WO2001004630A1 - Measurement of analytes in whole blood - Google Patents
Measurement of analytes in whole blood Download PDFInfo
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- WO2001004630A1 WO2001004630A1 PCT/IB1999/001923 IB9901923W WO0104630A1 WO 2001004630 A1 WO2001004630 A1 WO 2001004630A1 IB 9901923 W IB9901923 W IB 9901923W WO 0104630 A1 WO0104630 A1 WO 0104630A1
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5768—Hepatitis A
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/811—Test for named disease, body condition or organ function
Definitions
- This invention relates generally to methods to measurement of the level of an analyte in a sample of blood.
- Analytes may include infectious microorganisms, their toxic products, inflammatory mediators, hormones, acute phase proteins, toxins, drugs of abuse, markers of cardiac muscle damage, therapeutic drugs, cytokines, chemokines, and others.
- Analyte is defined as the specific substance of interest present in a bodily fluid sample and being analyzed by the methods of the present invention.
- these may include, for example, microorganisms and their components, including gram positive cell wall constituents and gram negative endotoxin, lipopolysaccharide, lipoteichoic acid, and the inflammatory mediators that appear in circulation as a result of the presence of these components, including tumor necrosis factor (TNF), interleukin-1 (IL-1) and other interleukins and cytokines.
- TNF tumor necrosis factor
- IL-1 interleukin-1
- Other analytes may include drugs of abuse, hormones, toxins, therapeutic drugs, markers of cardiac muscle damage, etc.
- Sepsis is defined as a pathological condition of the body resulting from the presence of infectious microorganisms, which clinically manifests as one or more of the following sequelae: pyrexia, hypotension, hypoxemia, tachycardia, hypothermia, neutrophilia, and neutropenia.
- Immunocomplexes is a synonym for antibody-antigen complexes.
- “Opsonized” refers to a particle to which immunoglobulin and complement factors are bound and which results in a more vigorous recognition of the particle by the immune system.
- the yeast polysaccharide zymosan, or latex particles may be opsonized by binding of immunoglobulin and complement factors to their surfaces; opsonized zymosan or latex will stimulate increased oxidant production by white cells after they are activated by exposure to immunocomplexes.
- Responsiveness is a measure of the patient's ability to respond to a maximum stimulatory dose of immunocomplex.
- Rapid quantitation of specific analytes in an individual's blood is critically important for the diagnosis of disease and its severity, often under emergency conditions, in the monitoring of the progression of pathological conditions and following the recovery process brought about by surgical and drug therapies. It is often important to know not only whether a specific analyte is present, but as well its level, in order to determine the present stage of a particular condition or disease in order to prescribe the most effective remedy at that particular stage. In the treatment of many diseases, a particular therapy may be ineffective or toxic if given at the wrong stage of the condition. For example, the levels of specific markers of cardiac muscle damage and the relationship among them may indicate that a patient has had or may be having a heart attack.
- the level of a therapeutic drug in the circulation may indicate whether the patient is being dosed optimally, and whether presumptive side effects are possibly due to excess levels of the drug.
- the circulating levels of infectious microorganism-derived toxins and inflammatory mediators produced by the patient's white blood cells in response to these toxins may indicate the severity and level or stage of sepsis and help identify the most efficacious course of therapy. Quantitation of analytes under emergency conditions and using the information to prescribe a particular therapy may mean the difference between saving a patient's life and contributing to the patient's death.
- nosocomial infections in the case of infection, hospital and particularly intensive care unit patients who have acquired nosocomial infections as a result of peri- or post-operative immunosuppression or infections secondary to other disease processes, such as pancreatitis, hypotensive or hypovolemic shock, physical trauma, burn injury, or organ transplantation, and subsequently develop septic shock syndrome, have a mortality which has been quoted to range from 30-70% depending upon other co-incident complications.
- pancreatitis peri- or post-operative immunosuppression or infections secondary to other disease processes
- hypotensive or hypovolemic shock physical trauma, burn injury, or organ transplantation
- septic shock syndrome a mortality which has been quoted to range from 30-70% depending upon other co-incident complications.
