US20200057077A1 - Methods for quantifying inter-alpha inhibitor proteins - Google Patents

Methods for quantifying inter-alpha inhibitor proteins Download PDF

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US20200057077A1
US20200057077A1 US16/608,531 US201816608531A US2020057077A1 US 20200057077 A1 US20200057077 A1 US 20200057077A1 US 201816608531 A US201816608531 A US 201816608531A US 2020057077 A1 US2020057077 A1 US 2020057077A1
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iaip
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agent
antibody
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Yow-Pin Lim
Denice Spero
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Prothera Biologics Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/38—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4704—Inhibitors; Supressors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • IAIP Inter alpha Inhibitor Proteins
  • IAIP Inter alpha Inhibitor Proteins
  • IAIP are a family of naturally occurring, immunomodulatory plasma proteins that circulate in high concentrations in the blood of all mammals. IAIP are primarily produced in the liver, released into the blood and a subunit (bikunin) is excreted in the urine. IAIP have an important role in modulating inflammation. They have extensive protective effects toward the serious inflammation caused by infection, trauma, and injury and importantly, the protective effects of IAIP are independent from the causative microbial agents or triggers.
  • Members of this family are composed of heavy and light polypeptide subunits that are covalently linked by glycosaminoglycan.
  • IAIP Inter-alpha-Inhibitor
  • H1 & H2 Inter-alpha-Inhibitor
  • L single light chain
  • P ⁇ I Pre-alpha-Inhibitor
  • IAIP When the body generates inflammatory signals, such as those elicited during injury or infection, IAIP traffic into the tissues and directly reach sites of inflammation.
  • the heavy chains of IAIP enhance the anti-inflammatory response by binding to proteins which are part of the inflammatory cascade. Also, when the heavy chains are cleaved, the light chain with its associated GAG (named Bikunin due to its two Kunitz domains) is released and the serine protease inhibitory activity of the light chain is activated.
  • Bikunin inhibits the activity of serine proteases such as trypsin, elastase, plasmin, cathepsin G, and furin. IAIP exert their anti-inflammatory effects through multiple mechanisms.
  • IAIP extra cellular matrix
  • ECM extra cellular matrix
  • IAIP have also been shown in multiple in vivo models to down regulate inflammatory cytokines, such as TNF- ⁇ and IL-6.
  • Bikunin-deficient (and therefore IAIP deficient) mice have been shown to have decreased inflammatory markers of cell adhesion, VCAM-1 and ICAM-1.
  • IAIP In healthy individuals, the amount of circulating IAIP in blood is relatively high (between 400-800 mg/L). However, IAIP levels rapidly decrease during systemic inflammation/sepsis in newborns and in adult patients (Baek Y W, et al. J Pediatr. 2003; 143:11-15; Lim Y P, et al. J Infect Dis. 2003; 188:919-926 and Opal S M, et al. Crit Care Med. 2007; 35:387-392), and decreased levels of IAIP have been shown to correlate strongly with disease progression.
  • IAIP has clinical utility as a prognostic and theranostic marker in assisting clinicians in monitoring disease progression and making informed treatment decisions for diseases such as severe inflammatory diseases such as severe pneumonia, sepsis and the associated organ damage, NEC, wound healing, burn, cancer, stroke, Alzheimer's disease, epilepsy and others.
  • severe inflammatory diseases such as severe pneumonia, sepsis and the associated organ damage, NEC, wound healing, burn, cancer, stroke, Alzheimer's disease, epilepsy and others.
  • a standardized competitive IAIP immunoassay has been used to measure IAIP in over blood samples from patients with systemic inflammation following bacterial and viral infections.
  • the competitive IAIP immunoassay provides a measure of IAIP that detects only the light chain, therefore both intact IAIP and cleaved bikunin are detected in this assay.
  • the competitive assay does not detect the other crucial parts of IAIP which are very important for its anti-inflammatory and tissues repair properties; namely the heavy chains and the glycosaminoglycan.
  • the competitive IAIP immunoassay has limitations in assessment of active IAIP in a patient sample.
  • IAIP as a crucial component of the body's protective innate immune defenses and its potential use as a prognostic biomarker, there exists a need for improved methods to quantitatively measure IAIP.
  • An ideal assay would measure both the light chain and heavy chain subunits to capture the complete molecule.
  • the present invention provides methods for quantifying IAIP in a sample from a subject through direct detection with agents that bind to and detect IAIP (e.g., agents that bind to intact IAIP, a heavy chain of IAIP, or a glycosaminoglycan (GAG) of IAIP).
  • agents that bind to and detect IAIP e.g., agents that bind to intact IAIP, a heavy chain of IAIP, or a glycosaminoglycan (GAG) of IAIP.
  • GAG glycosaminoglycan
  • the invention also features kits that can be used to quantify IAIP according to the methods described herein.
  • a method for quantifying inter-alpha inhibitor 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 a detection agent; and c) detecting an amount of the detection agent bound to the IAIP-binding agent complex to quantify IAIP in the sample.
  • IAIP inter-alpha inhibitor protein
  • the IAIP is intact IAIP.
  • 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.
  • the detection agent is or contains an IAIP ligand. In some embodiments, the detection agent further contains an antibody that binds to the IAIP ligand detection agent (e.g., an IAIP ligand). In some embodiments, the detection agent is an antibody that specifically binds to IAIP.
  • 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 a histone).
  • the binding agent is an an IAIP ligand that binds to IAIP (e.g., heparin, hyaluronic acid, LPS, or a histone) and the detection agent is an antibody that specifically binds to IAIP (e.g., MAb 69.26 or MAb 69.31).
  • IAIP an IAIP ligand that binds to IAIP
  • the detection agent is an antibody that specifically binds to IAIP (e.g., MAb 69.26 or MAb 69.31).
  • the IAIP is in an IAIP-IAIP ligand complex.
  • 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 IAIP of the IAIP-IAIP ligand complex.
  • the detection agent contains an IAIP ligand that binds to IAIP. In some embodiments, the detection agent further contains 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 detection 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 IAIP of the IAIP-IAIP ligand complex.
  • the antibody is a monoclonal antibody. In some embodiments, the antibody is MAb 69.26 or MAb 69.31.
  • the IAIP ligand is selected from the group consisting of endotoxin (LPS), heparin, a histone, hyaluronic acid, vitronectin, fibronectin, laminin, tenascin C, aggrecan, von Willebrand Factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), factor IX, a complement component, factor XIIIa, and tissue transglutaminase.
  • the complement component is C1q, C2, C3, C4, C5, C6, C8, properdin, or factor D.
  • the detection agent contains a label.
  • the label is biotin, an enzyme, an enzyme substrate, a radiolabel, a luminescent compound, colloidal gold, a particle, or a fluorescent dye.
  • the support is a plate, a particle, a nanoparticle, a resin, a membrane, a biochip, a container, a test strip, or a bead.
  • the method further includes a wash step between steps a) and b).
  • the method further includes a wash step between steps b) and c).
  • the method further includes a blocking step prior to step a) or step b).
  • the contacting in step a) and/or b) is performed at a pH of about 7.0 to about 3.5. In some embodiments, the pH is about 5.0 to about 3.5. In some embodiments, the pH is about 4.0.
  • the sample is a fluid.
  • 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.
  • the subject is a human subject. In some embodiments, the subject has been identified as having or at risk of developing an inflammatory disease or condition or an infection. In some embodiments, the subject has not been identified as having or at risk of developing an inflammatory disease or condition or an infection. In some embodiments, the method is performed before, after, or concurrent with diagnosis of the subject as having or at risk of an inflammatory disease or condition or an infection. In some embodiments, the method is performed substantially concurrent with treatment of the subject for an inflammatory disease or condition or an infection. In some embodiments, the method is performed prior to treatment of the subject for an inflammatory disease or condition or an infection. In some embodiments, the method is performed after treatment of the subject for an inflammatory disease or condition or an infection.
  • 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, renal disease, acute kidney injury, liver injury, acute circulatory failure, preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral artery disease, type 1 or type 2 diabetes, atherosclerotic cardiovascular disease, intermittent claudication, critical limb ischemic disease, myocardial infarction, carotid occlusion, umbilical cord occlusion, low birth-weight, premature birth, surgery-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
  • the infection is caused by a 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 species including Klebsiella pneumoniae, Salmonella species, Shigella species, Serratia species, Acinetobacter species; Haemophilus species including Haemophilus influenzae and Haemophilus ducreyi, Brucella species, Yersinia species including Yersinia pestis and Yersinia enterocolitica, Francisella species including Francisella tularensis, Past
  • the subject is a neonate, a child, an adolescent, or an adult.
  • 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.
  • the method is performed at least once, at least twice, at least three times, at least five times, or at least ten times.
  • the method further includes administering a treatment comprising IAIP or a therapeutic agent to the subject.
  • the subject has an IAIP concentration of 200 ⁇ g/mL or lower.
  • the sample from the subject has an elevated level of IAIP-IAIP ligand complex relative to a reference sample.
  • the subject has or is at risk of developing an inflammatory disease or condition or an infection.
  • the method includes administering IAIP and a therapeutic agent to the subject.
  • 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 anti-cancer agent, an anti-coagulant, an immunomodulatory agent, a bronchodilator agent, a complement inhibitor, a vasopressor, a sedative, or mechanical ventilation.
  • the subject has been ill for at least one day. In some embodiments, the subject has been ill for at least one week. In some embodiments, the subject has been ill for at least one month. In some embodiments, the subject has been ill for at least one year.
  • the method is for: a) evaluating the health status of the subject; b) monitoring the health status of the subject; c) diagnosing the subject as having or being at risk for an inflammatory disease or condition or an infection; d) evaluating efficacy of a treatment administered to the subject; or e) evaluating disease severity in the subject.
  • the method further includes comparing the amount of IAIP and/or an IAIP-IAIP ligand complex detected in the sample to the amount of IAIP and/or an IAIP-IAIP ligand complex found in a sample from a normal subject or to a cutoff value. In some embodiments, an amount of IAIP in the sample that is lower than an amount of IAIP in the sample from the normal subject or relative to the cutoff value indicates that the subject has or is at risk of developing an inflammatory disease or condition or an infection.
  • an amount of an IAIP-IAIP ligand complex in the sample that is greater than an amount of IAIP-IAIP ligand complex in the sample from the normal subject or relative to the cutoff value indicates that the subject has or is at risk of developing an inflammatory disease or condition or an infection.
  • the amount of IAIP in the sample from the normal subject, or the cutoff value is >250 ⁇ g/mL. In some embodiments, the amount of IAIP in the sample from the normal subject is about 260 to about 540 ⁇ g/mL.
  • a determination that the subject has an IAIP concentration of 250 ⁇ g/mL or less indicates that the subject has or is at high risk of developing an inflammatory disease or condition or an infection or is diagnosed as having an increased risk of morbidity and/or mortality.
  • the subject has an IAIP concentration of 200 to 300 ⁇ g/mL.
  • 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.
  • the subject previously had an inflammatory disease or condition or an infection.
  • the method is performed prior to the treatment and/or one or more times during the course of the treatment. In some embodiments, the method is performed after initiation of the treatment and/or after conclusion of the treatment. In some embodiments, the treatment is determined to be effective if the concentration of IAIP increases in the subject relative to a prior measurement of IAIP in the subject and/or if the concentration of an IAIP-IAIP ligand complex decreases in the subject relative to a prior measurement of an IAIP-IAIP ligand complex in the subject.
  • the treatment is determined to be ineffective if the concentration of IAIP decreases or remains constant in the subject relative to a prior measurement of IAIP in the subject and/or if the concentration of an IAIP-IAIP ligand complex increases or remains constant in the subject relative to a prior measurement of an IAIP-IAIP ligand complex in the subject.
  • the method further comprises modifying or changing the treatment.
  • a method of treating a subject e.g., a human, such as a neonate, a child, an adolescent, or an adult
  • a subject e.g., a human, such as a neonate, a child, an adolescent, or an adult
  • 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 anti-cancer agent, an anti-coagulant, an immunomodulatory agent, a bronchodilator agent, a complement inhibitor, a vasopressor, a sedative, or mechanical ventilation.
  • 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, renal disease, acute kidney injury, liver injury, acute circulatory failure, surgery-induced inflammation, abscess-induced inflammation, multiple sclerosis, preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral artery disease, type 1 or type 2 diabetes, atherosclerotic cardiovascular disease, intermittent claudication, critical limb ischemic disease, myocardial infarction, carotid occlusion, umbilical cord occlusion, low birth-weight, premature birth, pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia, bacterial infection, wounds, burns, lacerations, contusions, bone fractures, surgical procedures, tissue ischemia, rheumatoid arthritis
  • the infection is caused by a 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 species including Klebsiella pneumoniae, Salmonella species, Shigella species, Serratia species, Acinetobacter species; Haemophilus species including Haemophilus influenzae and Haemophilus ducreyi, Brucella species, Yersinia species including Yersinia pestis and Yersinia enterocolitica, Francisella species including Francisella tularensis, Past
  • kits for quantifying IAIP or an IAIP-IAIP ligand complex in a sample in which the kit comprises an IAIP binding agent and an IAIP detection agent and, optionally, one or more of the following: a wash buffer, a blocking agent, a substrate for detection of a label, and instructions for quantifying a level of IAIP or an IAIP-IAIP ligand complex in a sample.
