sample, where the sample is blood, plasma, serum, urine, cerebrospinal fluid, synovial fluid, amniotic fluid, interstitial fluid, follicular fluid, peritoneal fluid, bronchoalveolar lavage fluid, breast milk, sputum, lymph, bile or tissue homogenate; wherein the infarct IAIP comprises bikunin and at least one IAIP heavy chain selected from the group consisting of H1, H2 and H3. 23. The kit of claim 22, wherein the kit further comprises one or more of the following: a wash buffer, a blocking agent, and a substrate for label detection. 24. The kit according to claim 22 or 23, wherein the IAIP ligand is heparin, hyaluronic acid or histone. 25. The kit according to claim 24, wherein the IAIP ligand is heparin. 26. The kit according to any one of claims 22-25, where the label is a biotin, a gold nanoparticle, a magnetic particle, a latex particle, an enzyme, an enzyme substrate, a fluorescent dye, a luminescent compound or a radioactive label. 27. The kit of claim 26, wherein the label is an enzyme, and wherein the enzyme is horseradish peroxidase. 28. The kit according to claim 26, where the label includes a gold nanoparticle. 29. The kit according to any one of claims 26 to 28, wherein the detection agent comprises biotin and one of streptavidin or avidin. 30. The kit according to claim 26, where the enzyme is horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, acetylcholinesterase and catalase. 31. The kit of claim 26, wherein the enzyme substrate is PNPP, ABTS, OPD, TMB or DAB. 32. The kit according to any one of claims 22-31, where the sample is blood, cerebrospinal fluid, bronchoalveolar lavage fluid, synovial fluid or sputum. 33. The kit according to any one of claims 22-32, where the sample is blood. 34. The kit according to any one of claims 22-31, where the blood is plasma or serum. 35. The kit of claim 26, wherein the label is a gold nanoparticle and the IAIP ligand is heparin. 36. The kit according to any one of claims 22 to 35, wherein the support is a plate, particle, nanoparticle, resin, membrane, biochip, container, test strip or bead. 37. The kit according to claim 36, where the substrate is a test strip. -