- the incidence of nosocomial infections and, in particular, infections leading to sepsis or septicemia is increasing.
- the presence of bacteria, viruses or fungi or their cell wall components including gram- positive peptidoglycans, lipoteichoic and teichoic acids, and gram-negative endotoxin (lipopolysaccharide, LPS) in blood is indicative of an infection.
- the immune system's reaction to the presence of these foreign antigens by the production of pro-inflammatory cytokine mediators such as interleukin-1 (IL-1), tumor necrosis factor (TNF) and interleukin-6 (IL-6), is also indicative of an infection.
- the quantity of these analytes in circulation may be used to indicate the severity and level or stage of sepsis.
- LPS may be present at a concentration as low as 50 pg/ml of whole blood.
- TNF a mediator of sepsis
- TNF can be detected and measured using antibody against TNF.
- TNF may be present in smaller amounts since it is transitory and another transitory mediator, IL-1, may appear.
- IL-1 may increase and interleukin-6 (IL-6) may appear.
- IL-6 interleukin-6
- the endotoxin assay kit from BioWhittaker (Walkerville, MD., U.S.A.) or Seikagaku Kogyo Ltd. (Tokyo, Japan) is a Limulus Amebocyte Lysate (LAL) Assay technique which may be used as a comparison for the present invention.
- LAL Limulus Amebocyte Lysate
- a major clinical and diagnostic challenge is to identify and stage patients, ideally early in the progression of the septic response, or to identity those patients at high risk of developing fulminant sepsis syndrome.
- the same therapeutic agents given at the one stage in the septic process may have more significant beneficial effects than when given at another, since it is clear that an optimal window period may exist for the efficacy of any particular therapeutic agent.
- giving a patient antibodies or receptors directed against gram-negative endotoxins when the patient has no detectable levels of these agents present in the circulation and already has a maximally activated cytokine cascade is a waste of resources and of no benefit to the therapy of the patient.
- the potential market for these anti-sepsis strategies remains large (about 250,000 cases per year in the USA) and has been limited by the inability to identify and stage patients who could benefit from the appropriate pharmacologic interventions.
- the present invention is directed to a method for measuring the amount of a preselected analyte in a sample of a bodily fluid comprising the steps of (a), forming an immunocomplex between the analyte and an antibody thereto; (b) reacting the immunocomplex with an oxidant-producing phagocytic cell in the presence of an activator; and (c) measuring the amount of oxidant produced as compared with that produced by a maximal amount of immunocomplexes between the analyte and the antibody, in the presence of the activator, as an indicator of the amount of the analyte in the sample.
- the bodily fluid may be whole blood.
- the oxidant-producing phagocytic cells are neutrophils, lymphocytes, monocytes, or combinations.
- the activator may be, by way of non-limiting example, zymosan, latex particles, opsonized zymosan, opsonized latex particles, phorbol esters, N-formyl-met-leu-phe, or combinations.
- the amount of oxidant produced by the oxidant producing cells is achieved using a chemiluminescent compound which reacts with the oxidants to generate light. Non-limiting examples include luminol, lucigenin and pholasin.
- the antibody is monoclonal antibody of class IgM or IgG.
- the analyte is any substance or component present in a bodily fluid sample which may participate in the formation of an antigen-antibody complex (immunocomplex) with added, exogenous antibody.
- analytes may include gram-positive bacteria, gram-negative bacteria, fungi, viruses, gram-positive cell wall constituents, lipoteichoic acid, peptidoglycan, teichoic acid, gram-negative endotoxin, lipid A, hepatitis A, inflammatory mediators, drugs of abuse, therapeutic drugs, or cardiac markers, such as myoglobin, creatine kinase MB, troponin I or troponin T.
- Inflammatory mediators include but are not limited to tumor necrosis factor, interleukin-1, interleukin-6, interleukin-8, interferon, and transforming growth factor ⁇ .