  • a wash buffer such as a tube or vial
  • a form ready for use e.g., application to a support of the kit (e.g., a plate or test strip).
  • the binding agent is immobilized on a support.
  • the detection agent is labeled.
  • the IAIP binding agent is an IAIP-specific antibody or an IAIP ligand.
  • the kit further contains an IAIP ligand binding agent.
  • the IAIP ligand binding agent is an antibody that binds to an IAIP ligand.
  • the IAIP detection agent is an IAIP-specific antibody or an IAIP ligand.
  • the kit further contains an IAIP ligand detection agent.
  • the IAIP ligand detection agent is an antibody that binds specifically to an IAIP ligand.
  • the IAIP-specific antibody is a monoclonal antibody.
  • the monoclonal antibody is MAb 69.26 or MAb 69.31.
  • the support is a plate, a resin, a container, a membrane, a biochip, a particle, a nanoparticle, a test strip, or a bead.
  • the label is an enzyme, an enzyme substrate, biotin, a particle, a fluorescent dye, a luminescent compound, or a radiolabel.
  • the IAIP ligand is selected from the group consisting of endotoxin (LPS), heparin, a histone, hyaluronic acid, laminin, tenascin C, aggrecan, vitronectin, fibronectin, von Willebrand Factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), factor IX, a complement component, factor XIIIa, and tissue transglutaminase.
  • LPS endotoxin
  • heparin a histone
  • hyaluronic acid laminin
  • tenascin C tenascin C
  • aggrecan vitronectin
  • fibronectin fibronectin
  • von Willebrand Factor pentraxin-3
  • TNF-stimulated gene-6 TNF-stimulated gene-6
  • factor IX a complement component
  • factor XIIIa tissue transglutaminase
  • the term “about” refers to a value that is no more than 10% above or below the value being described.
  • the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.
  • administration refers to providing or giving a subject a therapeutic agent (e.g., IAIP), by any effective route. Exemplary routes of administration are described herein below.
  • a therapeutic agent e.g., IAIP
  • antibody refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes at least the variable domain of a heavy chain, and normally includes at least the variable domains of a heavy chain and of a light chain 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 quad-specific antibodies, diabodies, triabodies, and tetrabodies), single-domain antibodies (sdAb), epitope-binding fragments, e.g., Fab, Fab′ and F(ab′) 2 , Fd, Fvs, single-chain Fvs (scFv), rIgG, single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a V L or V H domain, fragments produced by an Fab expression library, and anti-idiotypic (anti-Id) antibodies.
  • heteroconjugate antibodies e.g., bi-tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies
  • Antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • class e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • subclass of immunoglobulin molecule e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • mAb monoclonal antibody
  • Fab and F(ab′) 2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than an intact antibody.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • the antibody fragments can be a Fab, F(ab′) 2 , scFv, SMIP, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody.
  • binding fragments encompassed of the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L , and C H 1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H 1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb including V H and V L domains; (vi) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a V H domain; (vii) a dAb which consists of a V H or a V L domain; (viii) an isolated complementarity determining region (CDR); and
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.
  • IAIPs inter-alpha inhibitor proteins
  • IAIPs inter-alpha inhibitor proteins
  • IAIP exert a broad range of anti-inflammatory mechanisms, in addition to serine protease inhibitory activity, such as binding to and inactivating complement, extracellular histones, and coagulation factors, down regulating pro-inflammatory cytokines such as TNF-a and IL-6, down regulating adhesion factors such as VCAM and ICAM, and down regulating NFkB.
  • IAIP are also importantly involved in promoting protection and repair of tissues where the heavy chains are transferred to matrix proteins to promote cellular migration and proliferation. In human plasma, IAIPs are found at relatively high concentrations (400-800 mg/L).
  • this family of inhibitors typically includes a combination of polypeptide chains (light and heavy chains) covalently linked by a chondroitin sulfate chain.
  • the heavy chains of IAIPs (H1, H2, and H3) are also called hyaluronic acid (HA) binding proteins.
  • the major forms of IAIPs found in human plasma are inter-alpha-inhibitor (I ⁇ I), which contains two heavy chains (H1 and H2) and a single light chain (L), and pre-alpha-inhibitor (P ⁇ I), which contains one heavy (H3) and one light chain (L).
  • IAIP is the light chain (also termed bikunin (bi-kunitz inhibitor) with two Kunitz domains), bound to the glycosaminoglycan, which is known to broadly inhibit plasma and tissue serine proteases.
  • Another IAIP is the heavy chain-related molecule H4, which circulates in the blood without linkage to bikunin.
  • Yet another IAIP is the heavy chain-related molecule H5.
  • I ⁇ I and P ⁇ I present in the plasma fraction have an apparent molecular weight of between about 60 kDa to about 280 kDa.
  • IAIP ligand refers to a molecule or a fragment thereof that binds to IAIP in vivo or in vitro (e.g., endotoxin (LPS), heparin, a histone, hyaluronic acid, matricellular proteins (e.g., vitronectin, fibronectin, tenascin C, laminin, aggrecan), von Willebrand Factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), coagulation factors (e.g., factor IX and factor XIIIa), a complement component, divalent cations such as Ca 2+ , and tissue transglutaminase).
  • LPS endotoxin
  • heparin e.g., heparin, a histone, hyaluronic acid
  • matricellular proteins e.g., vitronectin, fibronectin, tenascin C, laminin, aggre
  • IAIP ligands also include molecules that are predicted to bind to IAIP based on domain structure (e.g., proteins with RGD domains that would bind to the von Willebrand factor A domain of the IAIP heavy chains).
  • the IAIP ligands for use in the methods described herein include ligands that bind to the heavy chain of IAIP, to the IAIP complex, or to the GAG of IAIP (e.g., ligands that do not bind solely to bikunin).
  • IAIP-specific antibody or “antibody that specifically binds to IAIP” refers to any protein or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule, such as at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding 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.
  • CDR complementarity determining region
  • An antibody that binds specifically to IAIP will bind to IAIP and provide a signal that is it least twice the background signal or noise, and more typically more than 10 to 100 times background.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • a “pharmaceutical composition” or “pharmaceutical preparation” is a composition or preparation having pharmacological activity or other direct effect in the mitigation, treatment, or prevention of disease, and/or a finished dosage form or formulation thereof.
  • the composition is, for example, indicated for human use (e.g., according to drug or biologic regulatory guidelines, such as those promulgated by the F.D.A. and/or the E.M.A.).
  • 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 excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit/risk ratio.
  • the phrase “reducing the likelihood of developing” refers to prophylactic treatment of a patient (e.g., a human) who is susceptible to, or otherwise at risk of, a particular disease, syndrome, or condition (e.g., the conditions described herein, such as an inflammatory disease or an infection)) or is at risk of an increase in the degree or severity of a current disease, syndrome, or condition, for example, a patient having community acquired pneumonia (CAP) who is at risk of progressing to severe community acquired pneumonia (sCAP).
  • CAP community acquired pneumonia
  • sCAP severe community acquired pneumonia
  • a reference sample can be obtained from a healthy individual (e.g., an individual who does not have an inflammatory disease or infection).
  • a reference level can be the level (or an average thereof) of expression or concentration of an analyte (e.g., protein (e.g., an IAIP), nucleic acid, carbohydrate, etc.) determined from one or more reference samples.
  • an analyte e.g., protein (e.g., an IAIP), nucleic acid, carbohydrate, etc.
  • the reference can be an average level of an analyte (e.g., IAIP) (e.g., a mean level or median level) among a plurality of individuals (e.g., healthy individuals, or individuals who do not have an inflammatory disease or infection).
  • a reference level can be a predetermined threshold level, e.g., based on level or concentration of an analyte as otherwise determined, e.g., by empirical assays.
  • sample refers to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, lung lavage, cerebrospinal fluid, tissue (e.g., tissue biopsy or tissue homogenate), pancreatic fluid, synovial fluid, and cells) isolated from a subject (e.g., a mammal, such as a human).
  • a specimen e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, lung lavage, cerebrospinal fluid, tissue (e.g., tissue biopsy or tissue homogenate), pancreatic fluid, synovial fluid, and cells
  • a subject e.g., a mammal, such as a human.
  • the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an analyte (e.g., a protein, such as IAIP) in a heterogeneous population of proteins and other biological molecules.
  • the analyte may be an antigen that is recognized, e.g., by an antibody or antigen-binding fragment thereof.
  • a binding agent e.g., an antibody or antigen-binding fragment or ligand
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen binds to the antigen with a K D of up to 100 nM (e.g., between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof exhibits a K D of greater than 100 nM (e.g., greater than 500 nm, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for that particular antigen or epitope thereof.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate.
  • solid-phase ELISA immunoassays are routinely used to select antibodies 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.
  • infectious sepsis refers to a systemic response to an infection (referred to herein as “infectious sepsis”) or to a non-infectious process associated with acute tissue injury and innate immune activation (referred to interchangeably herein as “sterile inflammation” or “sterile sepsis”), which can lead to tissue damage, organ failure, and death.
  • infectious sepsis can result from an infection caused by bacteria, viruses, fungi, or other microorganisms such as parasites (e.g., protozoan parasites).
  • Sterile sepsis can 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.
  • the term “subject” refers to a mammal, including, but not limited to, a human or non-human mammal, such as a primate, bovine, equine, porcine, ovine, feline, or canine.
  • the subject may be a patient.
  • treating refers to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder or symptoms associated therewith be completely eliminated.
  • FIG. 1 is a schematic depicting the structure of circulating (e.g., in blood) IAIP (IaI and PaI) and free light chain (LC, bikunin), for example, excreted in urine. Heavy and light chains of IAIP are uniquely linked by glycosaminoglycan (GAG).
  • GAG glycosaminoglycan
  • FIG. 2A is a schematic depicting a competitive IAIP ELISA assay.
  • purified IAIP is immobilized to a support, such as a multi-well plate, and a biological sample and a labeled antibody directed to IAIP (e.g., MAb 69.26) are then added to the purified IAIP.
  • a biological sample and a labeled antibody directed to IAIP e.g., MAb 69.26
  • FIG. 2B is a graph showing a standard curve of data produced by a competitive IAIP ELISA assay. This assay provides an indirect measure of IAIP based on competitive antibody binding between IAIP in the sample and immobilized, purified IAIP. Lower signal indicates higher amounts of IAIP in the sample.
  • FIG. 3A is a schematic depicting the “sandwich-type” ELISA using labeled IAIP ligands (e.g., biotinylated heparin or LPS) and exemplary standard curves generated using said ELISA.
  • labeled IAIP ligands e.g., biotinylated heparin or LPS
  • an antibody specific for IAIP e.g., MAb 69.26
  • a 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 the addition of the labeled IAIP ligand.
  • FIGS. 3B and 3C are graphs showing standard curves for heparin-IAIP and endotoxin (LPS)-IAIP sandwich-type ELISAs, respectively. This assay provides a direct measure of IAIP concentration, with increasing signal indicating higher amounts of IAIP in the sample.
  • LPS endotoxin
  • FIG. 4 is a graph showing measurements of IAIP concentration obtained using a competitive ELISA as depicted in FIG. 2A .
  • IAIP was measured in plasma samples from patients with severe community acquired pneumonia (sCAP) on successive days during hospitalization and compared to IAIP levels in normal control subjects.
  • the competitive ELISA yielded an average IAIP concentration of 250 ⁇ g/mL in patients with sCAP and 330 ⁇ g/mL in healthy controls, and found significant differences between IAIP levels in subjects with sCAP and healthy controls on days 0, 1, and 3.
  • FIG. 5 is a graph showing measurements of IAIP concentration obtained using a “sandwich-type” ligand-IAIP ELISA as depicted in FIG. 3A .
  • the results depicted in FIG. 5 were generated using heparin as the IAIP ligand.
  • the same samples evaluated using the competitive ELISA in FIG. 4 were also measured using this assay.
  • the heparin-IAIP ELISA yielded an average IAIP concentration between 125 and 150 ⁇ g/mL in patients with sCAP and 422 ⁇ g/mL in healthy controls, and found differences between IAIP levels in subjects with sCAP and healthy controls that were more statistically significant than the differences observed using the competitive ELISA at all time points.
  • FIG. 6 is a graph showing measurements of IAIP concentration obtained using a “sandwich-type” ligand-IAIP ELISA as depicted in FIG. 3A .
  • the results depicted in FIG. 6 were generated using LPS as the IAIP ligand.
  • the same samples evaluated using the competitive ELISA in FIG. 4 and the heparin-IAIP ELISA in FIG. 5 were also measured using this assay.
  • the LPS-IAIP ELISA yielded an average IAIP concentration between 118 and 145 ⁇ g/mL in patients with sCAP and 338 ⁇ g/mL in healthy controls, and found differences between IAIP levels in subjects with sCAP and healthy controls that were more statistically significant than the differences observed using the competitive ELISA at all time points.
  • the LPS-IAIP ELISA performed comparably 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.
  • FIG. 7 is a series of blots showing the binding of heparin and an IAIP-specific antibody (MAb 69.26) to IAIP.
  • an IAIP-specific antibody MAb 69.26
  • biotinylated heparin bound to purified IAIP (250 kDa I ⁇ I and 125 kDa P ⁇ I), but did not bind to the IAIP light chain, bikunin, or to the negative control human serum albumin.
  • MAb 69.26 bound to both purified IAIP and bikunin.
