Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-02-28
2003-12-09
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S350000, C530S387100, C530S388100, C436S500000, C436S501000
Reexamination Certificate
active
06660482
ABSTRACT:
TECHNICAL FIELD
This invention relates to bacterial infections.
BACKGROUND
Sepsis is a systemic response to infection, e.g., a bacterial infection. It is commonly caused by endotoxins from Gram negative bacteria or exotoxins from Gram positive bacteria (which can trigger endotoxin-like responses). The systemic response can lead to septic shock, which is is characterized by a precipitous drop in blood pressure, cardiovascular collapse, and/or multiple organ failure. The mortality rate among patients diagnosed with septic shock can be as high as 35-45%. Rapidly and reliably detecting sepsis has been difficult using conventional diagnostic tools
SUMMARY
The invention provides a method of diagnosing sepsis in a mammal. The methods are also useful as a prognostic tool in predicting the outcome of those diagnosed with sepsis and those undergoing therapy for sepsis.
A method for diagnosis or prognosis sepsis in a mammal is carried out by contacting a bodily fluid, e.g., blood, plasma, or serum, from the mammal with a ligand which binds to an inter-alpha trypsin inhibitor (ITI) polypeptide under conditions sufficient to form an ITI-ligand complex and detecting the complex. The mammal is preferably a human patient; however, the assays are also applicable to veterinary use, e.g., to diagnose or prognose sepsis in animals such as dogs, cats, horses, cows and the like. Preferably, the ligand binds to an epitope of human ITI light chain. ITI is used as a marker for diagnosis or prognosis of sepsis in infants such as neonates as well as adult mammals.
A decrease in the level of ITI in a test sample compared to a normal level of ITI indicates a diagnosis of sepsis. A level of ITI that is at least 10%, more preferably at least 25%, more preferably at least 35% and most preferably at least 50% lower than the a normal level of ITI indicates a diagnosis of sepsis. For example, a normal level of ITI is approximately 1.2 mg/ml of ITI in plasma, and a level of 0.6 mg/ml ITI (or lower) is diagnostic of sepsis. For prognostic purposes, a level of ITI that is at least 10%, more preferably at least 25%, more preferably at least 35% and most preferably at least 50% lower than a diagnostic level of ITI indicates a high risk of mortality (i.e., a poor prognosis). For example, a diagnostic level of ITI is 0.6 mg/ml in plasma, and a level of 0.3 mg/ml ITI (or lower) is predictive of a poor outcome, e.g., septic shock and/or death.
The ITI-ligand used in the method is one that forms a detectable complex with ITI in a bodily fluid. For example, the ligand is an antibody or ITI-binding antibody fragment, e.g., a Fab or (Fab)
2
fragment; an engineered single chain Fv molecule; or a chimeric molecule such as an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin. Preferably, the antibody is a monoclonal antibody (mAb) such as mAb 69.31. Alternatively, the ligand is a synthetic or proteolytically-generated peptide that binds to an ITI light chain. For detection purposes, the ligand, e.g., ITI-specific antibody, is directly or indirectly labelled using, e.g., a colorimetric or radioisotopic marker. The amount of an immune complex (which contains ITI antigen bound to ITI-specific antibody) is quantitated to determine the level of ITI in the fluid, and the level of ITI in the fluid is compared to a normal control level of ITI (e.g., a previously determined baseline value or the level of ITI from a subject known to be healthy). The claimed assays accurately and reliably diagnose sepsis rapidly (in less time than results are obtained from a conventional bacteremia or sepsis workup).
Prognosis of sepsis is also determined by measuring ITI over time. For example, the method includes the steps of (a) contacting a bodily fluid from a mammal with a ligand which binds to an ITI polypeptide under conditions sufficient to form an ITI-ligand complex and detecting the complex; (b) quantitating the amount of complex to determine the level of ITI in the fluid; and (c) comparing the level of ITI in the fluid over time, wherein a decrease in the concentration of ITI in test samples taken over time indicates an adverse prognosis. Changes in the severity of the sepsis are monitored by comparing changes in the level of ITI in bodily fluids of the patient over time. Such temporal data is used to determine a course of treatment for the patient.