- the analyte may be one indicative of infection or indicative of sepsis.
- the analyte is lipopolysaccharide
- the antibody is anti- lipopolysaccharide antibody
- the sample is a whole blood sample
- the activator is zymosan.
- the present invention is directed to a method for measuring the level of a preselected analyte present in a sample of a bodily fluid comprising the following steps i) providing three aliquots of the sample, designated aliquots A, B, and C; ii) providing a source of oxidant-producing phagocytic cells and a source of complement proteins; iii) providing aliquot B with an amount of anti-analyte antibody sufficient to form an immunocomplex with the analyte in the sample, to provide reaction aliquot B; iv) providing aliquot A as a control to reaction aliquot B without added anti- analyte antibody, to provide reaction aliquot A; v) providing aliquot C with a equivalent amount of the anti-analyte antibody as in reaction aliquot B, and in addition containing a maximal stimulatory amount of analyte, to provide reaction aliquot C; vi) incubating reaction aliquots A,
- the aforementioned steps may be carried out following manual, semi-automated, or automated procedures.
- the test may provide results in a short period of time, such that the measurement of the analyte can be performed to aid in the rapid diagnosis of a patient's condition.
- Instrumentation may be provided that can be performed in the emergency room, at the bedside, or for home use.
- a test may be performed in around 20 minutes or less.
- the bodily fluid in the aforementioned method may be whole blood., in which oxidant-producing phagocytic cells are present.
- a source of oxidant-producmg phagocytic cells may be added, such as white blood cell fractions containing neutrophils, lymphocytes, monocytes, or combinations.
- An agent capable of increasing oxidant production by white blood cells on exposure to immunocomplexes may be included in reaction aliquots A, B, and C; this agent may be by way of non-limiting example, zymosan, latex particles, phorbol ester, N-formyl-met- leu-phe, opsonized zymosan, opsonized latex particles, or combinations thereof.
- the chemiluminescent compound may be, for example, luminol, lucigenin or pholasin.
- the anti-analyte antibody may be a monoclonal antibody of class IgM or IgG.
- the analyte is any substance or component present in a bodily fluid sample which may participate in the formation of an antigen-antibody complex (immunocomplex) with added, exogenous antibody.
- analytes may include gram-positive bacteria, gram-negative bacteria, fungi, viruses, gram-positive cell wall constituents, lipoteichoic acid, peptidoglycan, teichoic acid, gram-negative endotoxin, lipid A, hepatitis A, inflammatory mediators, drugs of abuse, therapeutic drugs, and cardiac markers, as described above.
- Inflammatory mediators include but are not limited to tumor necrosis factor, interleukin-1, interleukin-6, interleukin-8, interferon, and transforming growth factor ⁇ .
- the analyte may be one indicative of infection or indicative of sepsis.
- the analyte is lipopolysaccharide
- the antibody is anti- lipopolysaccharide antibody
- the sample is a whole blood sample
- the activator is zymosan.
- a diagnostic kit for measuring the level of a preselected analyte present within a sample of a bodily fluid comprising: a first container of IgM or IgG antibody specific to the preselected analyte; a second container of chemiluminescent compound; and a third container of analyte.
- a source of oxidant-producing phagocytic cells may be included in the kit for samples which do not contain them; the cells may be neutrophils, lymphocytes, monocytes, or combinations thereof.
- the diagnostic kit may also include additional container containing an agent capable of increasing oxidant production by white blood cells on exposure to immunocomplexes, for example, zymosan, latex particles, phorbol ester, N-formyl-met- leu-phe, opsonized zymosan, opsonized latex particles, or combinations.
- the chemiluminescent compound may be luminol, lucigenin or pholasin.
- the invention is directed to a method for determining the stage of sepsis of a patient from a sample of whole blood comprising the concurrent measurement of: (a) the level of microbial products or inflammatory mediators; (b) the maximum oxidant production by the patient's neutrophils; and (c) the level of responsiveness of the patient's neutrophils to a maximum stimulatory level of immunocomplexes.