  • FIGS. 8A-8C are a series of graphs showing measurements of IAIP concentration obtained using a competitive ELISA as depicted in FIG. 2A and “sandwich-type” ligand-IAIP ELISAs as depicted in FIG. 3A .
  • IAIP was measured in plasma samples from patients with severe pneumonia, severe sepsis, and in normal control subjects. Shown below each graph is the mean ⁇ SEM of IAIP concentration and the number of patient samples tested (in brackets). As shown in FIG.
  • the competitive ELISA yielded an average IAIP concentration of 246 ⁇ g/mL in patients with severe pneumonia, 250 ⁇ g/mL in patients with severe sepsis, and 330 ⁇ g/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 controls.
  • the LPS-IAIP ELISA yielded an average IAIP concentration of 141 ⁇ g/mL in patients with severe pneumonia, 150 ⁇ g/mL in patients with severe sepsis, and 338 ⁇ g/mL in healthy controls, and found significant differences between IAIP levels in subjects with severe pneumonia or severe sepsis and healthy controls.
  • the heparin-IAIP ELISA yielded an average IAIP concentration of 145 ⁇ g/mL in patients with severe pneumonia, 193 ⁇ g/mL in patients with severe sepsis, and 422 ⁇ g/mL in healthy controls, and found significant differences between IAIP levels in subjects with severe pneumonia or severe sepsis and healthy controls.
  • the LPS-IAIP ELISA performed comparably to the heparin-IAIP ELISA in terms of having increased sensitivity and providing more statistically significant results compared to the competitive ELISA.
  • FIG. 9 is a histogram showing the binding of biotinylated LPS to immobilized plasma-derived IAIP, bovine serum albumin (BSA), and IgG of IAIP-specific antibody MAb 69.26.
  • LPS demonstrated substantial binding to IAIP and little to 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 heavy chain of IAIP may facilitate binding to LPS.
  • FIG. 10 is a histogram showing the binding of biotinylated IAIP to LPS, BSA, IgG of IAIP-specific antibody MAb 69.26, and non-fat dried milk. As expected, IAIP bound to MAb 69.26 and LPS and showed minimal binding to BSA and non-fat dried milk. These data confirm the results of FIG. 9 .
  • FIG. 11 is a histogram showing the effect of pH on the binding of biotinylated IAIP to LPS, BSA, and IgG of IAIP-specific antibody MAb 69.26.
  • FIG. 12 is a histogram showing the effect of salt (NaCl) concentration on the binding of biotinylated IAIP to LPS, BSA, and IgG of IAIP-specific antibody MAb 69.26. These data show that the binding of IAIP to both LPS and MAb 69.26 was unaffected by salt, indicating strong and specific binding. No binding to BSA was observed at any of the salt concentrations tested.
  • salt NaCl
  • FIG. 13 is a histogram showing the effect of non-ionic detergent NP-40 on the binding of biotinylated IAIP to LPS, BSA, and IgG of IAIP-specific antibody MAb 69.26. Binding of IAIP to LPS was enhanced by the addition of 0.05% NP-40, and was still observed in the presence of 1% NP-40, indicating a strong binding interaction. No binding to BSA was observed at any of the NP-40 concentrations tested.
  • FIG. 14 is a histogram showing the effect of non-ionic detergent Tween-20 on the binding of biotinylated IAIP to LPS, BSA, and IgG of IAIP-specific antibody 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. No binding to BSA was observed at any of the Tween-20 concentrations tested.
  • Decreased IAIP levels were found in infants with proven NEC (mean ⁇ SD: 139 ⁇ 21 ug/mL) while the levels in healthy controls (276 ⁇ 110 ug/mL) or infants with SIP (319 ⁇ 72 ug/mL) were significantly higher (p ⁇ 0.05 and p ⁇ 0.005).
  • no statistically significant difference between IAIP levels in infants with SIP and controls p>0.4.
  • CRP levels no significant difference was found between SIP, NEC and control group (p>0.05).
  • FIGS. 16A-16B are graphs depicting ROC curves of IAIP and CRP in NEC infants.
  • the predictive value of IAIP level is superior with sensitivity of 100%, specificity of 88.2%, PPV 41% and NPV 100%, ( FIG. 16A ) compared to CRP ( FIG. 16B ) with sensitivity of 100%, specificity of 64.7%, PPV 18% and NPV 98%.
  • FIG. 18 is a schematic of a lateral flow immunoassay-based IAIP rapid test.
  • FIG. 19 is a graph depicting the standard curve of the rapid IAIP test using an ESEQuant reader. The value of each points were plotted as the mean+SD of a total of 13 independent analysis.
  • the rapid test standard curve is suitable for plasma samples with IAIP level that ranges from 17.5 to 1100 ⁇ g/mL.
  • FIGS. 20A-20C are a series of graphs depicting the correlations between the results obtained by an IAIP rapid test using ESEQuant reader, Detekt reader, iCalQ reader and the results obtained by the established competitive ELISA format.
  • FIGS. 21A-21B are a series of graphs depicting the quantification of IAIP using a “sandwich-type” ELISA in which hyaluronic acid is immobilized on a 96-well plate to capture IAIP, and IAIP is detected using biotin-conjugated MAb 69.26 (a monoclonal antibody against human IAIP).
  • the assay can be used to quantify IAIP in serial dilutions of both human plasma ( FIG. 21A ) and purified IAIP ( FIG. 21B ) using 50 ng, 100 ng, or 200 ng hyaluronic acid per well.
  • a sample e.g., a sample from a subject, such as a blood sample
  • 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 by using a detectable label.
  • the methods can be used to identify subjects having or at risk of developing an inflammatory disease or condition or an infection, to determine a health status of or disease severity in a subject, and to monitor treatment of a condition in a subject (e.g., an inflammatory disease or condition or an infection) administered a therapeutic agent (e.g., IAIP or an agent for treating an inflammatory disease or condition or an infection).
  • a condition in a subject e.g., an inflammatory disease or condition or an infection
  • a therapeutic agent e.g., IAIP or an agent for treating an inflammatory disease or condition or an infection
  • IAIP immunoassay-based competitive ELISAs
  • H1-H5 heavy chains
  • bikunin the light chain
  • the methods described herein use a reagent (e.g., an IAIP ligand) that detects IAIP in the sample directly by binding to an IAIP heavy chain, a heavy and light chain of an intact IAIP complex, or a GAG of an IAIP complex, but do not bind bikunin alone.
  • a reagent e.g., an IAIP ligand
  • the IAIP ligand-based assays described herein exhibit greater sensitivity and robustness than a competitive ELISA due to the detection of the IAIP heavy chain or intact IAIP complexes.
  • the present methods provide an improved readout of the amount of functional IAIP in a sample relative to antibody-based assays that detect cleaved or degraded IAIP light chain.
  • the methods described herein include the use of an IAIP ligand to detect IAIP, e.g., IAIP captured on a substrate (e.g., a solid support, e.g., a plate, a resin, a particle, a container, a membrane, or a bead), using, e.g., an IAIP-specific agent, such as an antibody, the use of an IAIP ligand to capture IAIP for subsequent detection with an IAIP-specific antibody, and the use of an IAIP ligand to capture IAIP for subsequent detection with a second, different IAIP ligand.
  • a substrate e.g., a solid support, e.g., a plate, a resin, a particle, a container, a membrane, or a bead
  • an IAIP-specific agent such as an antibody
  • a first method that can be used to detect intact IAIP in a sample involves an IAIP binding agent (e.g., an IAIP-specific antibody or an IAIP ligand) that is attached to a support and used to capture IAIP in the sample.
  • An IAIP detection agent e.g., an IAIP ligand or an IAIP-specific antibody
  • IAIP concentration can then be quantified, e.g., by detecting the presence of a label, which is either attached directly to the IAIP detection agent or is attached to a reagent that binds to the IAIP detection agent.
  • a second method that can be used to detect an amount of IAIP in a sample involves detecting IAIP-IAIP ligand complexes that have formed in vivo and that are present in the sample. This method also begins with the use of a binding agent that is attached to a support. The binding agent can bind to either IAIP or the IAIP ligand. A detection agent is then added, which binds to the other component of the IAIP-IAIP ligand complex (e.g., 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 concentration of the IAIP can then be measured, e.g., by detecting the presence of a label, which is either attached directly or indirectly to the detection agent.
  • IAIP ligands for use in the methods described herein include ligands that bind to one or more IAIP heavy chains, to an IAIP heavy chain(s) and the light chain, bikunin (e.g., intact IAIP), or to a GAG of IAIP (e.g., a GAG in intact IAIP).
  • Intact IAIP is a complex that contains at least one IAIP heavy chain (H1, H2, H3, H4, and/or H5) and the IAIP light chain (bikunin).
  • any ligand that binds to a heavy chain of IAIP or intact IAIP can be used in the methods described herein, including, e.g., 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 (e.g., factor IX and factor XIIIa), complement proteins (e.g., C1q, C2, C3, C4, C5, C6, C8, properdin, and factor D), divalent cations (e.g., Ca 2+ ), and tissue transglutaminase.
  • IAIP ligands can be labeled according to standard techniques known in the art (e.g., using one or more of the detectable labels described below).
  • 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 (e.g., interaction of an IAIP-specific antibody with non-IAIP proteins yields a signal similar to background).
  • the antibody that binds specifically to IAIP can bind to a heavy chain of IAIP, the light chain of IAIP, or both, or to a GAG of IAIP.
  • the antibody binds to a heavy chain of IAIP or to an intact IAIP containing heavy and light chains.
  • the antibody may be raised against human IAIP, or against IAIP from another mammal (e.g., non-human primate, cow, pig, sheep, goat, cat, dog, rat, mouse, rabbit, guinea pig, or any other non-human mammal that expresses IAIP).
  • the antibody that binds specifically to IAIP may bind only human IAIP, or may be capable of binding IAIP from humans and other mammals.
  • the antibody can be produced by immunization of an animal typically used to generate antibodies with IAIP (e.g., rabbit, guinea pig, rat, mouse, sheep, donkey, goat, hamster, and chicken).
  • the IAIP-specific antibody can be polyclonal (e.g., PAb R16, PAb R20, PAb R21), monoclonal (e.g., MAb 69.26 or MAb 69.31), chimeric, or recombinant.
  • polyclonal e.g., PAb R16, PAb R20, PAb R21
  • monoclonal e.g., MAb 69.26 or MAb 69.31
  • chimeric, or recombinant e.g., chimeric, or recombinant.
  • Labels for detecting the concentration of IAIP in the methods described herein can be attached or conjugated to IAIP ligands, IAIP-specific antibodies, or to other reagents described herein.
  • Labels suitable for detection of IAIP ligands, IAIP ligand-specific antibodies, and/or IAIP-specific antibodies include biotin, enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, acetylcholinesterase, and catalase), enzyme substrates, radiolabels (e.g., radioisotopes), luminescent compounds, particles (e.g., colloidal gold (e.g., gold nanoparticles), a magnetic particle, or a latex particle) and fluorescent dyes.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ -galactosidase acetylcho
  • the label can then be assessed directly (e.g., through imaging of fluorescent dyes, detection of radioactivity, or detection of particles), through the use of enzyme-conjugated avidin or streptavidin (e.g., avidin or streptavidin conjugated to AP or HRP for detection of biotin), and/or through use of a substrate, and can be visualized by known methods and devices, including, e.g., a spectrophotometer, fluorometer, luminometer, or liquid scintillation counter.
  • Substrates may be colorimetric (e.g., PNPP for detecting AP; or ABTS, OPD, or TMB for detecting HRP), chemiluminescent, or fluorescent.
  • Substrates also include liquid scintillators for detection of radioactivity. Standard detection methods known in the art can be used to detect the labels described herein.
  • the methods described herein can be performed using a sample from a subject (e.g., a human subject). Suitable samples include fluid samples.
  • IAIP can be measured in a sample of blood or plasma from a subject (e.g., a subject with an inflammatory disease or condition, such as sepsis, or a subject at risk for developing an inflammatory disease or condition, such as sepsis).
  • the methods described herein can also be performed using a sample of other bodily 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 e.g., biopsies
  • Tissue samples from a subject can be homogenized in an appropriate buffer to create a “fluid” for quantification of IAIP using these methods.
  • the volume of sample used in the assay will vary depending on the type of assay being performed (e.g., an ELISA, a lateral flow immunoassay, or another assay) and the support (e.g., a plate, a membrane, a test strip, or another support).
  • the volume of sample used can be from about 1 ⁇ L to about 500 ⁇ L (e.g., from about 1 ⁇ L to about 150 ⁇ L, e.g., from 1 ⁇ L to about 30 ⁇ L for an assay such as a lateral flow immunoassay, or from about 50 ⁇ L to about 200 ⁇ L for an assay such as an ELISA).
  • the sample can 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 a buffer used in the methods of the assay), and may be diluted 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:100 or more.
  • a buffer that will not interfere with binding to the binding or detection agent e.g., water, PBS, or a buffer used in the methods of the assay
  • a first IAIP quantification assay involves contacting a sample containing IAIP to an IAIP binding agent (e.g., an antibody that specifically binds to IAIP or an IAIP ligand) to form an IAIP-binding agent complex.
  • an IAIP binding agent e.g., an antibody that specifically binds to IAIP or an IAIP ligand
  • the IAIP binding agent may be attached to a support (e.g., a solid support).