Reagents, e.g., an ITI-specific antibody such as mAb 69.31, for carrying out the diagnostic or prognostic assay may be packaged together as a kit. A kit for diagnosis or prognosis of sepsis in a mammal contains a ligand, e.g., an antibody or antibody fragment, which binds to an ITI light chain and a means of detecting a complex containing the ligand bound to an ITI light chain. The means of detection is preferably a colorimetric marker or radioisotopic marker. For example, the antibody is immobilized on a solid phase and packaged together with other reagents suitable for detecting ITI-ligand complexes. Enzyme-conjugated reagents may be included in the kit. In some embodiments, the antibody or ITI ligand is immobilized on a solid phase such as an assay plate, an assay well, a nitrocellulose membrane, a bead, a dipstick, or a component of an elution column. The immobilized antibody acts as a capture antibody, and a secondary antibody is used to detect the immune complex (e.g., an ITI antigen bound to the mAb 69.31 antibody). The kit may optionally contain a purified ITI polypeptide or purified ITI complex as a control. The polypeptide or complex is purified from natural sources or recombinantly produced. The kit may also contain a second antibody or other detectable marker as described above. For example, the second antibody or marker is labelled, e.g., using a radioisotope, fluorochrome, or other means of detection.
Methods of treating sepsis or reducing the risk of mortality associated with sepsis are also within the invention. A method of treating sepsis in a mammal includes the steps of identifying a mammal suffering from or at risk of developing sepsis and administering to the mammal an ITI composition. The ITI compositions contain an ITI light chain polypeptide or a complex of an ITI light chain polypeptide and an ITI heavy chain polypeptide.
Alternatively, an ITI composition contains a nucleic acid encoding an ITI polypeptide, e.g., a a synthetic oligonucleotide encoding an ITI polypeptide. The ITI composition is preferably administered intravenously.
Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
REFERENCES:
patent: 5777081 (1998-07-01), Michalski et al.
patent: 5804370 (1998-09-01), Romaschin et al.
patent: WO 92/18160 (1992-10-01), None
Piette et al, “Urinary trypsin inhibitory activity for the diagnosis of bacterial infection: a prospective study in 690 patients”, Eur. J. Medicine 1(5):273-276, Sep. 1992.*
Byrajalsen et al, “Human inter alpha trypsin inhibitor and immunologically related inhibitors investigated by quantitative immunoelectrophoresis”, Clinica Chimica Acta 161:59-68, 1986.*
Trefz et al, “Establishment of an enzyme linked immuno sorbent assay for urinary trypsin inhibitor by using a monoclonal antibody”, J. Immunoassay 12(3):347-369, 1991.*
Mizon et al, “Development of an enzyme linked immunosorbent assay for human plasma inter alpha trypsin inhibitor (ITI) using specific antibodies against each of the H1 and H2 heavy chains”, J. Immunol. Methods 190:61-70 (1996).*
Daveau et al. (1993) “Human Inter-alpha-inhibitor Family in Inflammation: Simultaneous Synthesis of Positive and Negative Acute-Phase Proteins”,Biochem J, vol. 292, pp. 485-492.
Michalski et al. (1994) “Preparation and Properties of a Therapeutic Inter-alpha-trypsin Inhibitor Concentrate from Human Plasma”,Vox Sang,pp. vol. 67, No. 4, pp. 329-336.
Albani, et al. (1997). “Inter-&agr;-Inhibitor as Marker for Neutrophil Proteinase Activity: An In Vitro Investigation”Journal of Laboratory and Clinical Medicine 130(3):339-347.
Hixson Douglas C.
Lim Yow-Pin
Beattie Ingrid A.
Fredman Jeffrey
Mintz Levin Cohn Ferris Glovsky & Popeo P.C.
Rhode Island Hospital
LandOfFree
Inter-alpha-trypsin inhibitor as a marker for sepsis does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Inter-alpha-trypsin inhibitor as a marker for sepsis, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Inter-alpha-trypsin inhibitor as a marker for sepsis will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3143057