- aliquots A, B, and C are measured by the steps of i) providing three aliquots of said sample, designated aliquots A, B, and C; ii) providing aliquot B with an amount of anti-analyte antibody sufficient to form an immunocomplex with the analyte in the sample, to provide reaction aliquot B; iii) providing aliquot A as a control to reaction aliquot B without any added anti-analyte antibody, to provide reaction aliquot A; iv) providing aliquot C with a equivalent amount of anti-analyte antibody as in reaction aliquot B, and in addition containing a maximal stimulatory amount of analyte, to provide reaction aliquot C; v) incubating reaction aliquots A, B, and C with said oxidant-producing phagocytic cells and said source of complement proteins under suitable conditions and for a time sufficient for any immunocomplexes formed in the reaction aliquots to react with oxidant-producing
- C as an indicator of the level of microbial products or inflammatory mediators; the maximum oxidant production by the patient's neutrophils; and the level of responsiveness of the patient's neutrophils to a maximum stimulatory level of immunocomplexes; and ix) determining the patient's stage of sepsis from the aforementioned levels.
- FIGURE 1 depicts a typical, whole-blood chemilummescence profile of a sample from a patient with endotoxemia.
- Curve A represents whole blood plus zymosan;
- B whole blood plus zymosan plus anti-endotoxin antibody;
- C whole blood plus zymosan plus anti-endotoxin antibody plus exogenous endotoxin (800 pg/ml).
- the present invention relates to a method for measuring the level of a preselected analyte in a sample of a bodily fluid, such as whole blood of a mammal, including humans, by incubating the test sample with an antibody specific to the analyte to form an immunocomplex, which then interacts with phagocytic cells present in or added to the sample and result in the production of oxidants.
- Oxidants may be detected using chemiluminescent reagents added to the sample.
- the phagocytic cell oxidant response may be optionally enhanced by the inclusion of selected activator agents such as zymosan.
- the level of chemilummescence elicited from oxidant-producing phagocytic cells by a particular level of immunocomplexes is related to the maximal chemiluminescence elicited from phagocytic cells by a maximal amount of immunocomplexes.
- the present invention utilizes a separate measure of the maximal responsiveness of the phagocytic cells in the sample to immunocomplexes, and the ratio of oxidant production by immunocomplexes formed from the analyte to oxidant production by a maximal amount of analyte-antibody immunocomplexes provides a relative measure of the amount of analyte in the sample.
- This method may be used to determine levels of an analyte in a blood sample, such as endotoxin and other analytes related to sepsis, in order to assess severity and level or, in combination with other parameters derivable from the assay herein, the stage of sepsis of the patient, and to direct and monitor the proper therapeutic course.
- the assay may also be used to measure the level of other preselected analytes present in a blood sample, such as hormones, acute phase proteins, toxins, drugs of abuse, markers of cardiac muscle damage, therapeutic drugs, cytokines, chemokines, etc. Any analyte in the sample for which an antibody can be added that forms an immunocomplex capable of stimulating oxidant production by phagocytic cells can be measured in the assay of the present invention.
- the present invention utilizes a three-aliquot assay format for the measurement of the preselected analyte. All three aliquots include an optional activator or enhancer of oxidant production, such as zymosan. Two aliquots include antibodies to the analyte, of which one also contains added analyte to provide the maximal amount of immunocomplexes. Sample is added to all three tubes.
- Non-limiting examples of details as to preincubation and equilibration times, incubation times with the various components of the assays, preincubations and reaction times are provided below, and, with the benefit of this disclosure, can be readily determined by the skilled artisan to maximize the sensitivity and rapidity of the assay. Details as to the order in which the aliquots are prepared, samples are incubated and subdivided, are provided for illustration purposes only and can be modified.
- the aforementioned assay may be carried out following manual, semi-automated, or automated procedures.
- the test may provide results in a short period of time, such that the measurement of the analyte can be performed to aid in the rapid diagnosis of a patient's condition.