  • Suitable supports include plates (e.g., multi-well plates), particles (e.g., magnetic particles, nanoparticles, magnetic nanoparticles), biochips, resins, membranes (e.g., nitrocellulose membranes, PVDF membranes), containers (e.g., tubes), test strips (e.g., cellulose, glass fiber, nitrocellulose), and beads (e.g., protein A or protein G beads, magnetic beads, glass beads, plastic beads).
  • the support is preferably capable of being washed one or more times (e.g., 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 detection agent (e.g., an IAIP ligand or an antibody that specifically binds to IAIP).
  • an IAIP detection agent e.g., an IAIP ligand or an antibody that specifically binds to IAIP.
  • the IAIP detection agent may be conjugated to a label (e.g., one or more of the labels described above), which can then be detected using known detection methods. Alternatively, the IAIP detection agent can be directly detected without the use of a label.
  • an additional wash step e.g., one or more
  • IAIP can then be measured based on signal from the conjugated label or the attached detection agent (e.g., enzyme activity or fluorescence) using standard techniques known in the art. If an enzyme is used as the label, substrate can be added to produce the signal (for example, a color change) and can be read by a device suitable for detecting the signal, such as a spectrophotometer.
  • the signal (for example, absorbance or fluorescence) can be plotted against a standard with known concentration of IAIP to establish a standard curve or can be compared against a known reference concentration. The unknown concentration in the samples can be calculated and determined based on the established standard curve or reference concentration value.
  • Blocking agents for use in the methods described herein include, e.g., milk, BSA, casein, gelatin (e.g., fish gelatin), and serum (e.g., goat serum, donkey serum, horse serum, fetal bovine serum), among others.
  • the method steps can be performed at a pH of 7.0 to 3.5 (e.g., pH 5.0 to pH 3.5, e.g., pH 4.0).
  • a pH of 7.0 to 3.5 e.g., pH 5.0 to pH 3.5, e.g., pH 4.0
  • either or both of the binding step and the detection step can be performed at a pH of 7.0 to 3.5 (e.g., pH 5.0 to pH 3.5, e.g., pH 4.0).
  • the IAIP binding agent can be prepared in a buffer with a pH of 7.0 to 3.5 (e.g., pH 5.0 to pH 3.5, e.g., pH 4.0) and/or the IAIP detection agent (e.g., the IAIP ligand) can be prepared in a buffer with a pH of 7.0 to 3.5 (e.g., pH 5.0 to pH 3.5, e.g., pH 4.0).
  • a low pH buffer e.g., pH of 5.0 or lower, e.g., pH 4.0
  • divalent cations e.g., Ca2+, Mg2+, Mn2+, Fe2+, etc.
  • Divalent cations may be added to the sample before or during contact with the IAIP binding agent, and/or added to the buffer containing the detection agent before or during contact with the binding agent-IAIP complex.
  • Divalent cations can be provided in a concentration of 1 ⁇ M to 1 M (e.g., 100 ⁇ M to 100 mM, 1 mM to 10 mM).
  • the IAIP binding agent is an antibody that binds specifically 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).
  • the ligand can be unlabeled and detected using a labeled antibody that is specific for the IAIP ligand, or the unlabeled ligand can be detected using an unlabeled antibody that is specific for the IAIP ligand, which is then detected using a labeled secondary antibody (e.g., a labeled secondary antibody that binds to the antibody that is specific for the IAIP ligand but does not bind to the IAIP antibody, e.g., the IAIP antibody and the IAIP ligand antibody are generated in different host species).
  • a labeled secondary antibody e.g., a labeled secondary antibody that binds to the antibody that is specific for the IAIP ligand but does not bind to the IAIP antibody, e.g., the IAIP antibody and the IAIP ligand antibody are generated in different host species.
  • ligand-specific antibody a labeled ligand-specific antibody
  • additional wash steps may be performed before and/or after incubation with each antibody.
  • the IAIP binding agent is an IAIP ligand (e.g., heparin, hyaluronic acid, or LPS), and the detection agent is an antibody that is specific for IAIP (e.g., MAb 69.26 or MAb 69.31).
  • the 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 non-specific signal prior to IAIP measurement.
  • the binding agent is an IAIP ligand (e.g., hyaluronic acid)
  • the detection agent is a labeled IAIP ligand that binds to a different region of IAIP (e.g., heparin, hyaluronic acid, or LPS).
  • This method can be performed with any two IAIP ligands that do not bind to the same region of IAIP (e.g., two IAIP ligands that do not compete for binding to IAIP or for which the binding of one of the IAIP ligands does not sterically hinder the binding of the second IAIP ligand to IAIP).
  • the ligand that acts as the IAIP detection agent can be unlabeled and detected using a labeled antibody that is specific for the IAIP ligand that acts as the detection agent, or it can be detected by a labeled secondary antibody that binds to an unlabeled antibody that is specific for the IAIP ligand that acts as the detection agent. If labeled antibodies are used for detection of the IAIP ligand that acts as the detection agent, additional wash steps may be performed before and/or after incubation with each antibody.
  • the binding agent is an IAIP-specific antibody and the detection agent is a labeled IAIP-specific antibody.
  • the IAIP-specific antibodies is capable of binding to either intact IAIP or IAIP that includes at least one heavy chain (e.g., the antibody does not bind to bikunin unless at least one IAIP heavy chain is present, e.g., the antibody does not bind to cleaved or degraded IAIP lacking bikunin).
  • the two IAIP-specific antibodies used in the assay bind different epitopes of IAIP (e.g., the antibodies do not compete for binding to IAIP).
  • the IAIP-specific antibody used as the detection agent may be unlabled and detected using a labeled secondary antibody. If a labeled secondary antibody is used, additional wash steps may be performed before and/or after incubation with the labeled secondary antibody.
  • the methods described herein can be performed using a lateral-flow immunoassay-based test.
  • a small volume of a sample e.g., 1-30 ⁇ L e.g., 15 ⁇ L
  • a test strip e.g., a cellulose, glass fiber, or nitrocellulose
  • a diluted form e.g., diluted 1:2, 1:5, 1:10, 1:20, 1:100 or more, e.g., with a buffer (e.g., PBS) or water
  • a buffer e.g., PBS
  • the strip contains an IAIP binding agent (e.g., an IAIP-specific antibody or an IAIP ligand) to bind to IAIP in the sample.
  • IAIP can be detected using an IAIP detection agent (e.g., a labeled IAIP-specific antibody or a labeled IAIP ligand), which can be added to the strip before, after, or concurrent with the addition of the sample.
  • the test strip can be read using an appropriate reader for quantification of the label attached to the IAIP detection agent (e.g., a portable tabletop lateral flow reader, a handled PDA-based reader, or a smartphone/tablet based reader, among others).
  • the selection of the binding and detection agent combination can be made based on the parameters outlined herein.
  • This assay can be performed quickly (e.g., in 15 minutes or less, such as 15, 10, or 7 minutes or less) and provides a quantitative and rapid measurement of IAIP in a small sample volume.
  • IAIP-IAIP ligand complexes that have formed in vivo in a subject (e.g., a human subject) and that are present in a sample from the subject).
  • This method involves contacting a sample (e.g., a fluid sample, such as plasma, serum, blood, bronchoalveolar fluid, cerebrospinal fluid, sputum, urine or other bodily fluids) to a binding agent, e.g., a binding agent that is attached to a support (e.g., a solid support).
  • a sample e.g., a fluid sample, such as plasma, serum, blood, bronchoalveolar fluid, cerebrospinal fluid, sputum, urine or other bodily fluids
  • a binding agent e.g., a binding agent that is attached to a support (e.g., a solid support).
  • the binding agent can be an IAIP binding agent (e.g., an antibody that specifically binds to IAIP, for example, MAb 69.26 or MAb 69.31, or an IAIP ligand), or the binding agent can be an antibody that specifically binds to the IAIP ligand of interest (e.g., the IAIP ligand that is suspected to have formed an IAIP-IAIP ligand complex in vivo).
  • an IAIP binding agent e.g., an antibody that specifically binds to IAIP, for example, MAb 69.26 or MAb 69.31, or an IAIP ligand
  • the binding agent can be an antibody that specifically binds to the IAIP ligand of interest (e.g., the IAIP ligand that is suspected to have formed an IAIP-IAIP ligand complex in vivo).
  • Suitable supports include plates (e.g., multi-well plates), particles (e.g., magnetic particles, 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) and beads (e.g., protein A or protein G beads, magnetic beads, glass beads, plastic beads).
  • the support is preferably capable of being washed one or more times (e.g., using a buffer, such as TBS, TBS-T, PBS, or PBS-T) to remove material that does not bind to the binding agent.
  • the binding agent used in the assay is an IAIP binding agent (e.g., an antibody that specifically binds to IAIP, for example, MAb 69.26 or MAb 69.31, or an IAIP ligand)
  • the IAIP-IAIP ligand complex can be detected using a detection agent that is an antibody directed against the IAIP ligand of interest by contacting the antibody to the IAIP complex-binding agent complex.
  • 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 a different IAIP ligand) by contacting the detection agent to the IAIP complex-binding agent complex.
  • 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 a different IAIP ligand
  • a wash step (e.g., one or more) can be performed after incubation with the detection agent.
  • a blocking step can be performed before the addition of the sample to the IAIP or IAIP ligand binding agent to prevent or reduce non-specific binding.
  • Blocking agents for use in the methods described herein include, e.g., milk, BSA, casein, gelatin (e.g., fish gelatin), and serum (e.g., goat serum, donkey serum, horse serum, fetal bovine serum), among others.
  • the detection agent is an antibody (e.g., an IAIP-specific antibody or an IAIP ligand-specific antibody)
  • the detection agent can be directly conjugated to a label (e.g., a label described above), or the detection agent may be visualized by adding a labeled secondary antibody that does not bind to any other antibody used in the assay (e.g., the binding agent, if an antibody).
  • the detection agent is a ligand (e.g., an IAIP ligand, such as a different IAIP ligand than that being detected in the assay)
  • the detection agent can be directly conjugated to a label (e.g., a label described above), or detected using a labeled ligand-specific antibody, which is then detected.
  • 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 (e.g., the binding agent, if an antibody).
  • the detection agent is unlabeled and additional reagents (e.g., labeled primary or secondary antibodies) are used, one or more wash steps may be performed after incubation with the additional reagents to minimize non-specific signal.
  • the label 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 the sample can be compared to signal measured in sample(s) with a known concentration of the IAIP-IAIP ligand complex (e.g., to establish a standard curve).
  • the unknown concentration in the samples can be calculated based on an established standard curve or based on a known reference concentration value.
  • the method can be performed using an antibody that specifically binds to IAIP (e.g., an antibody that binds to intact IAIP and/or an IAIP heavy chain) as the binding agent.
  • the detection step can then be performed by adding labeled secondary antibodies specific to different IAIP ligands, washing after incubation with the labeled secondary antibodies, and detecting signal from the label to determine the identity of the IAIP ligand in the IAIP-IAIP ligand complex.
  • Labeled antibodies can be added and evaluated individually for IAIP ligand identification, or they can be added simultaneously if different labels are attached or conjugated to each antibody (e.g., different fluorescent dyes). Once the IAIP ligand is identified, the amount of the IAIP-IAIP ligand complex in the sample can be quantified as described herein.
  • IAIP or an IAIP-IAIP ligand complex can be quantified by performing the detection methods described herein using a sample of interest alongside samples containing known amounts of IAIP or an IAIP-IAIP ligand complex that are used to create a standard curve.
  • the sample from the subject can be measured at the same time as the known amounts of IAIP or an IAIP-IAIP ligand complex so that the concentration of IAIP or an IAIP-IAIP ligand complex in the sample can be determined.
  • the concentration in the sample from the subject can be compared to an average concentration of IAIP or an IAIP-IAIP ligand complex measured using the same assay in a control population, such as healthy controls, to determine whether the concentration of IAIP in the sample falls within a normal range, or diseased controls, to determine whether the concentration of IAIP in the sample falls within a range for the disease state.
  • IAIP or IAIP-IAIP ligand complex concentration in a sample from a subject can also be compared to IAIP or IAIP-IAIP ligand complex concentration in a healthy control by measuring IAIP or an IAIP-IAIP ligand complex in both the test sample and the control sample at the same time using the methods described herein.
  • Control samples include those that are derived from the same source material (e.g., both the test sample and the control sample are derived from the same bodily fluid or the same tissue type).
  • the test sample and control sample can also be collected from subjects of the same age and/or same sex to minimize possible variation between subjects. If IAIP concentrations are directly compared between a subject and a healthy control, a decrease in IAIP concentration in the subject of 25% or more compared to the healthy control would indicate that the subject has or is at risk of developing an inflammatory disease or condition or an infection.
  • IAIP or IAIP-IAIP ligand complex concentration in a sample from a subject can be compared to a predetermined cutoff value for the concentration of IAIP or IAIP-IAIP ligand complex under known conditions (e.g., a healthy state or a disease state).
  • the cutoff value may be an average concentration of IAIP or IAIP-IAIP ligand complex determined from a population of normal subjects or disease subjects.
  • healthy control subjects have been found to have 400 ⁇ 140 ⁇ g/mL IAIP in plasma, although higher concentrations have also been observed in healthy subjects.
  • Subjects with severe inflammatory disease have been found to have a mean concentration of IAIP below about 200 ⁇ g/mL.
  • An IAIP concentration of about 250 ⁇ g/mL can be used as a cut off to categorize subjects as having or at risk of developing a disease or condition (e.g., an inflammatory disease or condition or an infection). This categorization can then be used to recommend subjects for treatment or for further diagnostic testing.
  • Subjects with a moderate-to-low level of IAIP may benefit from repeated testing over time (e.g., once weekly, twice monthly, once monthly, once bi-monthly, three times annually, or biannually) to determine whether IAIP levels are constant or changing (e.g., increasing or decreasing), as these levels could indicate risk of developing an inflammatory disease or condition or an infection, the presence of an inflammatory disease or condition or an infection, or they could represent the normal baseline level for a subject.
  • the IAIP and IAIP-IAIP ligand complex detection methods described herein can be used to measure IAIP and/or IAIP-IAIP ligand complexes in a variety of subjects, such as a subject having, or suspected of having, a disease or condition (e.g., an inflammatory disease or condition or an infection (e.g., a bacterial infection)).
  • a disease or condition e.g., an inflammatory disease or condition or an infection (e.g., a bacterial infection)
  • the level of IAIP and IAIP-IAIP ligand complex in such a subject can be assessed using any of the above assays, e.g., for the purpose of diagnosing the presence of a disease or condition in the subject or the risk that the subject is developing or will develop a disease or condition or monitoring a subject for development or resolution of a disease or condition.
  • Inflammatory diseases or conditions that can be, e.g., diagnosed or monitored, using the assay methods include, e.g., 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 (e.g., ischemic stroke, hemorrhagic stroke), acute lung injury, acute respiratory distress syndrome (ARDS), pneumonia (e.g., severe pneumonia, severe or non-severe: community acquired pneumonia, hospital acquired pneumonia, nursing home acquired pneumonia), necrotizing enterocolitis, acute pancreatitis, renal diseases (e.g., acute kidney injury, liver injury, acute circulatory failure), preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral artery disease, type 1 or type 2 diabetes, atherosclerotic cardiovascular disease, intermittent claudication, critical limb ischemic disease, myocardial infarction, caroti
  • Infections that can be, e.g., diagnosed or monitored, using the assay methods include, e.g., infections with 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 species including Klebsiella pneumoniae, Salmonella species, Shigella species, Serratia species, Acinetobacter species; Haemophilus species including Haemophilus influenzae and Haemophilus ducreyi, Brucella species, Yersinia species including Yersinia pestis and Yersin
  • the assays described herein can be used to measure IAIP levels in a subject at risk for developing an inflammatory disease or condition or an infection.
  • Risk factors include immunosuppression, immunodeficiency (e.g., a subject that is immunocompromised), advanced age, burns (e.g., thermal burns), trauma, surgery, foreign bodies, cancer, premature birth (e.g., a newborn born prematurely), obesity, and metabolic syndrome.
  • the methods described herein can be performed as part of a routine physical examination or as a general assessment of health.
  • the assays described herein can be used alongside traditional diagnostic methods to determine whether a subject has or is at risk of developing an inflammatory disease or condition or an infection.
  • the IAIP measurement obtained using the assays can also be used to determine whether a patient is a candidate for treatment with IAIP or an anti-inflammatory or anti-infective therapy or for predicting response to administration of IAIP (e.g., a patient with low levels of IAIP can be treated with IAIP and/or may respond favorably to treatment with IAIP).
  • Measurement of IAIP can be followed by administration of IAIP an anti-inflammatory or anti-infective therapy to a subject, if deemed appropriate (e.g., if IAIP levels are determined to be low, e.g., at least 25% lower than what is considered to be a normal IAIP level in a healthy subject, or if IAIP levels are below 200 ⁇ g/mL).
  • Measurement of IAIP and/or an IAIP-IAIP ligand complex in a sample from a subject can be used to determine whether the subject has or is at risk of developing an inflammatory disease or condition or an infection.
  • the method includes measuring the level of IAIP and/or an IAIP-IAIP ligand complex using one of the methods described above, and comparing the level to a control value (e.g., a reference sample from a healthy patient or an average value obtained from measurements of a population of apparently healthy patients).
  • a control value e.g., a reference sample from a healthy patient or an average value obtained from measurements of a population of apparently healthy patients.
  • a reduced level of IAIP in a subject as 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
  • an IAIP concentration of about 250 ⁇ g/mL or lower indicates that the subject has or is at risk of developing an inflammatory disease or condition or an infection.
  • An elevated level of an IAIP-IAIP ligand complex in a subject as compared to a healthy control e.g., a level that is 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% higher or more in a subject compared to a control 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 naturally occurring IAIP-IAIP ligand complex may also provide insight into the type of inflammatory disease or infection that a subject has or is at risk of developing. For example, detecting an elevated level of an IAIP-LPS complex in a sample from a subject may indicate that the subject has or is at risk of developing an infection (e.g., a bacterial infection), and detecting an elevated level of an IAIP-histone complex in a sample from a subject may indicate that the subject has or is at risk of developing an acute systemic inflammatory disease (e.g., sepsis or stroke). Such measurements may be used in diagnosing subjects with particular inflammatory diseases or infections, or in recommending therapies or courses of treatment.
  • an infection e.g., a bacterial infection
  • an acute systemic inflammatory disease e.g., sepsis or stroke
  • Subjects with an IAIP concentration below about 200 ⁇ g/mL, as measured using the assays described herein, can be categorized as having or at high risk of developing severe inflammation or infection or as having a greater morbidity and/or mortality risk.
  • subjects with an elevated level of an IAIP-IAIP ligand complex e.g., subjects with a level of an IAIP-IAIP ligand complex, such as an IAIP-LPS complex, that is 10%, 20%, 30%, 40%, 50% higher or more compared to a healthy control or known reference concentration value
  • subjects with an elevated level of an IAIP-IAIP ligand complex can be categorized as having or at high risk of developing severe inflammation or infection or as having a greater morbidity and/or mortality risk. Once severity is assessed, a corresponding course of treatment can be recommended.
  • Subjects having an IAIP concentration that is indicative of severe inflammation could be selected for more frequent or more aggressive treatment than subjects whose IAIP concentration indicates moderate or low risk of having or developing an inflammatory disease or condition.
  • the assays described herein can be used to measure intact IAIP, and, thus, they can be used to detect life threatening conditions and assess the need for an appropriate therapeutic response.
  • a subject who has previously had or who is at risk of developing an inflammatory disease or condition or an infection e.g., a subject with a genetic predisposition, a subject who has been exposed to others with the disease or infection, or a subject having any of the risk factors described above
  • an inflammatory disease or condition or an infection e.g., a subject with a genetic predisposition, a subject who has been exposed to others with the disease or infection, or a subject having any of the risk factors described above
  • Monitoring may also be a suitable approach for subjects with moderate-to-low IAIP levels (e.g., 300 to 200 ⁇ g/mL), and/or subjects with slightly elevated levels of an IAIP-IAIP ligand complex (e.g., subjects with a level of an IAIP-IAIP ligand complex, such as an IAIP-LPS complex, that is 1%, 5%, or 10% higher compared to a healthy control or known reference concentration value), particularly if the subjects do not present with clear symptoms of inflammation or infection.
  • moderate-to-low IAIP levels e.g., 300 to 200 ⁇ g/mL
  • an IAIP-IAIP ligand complex e.g., subjects with a level of an IAIP-IAIP ligand complex, such as an IAIP-LPS complex, that is 1%, 5%, or 10% higher compared to a healthy control or known reference concentration value
  • IAIP and/or IAIP-IAIP ligand complex measurements can be taken at regular intervals (e.g., once a year, twice a year, once every three months, once monthly, bi-monthly, or once weekly) to determine whether IAIP and/or IAIP-IAIP ligand complex levels are constant or changing. Increasing levels of IAIP and/or decreasing levels of an IAIP-IAIP ligand complex could indicate improvement and lead to a discontinuation of monitoring and/or treatment.
  • the methods described herein can also be used to evaluate treatment efficacy in a subject being treated (e.g., with an antibiotic, anti-inflammatory agent, anti-infective agent, or IAIP) for an inflammatory disease or condition or an infection.
  • IAIP levels can be measured prior to or after the onset of treatment and then measured on an ongoing basis during treatment (e.g., once a day, once a week, bi-weekly, once a month, bi-monthly, once every three months, or twice a year).
  • IAIP levels e.g., an increase of 1%, 5%, 10%, 20%, 30% or more relative to a prior measurement
  • constant or decreasing IAIP levels e.g., one or more measurements that do not show a change or show a decrease of 1%, 5%, 10%, 20%, 30% or more relative to a prior measurement
  • the course of treatment should be modified or changed (e.g., increased in dose or frequency or both, changed to a different therapeutic, or modified to include additional therapeutic agents).
  • detection of an IAIP-IAIP ligand complex can be used to evaluate the efficacy of treatment with a therapy for treating or reducing 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 prior to or after the onset of treatment and then measuring on an ongoing basis during treatment (e.g., once a day, once a week, bi-weekly, once a month, bi-monthly, once every three months, or twice a year).
  • the level of the IAIP-IAIP ligand complex can be compared the level to a control value (e.g., a reference sample from a healthy patient or an average value obtained from measurements of a population of control subjects (e.g., healthy patients)) or to a prior measurement taken from the subject.
  • a control value e.g., a reference sample from a healthy patient or an average value obtained from measurements of a population of control subjects (e.g., healthy patients)
  • a prior measurement taken from the subject e.g., a decrease in the level of the IAIP-IAIP ligand complex toward a “normal level” or a decrease at later time points during treatment (e.g., a decrease of 1%, 5%, 10%, 20%, 30% or more relative to a prior measurement) would indicate that the therapy is efficacious.
  • IAIP-IAIP ligand complex e.g., an increase of 1%, 5%, 10%, 20%, 30% or more relative to a prior measurement or a “normal level” would indicate that treatment is ineffective and requires modification (e.g., a higher dose, more frequent administration, or both, or a different therapeutic or combination therapy).
  • the invention also features methods of treating, preventing, or reducing the risk of developing an inflammatory disease or condition or an infection (e.g., a severe infection) in a subject (e.g., a human) that has been determined to be in need according to the diagnostic methods described herein (e.g., a subject with low IAIP levels and/or elevated levels of an IAIP-IAIP ligand complex compared to a reference or compared to prior measurements).
  • the subject can be treated with a standard of care therapeutic appropriate for the disease or condition and/or IAIP.
  • the subject may be a neonate, a child, an adolescent, or an adult.
  • IAIP concentration Prior to administration of IAIP or another therapeutic agent to a subject in need thereof, IAIP concentration can be measured in a sample from the subject according to the methods described herein.
  • the method can include detecting an IAIP-IAIP ligand complex prior to administration of IAIP.
  • the method can include detecting an IAIP-LPS complex in a subject having or suspected of having a bacterial infection (e.g., a gram negative bacterial infection), and/or measuring IAIP concentration in a sample from the subject, and administering IAIP to the subject (e.g., administering IAIP to a subject with an increased level of the IAIP-LPS complex compared to a healthy control or a reference value (e.g., a level of the IAIP-LPS complex that is 1%, 5%, 10%, 20%, 30% higher or more than the level in a healthy control), or administering IAIP to a subject at risk of systemic inflammation or shock syndrome (e.g., a subject with IAIP levels at least 25% below those of a healthy control)).
  • a bacterial infection e.g., a gram negative bacterial infection
  • IAIP concentration e.g., administering IAIP to a subject with an increased level of the IAIP-LPS complex compared to
  • IAIP or compositions containing IAIP can be administered to a subject in need thereof (e.g., as determined by using one or more of the methods described herein).
  • Subjects who can be treated with IAIP include subjects having an infection (e.g., a gram negative bacterial infection) or subjects with an elevated risk of developing an infection (e.g., subjects with one or more risk factors including immunosuppression, immunodeficiency (e.g., a subject that is immunocompromised), advanced age, burns (e.g., thermal burns), trauma, surgery, foreign bodies, cancer, recent birth (e.g., newborns), premature birth (e.g., newborns born prematurely), obesity, and metabolic syndrome).
  • an infection e.g., a gram negative bacterial infection
  • subjects with an elevated risk of developing an infection e.g., subjects with one or more risk factors including immunosuppression, immunodeficiency (e.g., a subject that is immunocompromised), advanced age, burns (e
  • the infection can result from endotoxins triggered by the release of lipopolysaccharide (LPS) molecules from infecting gram negative bacteria. Severe infection 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.
  • LPS lipopolysaccharide
  • Infections suitable for treatment with IAIP include infections with 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 species including Klebsiella pneumoniae, Salmonella species, Shigella species, Serratia species, Acinetobacter species; Haemophilus species including Haemophilus influenzae and Haemophilus ducreyi, Brucella species, Yersinia species including Yersinia pestis and Yersinia enterocolitica, Francisella species including Francisella tularensis
  • Subjects determined to be in need of treatment (e.g., by using one or more of the methods described herein) or who can be treated with IAIP, after such a need is determined also include subjects having or at risk of developing an inflammatory disease or condition such as acute inflammatory disease, sepsis, septic shock, sterile sepsis, systemic inflammatory response syndrome (SIRS), trauma/injury (e.g., wounds, burns, lacerations, contusions, bone fractures, surgical procedures), stroke (e.g., ischemic stroke, hemorrhagic stroke), acute lung injury, acute respiratory distress syndrome (ARDS), pneumonia (e.g., severe pneumonia, severe or non-severe: community acquired pneumonia, hospital acquired pneumonia, nursing home acquired pneumonia), necrotizing enterocolitis, acute pancreatitis, renal diseases including acute kidney injury, liver injury, acute circulatory failure, preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral artery disease, type 1 or type 2 diabetes, atheros
  • IAIPs e.g., I ⁇ I and/or P ⁇ I
  • a composition containing such proteins and a pharmaceutically acceptable excipient, diluent, or carrier can be administered to a subject (e.g., a human) having or at risk of developing an inflammatory disease or condition or an infection that has been determined to be in need according to the diagnostic methods described herein (e.g., a subject with low IAIP levels and/or elevated levels of an IAIP-IAIP ligand complex compared to a reference or compared to prior 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.