- Instrumentation may be provided that can be performed in the emergency room, at the bedside, or for home use, among others.
- a test may be performed in around 20 minutes or less.
- the assay is carried out as follows.
- the assay readout for a particular analyte is provided in units of activity.
- the assay readout is endotoxin activitry, or EA.
- the assay may be modified for automation, or semi- automation, and performed in any order or sequence which provides equivalent data. For example, two aliquots of whole blood are dispensed into suitable tubes that are free of any contaminants or other materials that may adversely influence the outcome of the test. The tubes are placed in a incubator or other equivalent device in which the temperature of all three tubes may be maintained similarly, to keep the conditions the same among the tubes.
- a thermostatted aluminum block pre-heated to about 37 °C may be used.
- One tube contains an amount of the analyte to maximally stimulate oxidant production when combined, in a subsequent step, with antibody to the analyte.
- the other tube contains no additives.
- These tubes are incubated for about 10 min. at 37°C. During the last 5 minutes of this incubation assay three tubes per assay tubes are loaded into the heating block.
- One tube, herein referred to as Tube A contains a control reagent used for antibody stabilization or no reagent at all, Tubes B and C contain the same amount of antibody to the analyte.
- each tube a mixture of buffer containing the chemuluminescent compound, optionally with the stimulant, such as non-opsonized zymosan, is added. This mixture is temperature equilibrated for at least about 5 min. After the blood has incubated for a total of about 10 min. at about 37 °C, an equal volume, for example 20 ⁇ l, is transferred into assay tubes A and B from the blood tube with no LPS and the same volume, in this example, 20 ⁇ l, is transferred from the blood tube containing LPS into assay tube C. All tubes are mixed well and placed in the chemiluminometer for reading. The luminometer is thermostatted at about 37°C and the assay is read for a total of about 20 min. Individual tube light integrals are calculated and the analyte activity, for example, EA is determined by the calculation:
- the various components and conditions described above may be tailored to the specific analyte being measured.
- the amount of components, the lengths of preincubation and incubation, the period of time over which the luminescence is read, the temperatures and other parameters of the assay may be adjusted by the skilled artisan within the range of operability of the particular assay. Numerous variations in the examples provided are embraced herein.
- the various components of the assay are as follows.
- analyte is any substance or component present in a bodily fluid sample which may participate in the formation of an antigen-antibody complex (immunocomplex) with added, exogenous antibody.
- analytes may include gram-positive bacteria, gram-negative bacteria, fungi, viruses, gram-positive cell wall constituents, lipoteichoic acid, peptidoglycan, teichoic acid, gram-negative endotoxin, lipid A, hepatitis A, inflammatory mediators, drugs of abuse, therapeutic drugs, or cardiac markers, such as myoglobin, creatine kinase MB, troponin I or troponin T.
- Inflammatory mediators include but are not limited to tumor necrosis factor, interleukin-1, interleukin- 6, interleukin-8, interferon, and transforming growth factor ⁇ .
- the analyte may be one indicative of infection or indicative of sepsis.
- the analyte is lipopolysaccharide
- the antibody is anti- lipopolysaccharide antibody
- the sample is a whole blood sample
- the activator is zymosan.
- Anti-analyte antibody The antibody against the preselected analyte of the present method is preferably of the IgM class. IgM-analyte immunocomplexes trigger a reaction sequence which results in the stimulation of white blood cell oxidant production via complement pathway activation.
- the antibody against the analyte can also be of the IgG class.
- Xomen-E5 a commercially-available murine monoclonal IgM pentamer directed against a lipid A component of gram-negative endotoxin, produced by Xoma, Palo Alto, CA, is suitable.
- the antibody may be provided in a stabilized liquid or solid form, for example, lyophilized in bead form with the stabilizing agent trehalose.
- a stabilized liquid or solid form for example, lyophilized in bead form with the stabilizing agent trehalose.