  • parenterally by inhalation spray, topically, nasally, buccally, sublingually, intranasally, by oral administration, inhalation, suppository, rectally, va
  • Administration by injection includes, for example, intravenous, intraperitoneal, subcutaneous, intradermal, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intravitreous, and intracranial injection. If the patient is hospitalized, the preferred method of administration is by intravenous injection.
  • the IAIPs e.g., I ⁇ I and/or P ⁇ I
  • the IAIPs may be administered to the 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.
  • the IAIPs e.g., I ⁇ I and/or P ⁇ I
  • the composition containing such proteins are administered as a continuous infusion.
  • IAIPs for use in the compositions of the invention can be obtained from, e.g., human plasma and blood by methods known in the art (see, e.g., U.S. Pat. No. 9,139,641, which is incorporated herein by reference in its entirety).
  • the IAIPs can be obtained at a purity of 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 (e.g., blood) and used to prepare a composition of the invention (see, e.g., U.S. Pat. No. 7,932,365, which is incorporated herein by reference in its entirety).
  • the IAIPs for use in the compositions of the invention can also be exposed to low pH conditions (e.g., 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 U.S. Pat. No. 9,139,641).
  • compositions may include any suitable IAIP, for example, I ⁇ I, P ⁇ I, a heavy chain, a light chain, or any combination thereof.
  • the composition may include I ⁇ I, P ⁇ I, and/or bikunin.
  • the composition may include I ⁇ I and P ⁇ I.
  • the heavy chain can be H1, H2, H3, H4, or H5.
  • the light chain can be bikunin.
  • the proportion or concentration of IAIPs (e.g., I ⁇ I and/or P ⁇ I) in the compositions can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration.
  • the IAIPs (e.g., I ⁇ I and/or P ⁇ I) may be present in the composition in a physiological proportion.
  • Physiological proportions may be, for example, the proportions found in a person or animal that is healthy and/or the ratio of I ⁇ I and P ⁇ I that appears naturally in human plasma. Physiological proportions are typically from between about 60% to about 80% I ⁇ I and between about 20% to about 40% P ⁇ I.
  • IAIPs e.g., I ⁇ I and/or P ⁇ I
  • IAIPs can have a half-life of, for example, greater than about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7.5, or 10 hours.
  • IAIPs e.g., I ⁇ I and/or P ⁇ I
  • compositions thereof can 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 be administered to a subject over time.
  • a pharmaceutically acceptable composition of the invention for administration to a subject having or at risk of developing an inflammatory disease or condition or an infection that has been determined to be in need according to the diagnostic methods described herein includes IAIPs (e.g., I ⁇ I and/or P ⁇ I) in a dosage known in the art (see, e.g., U.S. Pat. No. 7,932,365, International Patent Application Publication No. WO2009154695, and U.S. Patent Application Publication No. 2009/0190194, each of which is incorporated herein by reference in its entirety).
  • compositions of the invention can be administered in a dosage ranging from about 1 mg/kg to 50 mg/kg, preferably dosages between 10 mg/kg and 30 mg/kg.
  • the dose can 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 recited above may be advantageous.
  • Specific dosage and treatment regimens for any particular subject will depend upon a variety of factors, including the activity of the specific composition employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease (e.g., the patient's condition and/or symptoms), the subject's disposition to the disease, and the judgment of the treating medical professional (e.g., the physician).
  • the IAIPs may be combined with a carrier material to produce a single dosage form.
  • a maintenance dose of an IAIP composition or combination therapy may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the reduction in symptoms, to a level at which the improved condition is retained.
  • treatment may cease. Subjects may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms or decrease in IAIP levels.
  • Improvement of the condition may also be judged based upon the level of I ⁇ Ip in a biological sample derived from the patient (e.g., blood (e.g., whole blood, plasma, or serum), bronchial lavage fluid (BALF), sputum, urine, cerebrospinal fluid (CSF), or a tissue homogenate (e.g., a homogenate of a liver biopsy).
  • a biological sample derived from the patient e.g., blood (e.g., whole blood, plasma, or serum), bronchial lavage fluid (BALF), sputum, urine, cerebrospinal fluid (CSF), or a tissue homogenate (e.g., a homogenate of a liver biopsy).
  • the level of I ⁇ Ip and/or an IAIP-IAIP complex in a biological sample can be determined using one or more of the assays described herein.
  • the invention provides methods of administering IAIP to a subject having or at risk of developing an inflammatory disease or condition or an infection that has been determined to be in need according to the diagnostic methods described herein (e.g., a subject found to have low levels of IAIP and/or elevated levels of an IAIP-IAIP ligand complex, e.g., an IAIP-LPS complex, as measured using the methods described herein).
  • the methods include administration of IAIPs (e.g., I ⁇ I and/or P ⁇ I), a composition that includes IAIPs (e.g., I ⁇ I and/or P ⁇ I) and a pharmaceutically acceptable excipient, carrier, or diluent, or such compositions combined with a secondary treatment, as is described herein.
  • the compositions can be formulated as a solid or a liquid.
  • the compositions can be formulated for administration by any suitable means including those described herein.
  • IAIPs for administration are particularly preferred.
  • IAIPs and compositions containing the same may be formulated for intravenous, intraperitoneal, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intradermal, intravitreous, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, TWEEN® 80) and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
  • 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.
  • the IAIPs may be mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogeneous mixture. This solid pre-formulation can then be subdivided into unit dosage forms of the type described above containing from, for example, 1 mg/kg to about 50 mg/kg of IAIPs (e.g., I ⁇ I and/or P ⁇ I).
  • the solid pre-formulation can contain about 10 mg/kg to 30 mg/kg of IAIPs (e.g., I ⁇ I and/or P ⁇ I).
  • IAIPs e.g., I ⁇ I and/or P ⁇ I.
  • the tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • aqueous solutions suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein and/or known in the art.
  • the compositions can be administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions can be nebulized by use of inert gases.
  • Topical administration of the compositions is useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
  • the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active composition suspended or dissolved in a carrier with suitable emulsifying agents.
  • compositions may also be administered in the form of suppositories for rectal administration.
  • These compositions can be prepared by mixing a composition of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • Topically-transdermal patches are also included in this invention.
  • compositions administered to a subject can be in the form of one or more of the pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of compositions of the invention, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as those described in (U.S. Pat. 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).
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Delivery systems also include non-polymer systems that are: lipids; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Methods for preparation of such formulations will be apparent to those skilled in the art (see, e.g., U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660, 3,832,253, and 3,854,480).
  • the methods of the invention also include administering or co-administering a second treatment (e.g., as a standalone therapy or in addition to IAIPs (e.g., I ⁇ I and/or P ⁇ I) or a composition thereof) for the treatment of an inflammatory disease or condition (e.g., sepsis, septic shock, sterile sepsis, SIRS, trauma/injury (e.g., wounds, burns, lacerations, contusions, bone fractures, surgical procedures), stroke (e.g., ischemic stroke, hemorrhagic stroke), acute lung injury, ARDS, pneumonia (e.g., severe pneumonia, severe or non-severe: community acquired pneumonia, hospital acquired pneumonia, nursing home acquired pneumonia), necrotizing enterocolitis, acute pancreatitis, renal diseases including acute kidney injury, liver injury, acute circulatory failure, preeclampsia, cancer, cancer metastasis, tumor invasion, peripheral artery disease, type 1 or type 2 diabetes, atherosc
  • the second treatment may include administering an antibiotic agent if the subject has or is at risk of developing an bacterial infection, an antiviral agent if the subject has or is at risk of developing an viral infection (e.g., 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 anti-cancer agent if the subject has or is at risk of developing cancer or cancer metastasis, an anti-coagulant if the subject has or is at risk of stroke or myocardial infarction, an immunomodulatory agent if the subject has cancer or an autoimmune disease or condition (e.g., inflammatory bowel disease or rheumatoid arthritis), and a bronchodilator agent, a complement inhibitor, a vasopressor, a sedative,
  • each agent is present at a dosage level of between about 1 to 100%, and more preferably between about 5 to 95%, of the dosage normally administered in a monotherapy regimen.
  • the agent(s) of the second treatment may be administered separately, as part of a multiple dose regimen, from the IAIPs (e.g., I ⁇ I and/or P ⁇ I) or the composition thereof.
  • the IAIPs and agent(s) of the second treatment can be administered simultaneously or sequentially in any order.
  • the agent(s) of the second treatment may be part of a single dosage form, e.g., mixed together with the IAIPs (e.g., I ⁇ I and/or P ⁇ I) in a single composition.
  • Agents that can be administered in combination with IAIPs include dideoxynucleosides, e.g. zidovudine (AZT), 2′,3′-dideoxyinosine (ddl) and 2′,3′-dideoxycytidine (ddC), lamivudine (3TC), stavudine (d4T), and TRIZIVIR (abacavir+zidovudine+lamivudine); non-nucleosides, e.g., 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, e.g., furin inhibitors, indinavir (Merck), ritonavir (Ab
  • IAIPs e.g., I ⁇ I and/or P ⁇ I
  • compositions thereof are discussed below.
  • the second treatment may include an antibiotic agent that is used to treat a bacterial infection.
  • antibiotic agents include amoxicillin, penicillin, doxycycline, clarithromycin, benzylpenicillin, azithromycin, daptomycin, linezolid, levofloxacin, moxifloxacin, gatifloxcin, gentamicin, macrolides, cephalosporins, azithromycin, ciprofloxacin, cefuroxime, amoxillin-potassium clavulanate, erythromycin, sulfamethoxazole-trimethoprim, doxycycline monohydrate, cefepime, ampicillin, cefpodoxime, ceftriaxone, cefazolin, erythromycin ethylsuccinate, meropenem, piperacillin
  • the second treatment may include an antiviral agent that is used to treat a viral infection.
  • antiviral agents include zanamivir, oseltamivir, permivir, ribavirin, acyclovir, ganciclovir, foscarnet, cidofovir, and others known in the art.
  • the second treatment may include an antifungal agent that is used to treat a fungal infection.
  • antifungal agents include amphotericin, caspofungin, voriconazole, itraconazole, posaconazole, fluconazole, flucytosine, and others known in the art.
  • the second treatment may include an antiparasitic agent that is used to treat a parasitic infection (e.g., a parasitic protozoan infection.
  • antiparasitic agents include nitazoxanide, melarsoprol, eflornithine, metronidazole, tinidazole, miltefosine, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, albendazole, praziquantel, rifampin, and others known in the art.
  • the second treatment may include an anti-inflammatory agent that is used to treat or reduce inflammation.
  • anti-inflammatory agents include corticosteroids, statins, steroids, nonsteroidal anti-inflammatory drugs, glucocorticoids, and others known the art.
  • the second treatment may include a bronchodilator that is used to relax the bronchial muscles allowing airways to be larger and air to pass through the lungs.
  • bronchodilators include beta 2 agonists, xanthines, ipratropium, oxitropium, muscarinic receptor antagonists, ipratropium, oxitropium, theophylline, theobromine, caffeine, salbutamol, isoproterenol, albuterol, levalburerol, pirbuterol, metaproterenol, terbutaline, salmeterol, formoterol, and others known in the art.
  • the second treatment may include a vasopressor that causes vasoconstriction and/or an increase in blood pressure.
  • vasopressors include epinephrine, isoproterenol, phenylephrine, norepinephrine, dobutamine, ephedrine, droxidopa, and others known in the art.
  • the second treatment may include a sedative.
  • sedatives include propofol, diprivan, morphine, fentanyl, midazolam, lorazepam, precede, infumorph, dexmedetomidine, alfentanil, and others known in the art.
  • the second treatment may include an inhibitor of complement activation.
  • the composition may inhibit activation of one or more complement components such as C1, C2, C3 (e.g., C3a and C3b), C4 (e.g., C4b), C5 (e.g., C5a and C5b), C6, C7, C8, C9, membrane attack complex, Factor B, Factor D, MASP-1, and MASP-2, or fragments thereof.
  • complement components such as C1, C2, C3 (e.g., C3a and C3b), C4 (e.g., C4b), C5 (e.g., C5a and C5b), C6, C7, C8, C9, membrane attack complex, Factor B, Factor D, MASP-1, and MASP-2, or fragments thereof.
  • the complement inhibitors may include protease inhibitors such as C1-INH and Rhucin/rhC11 NH, soluble complement regulators such as sCR1/TP10, CAB-2/MLN-2222, therapeutic antibodies such as eculizumab/SOLIRIS®, Pexelizumna, ofatumumab, complement component inhibitors such as compstatin, receptor antagonists such as PMX-53 and rhMBL.
  • protease inhibitors such as C1-INH and Rhucin/rhC11 NH
  • soluble complement regulators such as sCR1/TP10, CAB-2/MLN-2222
  • therapeutic antibodies such as eculizumab/SOLIRIS®, Pexelizumna, ofatumumab
  • complement component inhibitors such as compstatin
  • receptor antagonists such as PMX-53 and rhMBL.
  • kits for use in measuring IAIP in a sample e.g., a fluid sample
  • a patient e.g., a human patient, such as a neonate, a child, an adolescent, or an adult.
  • the kit may include one or more of the following: a support (e.g., a plate (e.g., a multi-well 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 an immobilized IAIP binding agent (e.g., an IAIP-specific antibody or an IAIP ligand), a labeled IAIP detection agent (e.g., an IAIP ligand or IAIP-specific antibody), a wash buffer, a blocking agent, a substrate for detection of the label, a dilution agent, and instructions for performing the detection assay.
  • the binding agent and detection agent may be provided
  • kits for use in measuring an IAIP-IAIP ligand complex in a sample e.g., a fluid sample from a patient.
  • the kit may include one or more of the following: a support (e.g., a plate (e.g., a multi-well 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 an immobilized binding agent (e.g., an IAIP-specific antibody, a different IAIP ligand, or an antibody that binds specifically to the IAIP ligand), a labeled detection agent (e.g., a different IAIP ligand),
  • Heparin Heparin Sodium Injection USP, Sagent Pharmaceuticals, Cat # NDC 25021-400-30
  • Biotin Hydrazide reagent ApExBIO, Cat # A87007
  • 1000 IU heparin solution was mixed with 0.25 mg crosslinker reagent EDC (1-(3-Dimethylaminopropyl)-3-3ethylcarbodiimide hydrochloride, Alfa Aesar Cat # A10807) and 0.5 mM Biotin hydrazide that had been previously dissolved in DMSO in 0.1 M MES buffer pH 4.7 with gentle mixing at room temperature for 3 hrs.
  • the unconjugated biotin and buffer exchange was carried out by ultrafiltration on an Amicon Ultra centrifugal filter device with 5 kDa cut off filter membrane (Millipore). Following dilution in d-H2O, the biotinylated Heparin was ready for use in the assay.
  • Purified mouse monoclonal antibody against the light chain of human IAIP was immobilized on a 96-well microplate (Immulon 600, Greiner BioOne) at 200 ng/well at 32° C. for 2 hrs. After blocking with 5% non-fat dried milk for 1 hr 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 uL/well). The samples and the serially diluted IAIP standard solution were incubated for 1 hr at 32° C.
  • biotinylated heparin was diluted in a buffer containing 20 mM Acetic acid+25 mM NaCl, pH 4.0 (1:2500) and 50 ⁇ L was added per well. The biotinylated heparin was incubated for 30 minutes at 32° C. and the microplate was then washed at least three times using TBS-T. Finally, HRP-conjugated Streptavidin (Pierce) diluted at 1:5000 (50 uL/well) was added to the microplate.
  • the substrate TMB was added (Neogen Enhanced K-Blue TMB substrate) and the reaction was stopped with the addition of 50 ⁇ L 1 M HCl and the color change was read on spectrophotometer (Molecular Devices) at 450 nm wavelength.
  • the standard curve was generated using four-point logistic regression (SoftMax Pro software, Molecular Devices) and a seven-point curve was plotted from maximum IAIP concentration of 2.0 pg/mL to 0.03125 pg/mL with serial two-fold dilution as shown in FIG. 3B .
  • the IAIP concentration of the unknown samples was calculated based on the generated standard curve.
  • Lipopolysaccharide from Escherichia coli 055:B5 (Sigma Catalog # L2280) was labeled with biotin using Biotin Hydrazide reagent (ApExBIO, Cat # A87007) according to the manufacturer's instructions and similarly 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 # A10807) were gently mixed for 3 hrs at room temperature.
  • mouse monoclonal antibody against the light chain of human IAIP (MAb 69.26) was immobilized on 96-well microplate (Immulon 600, Greiner BioOne) at 50 ng/well at 32° C. for 2 hrs. After blocking with 5% non-fat dried milk for 1 hr 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 uL/well). The samples and the serially diluted IAIP standard solution were incubated for 1 hr at 32° C.
  • biotinylated LPS was diluted in a buffer containing 20 mM Acetic acid+25 mM NaCl, pH 4.0 (1:32,000) and 50 uL was added per well.
  • the biotinylated LPS was incubated for 30 minutes at 32° C. and the microplate was then washed at least three times using TBS-T.
  • HRP-conjugated Streptavidin (Pierce) diluted at 1:10,000 (50 ⁇ L/well) was added to the microplate.
  • IAIP levels were significantly lower in sCAP patients compared to healthy controls at the time of hospitalization and during disease progression (up to 7 days). Therefore, IAIP levels can be used to guide physicians in evaluating prognosis and making therapeutic decisions.
  • the competitive ELISA showed decreased IAIP levels in sCAP patients
  • the “sandwich-type” ELISA assays using biotin-conjugated heparin and/or LPS also yielded a lower concentration of IAIP in sCAP patients, giving rise to a greater difference between IAIP levels measured in patients with sCAP and healthy controls. These data indicate that the “sandwich-type” ELISA assays using labeled IAIP ligands have increased sensitivity and potentially greater accuracy than competitive ELISA assays.
  • HRP-conjugated streptavidin (1:15,000) was added and incubated for 1 hr at room temperature. Following washing, the substrate Metal-enhanced DAB (Pierce) was added to visualize the reactive bands.
  • the biotin-conjugated heparin bound specifically to purified IAIP (250 kDa I ⁇ I and 125 kDa P ⁇ I) as shown in Lane 2 ( FIG. 7 ), similar to MAb 69.26 (monoclonal antibody against human IAIP).
  • MAb 69.26 monoclonal antibody against human IAIP
  • biotin-conjugated heparin did not bind to the light chain of IAIP, suggesting that heparin binds to IAIP via the heavy chain.
  • the heavy chains of IAIP appear to be bound specifically to heparin.
  • BSA bovine serum albumin
  • bikunin the light chain of IAIP
  • purified IgG of monoclonal antibody MAb 69.26 at 1000 ⁇ g/well were immobilized on a 96-well microplate (Greiner Microlon 600) and blocked with non-fat dried milk (5% in TBS+0.1% Tween 20).
  • biotinylated LPS Lipopolysaccharides from Escherichia coli 055:B5, purchased from Sigma, Cat # L2880
  • TBS+150 mM CaCl2 100 ⁇ g was added to each well and incubated in TBS+150 mM CaCl2) buffer for 1 hr at room temperature.
  • HRP-conjugated Streptavidin was added and incubated for 1 hr.
  • TMB substrate was added and the reaction was stopped by adding 1 M HCl solution. The color change and absorbance were measured spectrometrically at 450 nm.
  • biotinylated IAIP (4 ⁇ g/well) was added and incubated for 1 hr to immobilized LPS (100 ⁇ g/well), BSA (2 ⁇ g/well), IgG of MAb 69.26 (2 ⁇ g/well) as a positive control, and non-fat dried milk (2 ⁇ g/well) as negative control (blank).
  • HRP-conjugated streptavidin was added and subsequently TMB substrate was added to the microplate wells.
  • Significant binding of biotinylated IAIP to immobilized LPS was detected, while significantly lower binding of IAIP to immobilized BSA or non-fat dried milk was observed FIG. 10 ).
  • IgG of MAb 69.26 monoclonal antibody specific to human IAIP
  • the methods described herein can be used for the rapid quantification of IAIP using a lateral-flow immunoassay (LFIA).
  • LFIA lateral-flow immunoassay
  • the “sandwich type” IAIP ELISA described and used in Examples 1-3 can be adapted to LFIA, a point-of-care (POC), rapid, reliable, quantitative and user-friendly test that can be used to identify high-risk subjects (e.g., infants, adolescents, or adults) with life threatening, severe inflammatory conditions (e.g., neonatal sepsis (NS) and necrotizing enterocolitis (NEC)).
  • high-risk subjects e.g., infants, adolescents, or adults
  • severe inflammatory conditions e.g., neonatal sepsis (NS) and necrotizing enterocolitis (NEC)
  • the LFIA can a) measure a linear range between 20 and 700 ⁇ g/ml IAIP; b) exhibit high precision (e.g., variability or error of ⁇ 5%) for IAIP levels near 150 ⁇ g/ml (e.g., about 100 to about 200 ⁇ g/mL); c) obtain results in an hour or less (e.g., 15 minutes or less, such as less than 10, 7, or 5 minutes or less); and d) require a small sample volume ⁇ 150 ⁇ L of plasma or whole blood samples (e.g., 150, 100, 75, 50, 25, 15, ⁇ L or less).
  • the test can be used to quickly identify a dysregulated inflammatory response in subjects (e.g., preterm newborns, infants, adolescents or adults). It is known that early intervention is important for improving survival in patients experiencing an inflammatory disease or condition or an infection.
  • IAIP In newborns, for example, the ability to apply early intervention protocols is often limited by the difficulty in identifying such conditions (e.g., NS and NEC) from other less serious diseases.
  • the excellent negative predictive value of IAIP can help guide clinicians to make difficult decisions, such as, e.g., early termination of antibiotic treatment in a subject (e.g., an infant, an adolescent, or an adult) in whom current tests are uninformative.
  • IAIP might be beneficial in critically ill subjects (e.g., infants, adolescents, or adults) and the blood levels can serve as a useful theranostic marker to help physicians make informed decisions about a replacement therapy with IAIP or other adjunctive therapeutic agents and to 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 (e.g., antibiotic treatment), using a simple, user-friendly and portable device with accurate quantitative results readable within a short time period (e.g., 15 min. or less) is not only innovative, but also clinically useful to help reduce morbidity and mortality.
  • the LFIA involves adding a sample from a subject to a test strip (e.g., cellulose, glass fiber, or nitrocellulose), which is then pushed into the strip using a buffer.
  • a test strip e.g., cellulose, glass fiber, or nitrocellulose
  • the strip contains an IAIP binding agent (e.g., an IAIP-specific antibody or an IAIP ligand).
  • the labeled detection agent e.g., an IAIP-specific antibody or an IAIP ligand
  • Gold nanoparticles can be used to label the reagents in the (LFIA) format due to its superior colloidal stability.
  • a stable formulation of gold nanoparticles that are functionalized with hydrazides are commercially available (Innova Bioscience). While usually antibodies are covalently attached to colloidal gold as detecting reagents, heparin or LPS can be similarly conjugated via their sugar chains using the InnovaCoat Gold nanoparticles. The gold conjugation of LPS and heparin can be performed in a small scale and tested for their stability and performance in the LFIA.
  • the formulation and coating conditions of MAb 69.26 on the capture line and other factors, such as blocking agents, surfactants, and carbohydrates, can be selected to reduce non-specific binding and to improve flow characteristics.
  • the optimal dilution factor and suitable diluent that reduces interference and non-specific binding can also be determined.
  • the test strip can be housed in a plastic cassette with sample well and test read windows placed over the appropriate areas of the strip.
  • a variety of standardized strip dimensional configurations can be used.
  • a suitable housing can be chosen, such as one that is adaptable to the Detekt or ESEQuant reader.
  • the composition of sample and conjugate pad, as well as the pore size of the nitrocellulose membrane, can be selected to achieve a desired rate of capillary action, and hence, reaction time of the sample with detector reagent.
  • LFIA performance goals can be optimized by changing, e.g., the formulations and processes for the striping conditions of the Test line reagent, the conjugation method of the detecting molecules (e.g., LPS or heparin), and the titration of the detection agents, as well as the optimal material and dimensions of pads and wicks, as described below:
  • Membrane stripping conditions can be optimized for both Test Line (MAb 69.26) and Control Line (LPS-binding proteins or heparin-binding proteins (for e.g. clotting factor IX) of the test strip.
  • a 30 minute at 37° C. drying period can be used to immobilize the Test and Control Lines, as robust manufacturing procedures for LFIA membranes.
  • Multi-factorial DoE can also be used to determine the efficacy of membrane co-coating with various concentrations (0-1 ⁇ g/strip) of protein blockers, surfactants, carbohydrates, and other agents which may reduce gold particle non-specific binding or improve flow characteristics of the membrane. Based on our solid phase binding studies, LPS and heparin bind significantly stronger to IAIP at pH lower than 5. Similar conditions can be used in the LFIA.
  • composition of the sample pad and conjugate pad, as well as the pore size of the nitrocellulose membrane, can affect the rate of capillary action, and hence, reaction time of the sample with gold detector reagent, and thus, can be selected to achieve desired results.
  • a fast flow NC HF090, Millipore
  • DoE can be used to assess types of filter, and pad and wick materials for the assay.
  • the size and geometry of the sample pad and conjugate pad can be adjusted to achieve desired results.
  • the sample pad can be optimized to: 1) accept undiluted sample, 2) provide the necessary formula to minimize non-specific binding and 3) accept whole blood samples.
  • the competitive IAIP prototype rapid test discussed below utilized a diluted sample (1:5), which mitigated the need for raw sample volume entering the test strip and contained blocking proteins and surfactants that mitigate non-specific binding issues.
  • a formulation-based sample pad e.g., dried formula
  • components e.g., protein blockers, surfactants, etc.
  • concentrations e.g., concentration of components
  • immunoassay additives that may be used to effectively and reliably reduce interferences and non-specific binding. Screening studies of candidate agents can be followed by multi-factorial DoE to determine the optimal concentrations of, e.g., blocking agents.