- a non-limiting example of such a bead form is described in copending application Serial No. (Attorney's Docket Number 1112-1-999), incorporated herein by reference.
- Optional white cell stimulant Although stimulants such as zymosan or latex beads are not required additions to the test procedure, the chemiluminescence produced by immunocomplexes in the test sample is enhanced by such inclusion. Zymosan and latex beads enhance the chemiluminescent response by stimulating concerted white cell oxidant production and phagocytosis. This stimulation may be further enhanced if the zymosan or latex beads are opsonized, through the binding of immunoglobulin G and complement factors (iC3b and C3b). The addition of zymosan or latex acts as an amplification process to increase oxidant production and is preferred in the practice of the present invention, but is not obligatory for the recognition of immunocomplexes by white blood cells.
- stimulants such as zymosan or latex beads are not required additions to the test procedure, the chemiluminescence produced by immunocomplexes in the test sample is enhanced by such inclusion.
- Zymosan and latex beads enhance the chemiluminescent
- latex beads There are many kinds of latex beads depending upon the polymer from which they are prepared which can be utilized in the practice of this invention. These include, for example, polystyrene, styrene divinylbenzene, and acrylic acid polymers; polystyrene is preferred.
- phorbol esters such as phorbol 12- myristate 13-acetate
- peptides such as N-formyl-methionine-leucine-phenylalanine, abbreviated fMLP or N-formyl-met-leu-phe.
- Chemiluminescent indicator The phenomenon of chemiluminescence resulting from the production of neutrophil oxidants is described by Allen, R.C. Methods in Enzymology 133:449 (1986) using the acyl azide dye luminol as a light emitting agent. This technique permits the sensitive measurement of neutrophil respiratory burst activation using small numbers of polymorphonuclear leukocytes or later, even white cells in whole blood. Other chemiluminescent dyes which produce light as a result of neutrophil oxidant production have also been identified including lucigenin and pholasin; others will be known by the skilled artisan.
- Oxidant-producing phagocytic cells If whole blood samples are used, or other bodily fluid samples containing white blood cells, oxidant-producing phagocytic cells are already present in the sample. Whole blood is the preferred bodily fluid. For samples without such cells or too little to provide a means for producing oxidants proportional to the amount of immunocomplexes, phagocytic cells can be added to the sample. Appropriate cells include but are not limited to neutrophils, lymphocytes, and monocytes. These cells may be derived from another sample, cell culture, artificially prepared cells, and other source.
- Immunocomplexes to maximally stimulate white blood cells Inclusion in the assay described herein of a measure of the maximal response to a maximal stimulatory level of immunocomplexes, by the white blood cells present in or added to the sample, enables the present invention to provide a measure of the amount of analyte in the sample. This measure may be achieved with any antigen and corresponding anti-antigen antibody that achieves the desired stimulation, such as endotoxin and anti-endotoxin antibody. Antibodies of the IgM class are preferred. For simplification of the assay, the same analyte as the preselected analyte is preferably used.
- all three tubes contain zymosan; two tubes contain the same amount of anti-LPS antibodies, of which one tube also contains an amount of LPS which forms a maximal amount of immunocomplexes with the anti-LPS.
- the sample whole blood containing an unknown amount of LPS, is added in equal amounts to all three tubes. All tubes contain a reagent which produces light in response to oxidants. White blood cells in the sample provide the oxidants in response to immunocomplexes.
- the first tube containing sample and zymosan only, produces a background level of chemiluminescence.
- the second tube containing zymosan, sample LPS, and anti-LPS antibodies, produces a level of chemiluminescence proportional to the amount of analyte, LPS, in the sample.
- the third tube containing zymosan, sample LPS, added maximal LPS, and anti-LPS antibodies, produces a maximal amount of chemiluminescence.
- the ratio of the integrated chemiluminescence of the second tube, minus that of the control tube, to the integrated chemiluminescence of the third tube, minus that of the same control provides the readout of the relative amount of LPS in the sample.
- suitable white blood cells or equivalent cells from another source, such as a whole blood sample, cultured white blood cells, or other white cells, artificially prepared cells, and other source.
- Assay procedure The following procedure is a non-limiting example of a protocol that may be followed to provide a rapid and sensitive assay for endotoxin (LPS).
- the assay readout is endotoxin activity, or EA.
- the assay may be modified for automation, or semi-automation, and performed in any order or sequence which provides equivalent data.
- two aliquots of blood 500 ⁇ l are dispensed into depyrogenated glass tubes into a thermostatted aluminum block pre-heated to 37°C.
- One tube contains LPS as a maximum calibrator the other tube contains no additives. These tubes are incubated for 10 min. at 37 °C.
- Tube A contains a control reagent used for antibody stabilization or no reagent at all
- Tubes B and C contain antibody.
- Luminol Buffer with unopsonized zymosan is added (500 ⁇ l per tube). This mixture is temperature equilibrated for at least 5 min.
- 20 ⁇ l is transferred into assay tubes A and B from the blood tube with no LPS and 20 ⁇ l is transferred from the blood tube containing LPS into assay tube C.
- EA Light Integral Tube C - Light Integral Tube A.
- the clinical cutoff value may be chosen on the basis of epidemiological studies on the particular analyte of interest, and the sensitivity and specificity of the particular assay compenents, to provide normal and abnormal ranges, cutoff values, as well as the positive and negative predictive values of the assay. Under a particular set of operating conditions provided herein by way of non-limiting example, a value of > 35 EA, would indicate clinically significant endotoxemia.
- analytes may be determined utilizing the methods of the present invention.
- the levels of such other analytes may also be expressed in relative units, expressed as a ratio to maximal chemiluminescence, as corrected by background.
- cardiac markers to identify the cause of chest pain and distinguish unstable angina from a heart attack using combinations of markers including myoglobin, creatine kinase MB, troponin I or troponin T, and other cardiac specific and skeletal muscle specific markers for ruing in or ruling out heart attack, are embraced within the present invention.
- chemiluminescence of the three tubes may be determined serially, in an instrument that reads a single channel, by the appropriate timing and addition of reagents to the appropriate tubes, with the appropriate preincubation times.
- a single whole blood sample may be used successively for determining the background chemiluminescence, that of the sample analyte, and lastly, the maximal stimulatory dose.
- a multichannel luminometer may subtract the background values during measurement.
- the assays also may be adapted to a thin film format, rather than liquid in tubes, to facilitate the automation and simplification of assay.
- a test strip-like device may be prepared with three separate fluid paths, to conduct a whole blood sample, such as that obtained by fmger prick, to three separate regions, each of which has the appropriate reagents corresponding to tubes A, B, and C as described above.
- the strip may be inserted into a luminometer device, such as a handheld device.
- the reagents used to detect the level of the phagocyte oxidant burst yields a chromogen, visible or fluorescent, the density of which is proportional to the oxidant level and, proportionately, the immunocomplex level. Reflectometry may be used to quantitate the color and calculate the analyte level.
- the quantity of infection or sepsis related analytes in circulation may be used to indicate the severity and level or stage of sepsis.
- the present assay may be used to add additional parameters to the diagnosis of stage of sepsis.
- the maximum oxidant production of neutrophils is a measure of the ability of the white blood cell to respond to programmed opsonic challenge.
- the responsiveness of the patient's neutrophils to immunocomplexes termed responsiveness, is a measure of the maximal ability of the white blood cell to bind and respond to opsonized immunocomplexes. A large responsiveness is indicative of a large response reserve for processing opsonized immunocomplexes and is viewed as the normal healthy state.
- a small responsiveness represents a diminished reserve for processing opsonized immunocomplexes and is indicative of an immunocompromised or diseased state. Lack of a robust response in an indication of anergy, the inability of the immune system to mount an effective immune response. In the final stage of anergy in sepsis, during the terminal stage of the condition, the patient's immune system is progressively weakening in its ability to combat microbial infection. Patients progressing to this latter stage suffer a very poor prognosis.