  • Conjugation techniques for LPS and heparin can be evaluated for use with gold colloid (nanoparticles) and dyed latex particle-based conjugation techniques.
  • an in-line blood separation filter membrane suitable for excluding red blood cells can be adopted for whole blood samples.
  • the goal is to simplify the process and make the testing of the rapid test format user friendly, especially for the off-laboratory applications at point-of-care, such when using directly applied whole blood collected from finger prick or via collection tube.
  • Multiple types of these blood filters are commercially available, such as VividTM or CytosepTM membranes (Pall) or other manufacturers (GE Lifesciences) and can be used in the LFIA.
  • the plastic housing dimensions for the test strip can be selected for use with a reader (e.g., a Detekt reader).
  • DetektTM reader model RDS-150 PRO (Detekt Biomedical, Austin, Tex.) can be used in the LFIA.
  • Detekt's optical lateral flow reader technology for rapid test readers is currently used in commercial diagnostic devices in food and beverage safety testing, bio-threat detection, environmental monitoring and animal health.
  • the reader is a hand-held device that optically scans the test strip and compares the Test and Control line signal strength to a programmed calibration algorithm.
  • the signal integration software can be customized to interpret the IAIP dose-response curve, and this algorithm can be resident on DetektTM units.
  • the Vision Suite Pro developer's kit software program can be used in collaboration with the manufacturer, Detekt Biomedical LLC in Austin, Tex., to integrate the interpretation of the IAIP test strip with the Detekt reader.
  • a software algorithm based on IAIP binding curve shape and internal controls can be developed to perform the following functions: a) to establish the shape of the IAIP binding curve relative to Test Line signal intensity; b) to establish an internal control, possibly based on Control Line signal, to correct for day-to-day variability of test strip runs and c) to establish the software for kit lot-specific information to be incorporated into the reader (e.g. via bar code scanning).
  • the performance of a developed and optimized sandwich-type IAIP rapid test (according to the parameters discussed above and herein) can be evaluated in a pre-clinical setting.
  • the LFIA cassette can be tested as follows:
  • Data can be analyzed to establish precision at all spots of the IAIP binding curve, including precision of the assay cut-off point (set at 150 ⁇ g/mL).
  • Sample pools of known IAIP concentrations can be spiked with potentially interfering substances to determine effects on dose recovery.
  • the Clinical and Laboratory Standards Institute (CLSI) EP-07A protocol can be used for reference.
  • Stock solutions of substances e.g. hemoglobin, bilirubin, human IgGs, etc.
  • substances e.g. hemoglobin, bilirubin, human IgGs, etc.
  • Sample pools of known IAIP concentration can be used to verify the robustness of the test strip to various scenarios/errors produced by end-users. Parameters tested include: 1) incorrect sample volume added (+25%), 2) laboratory environmental conditions (temperature 65-85° F.; humidity 10-70% RH) and 3) incorrect test strip read time (+50-100% recommended).
  • IAIP test kits can be stored under different temperature regimens (e.g., ambient and 37° C.) and tested using a set of IAIP controls of known IAIP concentration at various intervals post-manufacture (>1 year) to determine the stability of the product.
  • temperature regimens e.g., ambient and 37° C.
  • IAIP controls of known IAIP concentration at various intervals post-manufacture (>1 year) to determine the stability of the product.
  • the product can be tested using clinical samples from in subjects (e.g., infants with suspected and confirmed diagnoses of NS and NEC).
  • samples can be collected from subjects (e.g., subjects having or at risk for an inflammatory disease or condition or an infection) undergoing routine clinical evaluation and management. Acquisition of data from multiple centers caring for a broad, heterogeneous population of subjects with the same inflammatory disease or condition or infection can be used to provide an adequate number of study subjects and strengthen the association between IAIP and the disease or infection.
  • the rapid, point-of-care bedside device that can provide IAIP levels in real time can aid in obviating the use of unnecessary interventions or the prolongation of unnecessary treatment (e.g., antibiotic therapy) and can reduce the length/cost of hospitalization.
  • the IAIP LFIA can be used for the clinical evaluation of subjects as follows:
  • Samples can be collected from subjects having or at risk of developing an inflammatory disease or condition at a variety of 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 collected at various time points (e.g., 0, 24, 48, 72 hrs. and 7d) can also be used in the LFIA. The LFIA could be used to determine whether IAIP levels correlate with the severity and progression of the disease, whether the IAIP level predicts risk or subsequent disease, and whether subjects will develop more severe disease or improve following therapy.
  • the collected clinical plasma samples can be transferred into study vials, labeled (deidentified), and stored in a frozen state until testing.
  • IAIP levels are stable for 24 hours at room temperature, for up to 14 days under routine refrigerated clinical storage, and for an unlimited time at 20° C.
  • IAIP levels can be analyzed using the LFIA rapid IAIP test and the sandwich-type IAIP ELISA for a comparison study.
  • test strip cassettes designed to measure IAIP using a competitive LFIA similar to that shown in FIG. 2A The data produced using this immunoassay format, described below, confirm that a LFIA based on a sandwich-type format (similar to that shown in FIG. 3A ) can be used, as well.
  • the competitive LFIA is described below.
  • VLBW very low birth weight
  • NEC necrotizing enterocolitis
  • NEC pathogenesis results when infectious agents translocate across the intestinal epithelial layer, evade innate immune defenses and cause subsequent inflammation and tissue necrosis.
  • Both NS and NEC are associated with systemic inflammatory responses. Their clinical presentation, which is non-specific and subtle at the initial stage, is very similar.
  • NS and NEC often coexist in the same disease episode (for example, NEC with sepsis occurred in one-third of cases of NEC in the case-control studies). Immediate medical management with prompt antimicrobial treatment and supportive care are 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 opportunity.
  • a biomarker for early and accurate identification of an inflammatory disease or infection would be very useful to help physicians to make challenging decisions on initial use and continuation or early termination of antibiotic treatment in subjects (e.g., infants) in whom conventional tests are uninformative.
  • an inflammatory disease or infection e.g., NS and/or NEC
  • PCT procalcitonin
  • IAIP is an excellent severity biomarker and that it can differentiate NEC from a more focal inflammatory disease, Spontaneous Intestinal Perforation (SIP).
  • SIP Spontaneous Intestinal Perforation
  • IAIP can discern disease severity, e.g., a local inflammatory response (SIP) vs systemic and potentially life threatening inflammatory responses (NEC and NS).
  • SIP local inflammatory response
  • NEC Necrotizing enterocolitis
  • SIP Spontaneous intestinal perforation
  • the predictive value of IAIP was superior compared to that of CRP with sensitivity of 100%, specificity of 88.2%, PPV of 41, and NPV of 100 ( FIGS. 16A-16B ).
  • 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 appear to also distinguish NEC from the less life-threatening conditions in SIP patients.
  • the IAIP test demonstrated excellent NPV both in NEC (100%) and NS (98%).
  • the IAIP test can be used to guide treatment (e.g., antibiotic treatment) decisions, such as early termination in infants in whom conventional tests are uninformative.
  • LFIA Quantitative Competitive Lateral Flow Immunoassay
  • a lateral flow immunoassay-based test was chosen for the format of IAIP rapid test (see FIG. 18 ).
  • An LFIA offers many advantages when developing a rapid point-of-care assay as they are designed to: 1) use small sample volumes; 2) follow well characterized kinetics and offer rapid intervals to test results; 3) be analytically sensitive and precise; 4) commonly contain an internal control to verify proper performance of the device; 5) be made from raw materials that are well characterized and widely available; and 6) produce signals that can be quantitatively analyzed using a strip reader.
  • LFIA as an IAIP rapid test, can be used, e.g., at a point-of-care setting.
  • Both reagents are key components used in the competitive assay format.
  • the hybridoma cells were grown in scalable CELLine culture flasks (Integra Bioscience) for large scale production of antibodies in vitro.
  • the IgG was isolated from the hybridoma supernatant by affinity Protein A chromatography.
  • a small volume of plasma (15 ⁇ l of 1:5 diluted samples) was added to strip then 115 ⁇ l chase buffer to push sample through strip.
  • the chase buffer had been optimized: 2 mM Tris pH 8.0+100 mM NaCl+0.5% Brij+0.05% Tween 20+1.0% Fetal Calf Serum.
  • Brij surfactant was added to the buffer to facilitate a rapid clearing of the conjugate from the upstream portions of the strip
  • Strips were run with plasma samples or IAIP calibrators (at three different concentrations) and then quantified by the Qiagen reader at 10, 15, 20, 25, 30, 45 and 60 minutes after sample addition.
  • the strip ran in about 7 min (i.e., nitrocellulose had cleared of extraneous gold) and could be read any time after that. We found that after 15 min the signals did not change dramatically. Thus, we read the strips uniformly at 15 min after the samples were added.
  • IAIP rapid test is intended to be quantitative, we carefully selected a reader to capture the signal generated by the test strips.
  • Various types of test strip readers are commercially available. The choice of reader was based on features and specifications such as engineering, ergonomics, and software robustness. Since this is an important part of the rapid test, we tested three different readers with different technologies and features for IAIP rapid test strips: 1) a portable tabletop lateral flow reader (ESEQuant LFR by Qiagen); 2) a handheld PDA based reader Detekt RDS 1500 Pro (Detekt Biomedical, Austin, Tex.) and 3) Smart Phone/tablet based reader system (iCalq reader—iCalq, Salt Lake City, Utah). We found that the Detekt reader produced a better linear curve ranging from 5 to 700 ⁇ g/mL than the other readers tested.
  • the CV of the IAIP rapid test using ESEQuant reader ranged between 4 to 16% with an average of 13%, while the Detekt reader demonstrated an average CV of 10% (ranging from 5-16%) and the iCalQ smart phone based reader resulted in an average CV of 16% (ranging from 10-23%).
  • the Detekt reader performed better with a relatively lower CV than the other two readers we tested.
  • the signal obtained from each sample was read three times on the various readers and IAIP values were calculated individually based on the established standard curves. Subsequently, the CV was determined from the triplicate readings and was found to range between 2 to 8% with an average CV of 5% for all three readers tested.
  • the “sandwich-type” IAIP ELISA described herein can also be adapted to LFIA format. Rapid quantification of IAIP can lead to early identification of subjects (e.g., human subjects, such as infants, children, adolescents, or adults) having or at risk of developing an inflammatory disease or condition or an infection (e.g., sepsis, NEC, bacterial infection, or another disease or condition) and timely initiation of optimal therapy.
  • subjects e.g., human subjects, such as infants, children, adolescents, or adults
  • an infection e.g., sepsis, NEC, bacterial infection, or another disease or condition
  • Hyaluronic Acid sodium salt (Sigma-Aldrich), was dissolved in dH 2 O (stock concentration of 1 mg/mL) and further diluted into 20 mM NaHCO 3 /Na 2 CO 3 buffer at pH 9.0. Fifty, 100 and 200 ng hyaluronic acid per well was immobilized on a 96-well microplate (Greiner BioOne, Microlon 600) at 37° C. for 120 minutes. Following blocking with 5% Non-fat Milk Powder in TBS-T (20 mM Tris-buffered saline solution at pH 7.3+0.05% Tween-20 (v/v)) at 37° C.
  • TBS-T 20 mM Tris-buffered saline solution at pH 7.3+0.05% Tween-20 (v/v)
  • IAIP human plasma and highly purified IAIP in TBS
  • Human plasma was prepared by cryo-precipitation of fresh frozen plasma obtained from a local blood bank. The cryo-supernatant was used in this experiment and had an IAIP concentration of 250 ⁇ g/mL.
  • the purified IAIP was at a starting concentration of 1 mg/mL.
  • microplate was then washed 3 times with TBS-T, and biotin-conjugated MAb 69.26 (monoclonal antibody against human IAIP) at 1:1000 dilution in TBS was added and incubated at 37° C. for 30 minutes.
  • MAb 69.26 monoclonal antibody against human IAIP
  • HRP horseradish peroxidase
  • Inova Bioscience horseradish peroxidase-conjugated Streptavidin diluted to 1:5000 in TBS was added and incubated at 37° C. for 30 minutes.
  • 50 ⁇ L Enhanced K-Blue TMB Substrate (Neogen) was added to each well and the reaction was stopped by adding 50 ⁇ L 1M hydrochloric acid (HCl).
  • FIGS. 21A-21B The color change was read using a spectrophotometer (Molecular Devices SpectraMax Plus microplate reader) at 650 nm wavelength, and the standard curve for human plasma or purified IAIP was plotted at 50, 100 and 200 ng/well immobilized hyaluronic acid ( FIGS. 21A-21B ).
  • a spectrophotometer Molecular Devices SpectraMax Plus microplate reader
  • FIGS. 21A-21B show that IAIP can be quantitatively measured in biological mixtures by capturing IAIP complex molecules using hyaluronic acid and detecting the captured IAIP using a monoclonal antibody specific for the light chain of IAIP (e.g., MAb 69.26).
  • a standard curve can be optimized and established using purified IAIP or human plasma with a known amount of IAIP.
  • this assay can be used to quantify an unknown amount of IAIP in a biological sample from a subject.
  • An alternative approach to this method can be employed, in which an IAIP-specific monoclonal antibody is used to capture IAIP and biotin-labeled hyaluronic acid is used as a ligand to detect the bound IAIP, similar to the approaches used in Examples 1 and 2.

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