- reaction aliquots contained zymosan in order to optimize oxidant production of the patient's white blood cells in response to immunocomplexes.
- tube B contained antibody against the analyte to be measured, in this case endotoxin. Tube A served as a control to tube B.
- tube C contained a maximal stimulatory amount of immunocomplexes, derived from the same amount of anti-endotoxin antibody as in tube B, with the addition of LPS from K coli 055 :B5 (determined to be 800 pg/ml or 0.67 EU/ml at an antibody concentration of 0.4 ⁇ g/assay). While in this example the antigen used to form immunocomplexes to determine maximal response (endotoxin-anti-endotoxin) was identical to the analyte, this does not need to be the true for all analytes, although it is most convenient to do so. The following materials were used and methods followed in carrying out the assay. Variations in the components described here as well as the procedures may be modified by standard procedures without deviating from the invention.
- All glass surfaces used for endotoxin assay or storage of reagents for endotoxin assay o including assay tubes were depyrogenated by heating to 300 C for at least 6 hours.
- All polystyrene and polyethylene surfaces used for storage of antibodies, HBSS-luminol or blood products were sterile and essentially endotoxin free as determined by chromogenic LAL assay of pyrogen free water left in contact with the surface of interest.
- All pipette tips used for fluid transfer were sterile and pyrogen free (Diamed, Mississauga, Ontario, Canada).
- Blood samples used for the assay were drawn by venipuncture or through indwelling arterial lines into sterile 3 ml EDTA anti-coagulated Vacutainer tubes (Becton Dickenson, Franklin Lakes, New Jersey) which were pretested for LPS content (less than 0.005 EU/ml).
- Luminol (5-amino-2,3-dihydro-l,4-phthalazinedione, free acid), zymosan A (Saccharomyces cerevisiael lipopolysaccharides from Escherichia coli (E. coli) serotypes (026:B6, 055:B5, 0111:B4) (gram-negative endotoxin), and lipoteichoic acids from Streptococcus spp. (Gram-positive cell wall constituent) were purchased from Sigma ("Sigma Chemical Co., St. Louis, Mo).
- Buffer for measurement of whole blood or white cell chemiluminescence studies was HBSS (pyrogen free, endotoxin less than 0.005 EU/ml) containing 1.5 mM calcium salt and 0.9 mM magnesium salt (Gibco BRL, Grand Island, New York). This buffer (500 o ml) was vigorously mixed overnight at 25 C with luminol to yield a saturated solution (150 ⁇ M, HBSS-luminol) and then supplemented with 4 U/ml of lithium heparin. All chemiluminescence experiments were assayed in triplicate and the results expressed as the mean luminometer counts per minute ⁇ 1 SD. Assays may also be prepared using duplicate or single tubes for reaction tubes A, B and C.
- the following assay protocol was followed. Two aliquots of blood (500 ⁇ l) are dispensed into depyrogenated glass tubes into a thermostatted aluminum block pre-heated to 37°C. One tube contained a maximal dose of LPS; the other tube is empty. These tubes are incubated for 10 min. at 37 °C. During the last 5 minutes of this incubation glass or polystyrene assay tubes are loaded into the heating block. Three tubes are used per assay. Tube A contains control reagent used for antibody stabilization or no reagent at all, Tubes B and C contain antibody. To each tube a mixture of Luminol Buffer with unopsonized zymosan is added (500 ⁇ l per tube).
- This mixture is temperature equilibrated for at least 5 min. After the blood has incubated for a total of 10 min. at 37°C, 20 ⁇ l is transferred into assay tubes A and B from the blood tube with no LPS and 20 ⁇ l is transferred from the blood tube containing LPS into assay tube C. All tubes are vortexed and placed in the chemiluminometer for reading. The luminometer is thermostatted at 37 °C and the assay is read for a total of 20 min.
- FIG. 1 A typical whole blood chemilummescence profile of a patient with endotoxemia is shown in Figure 1.
- the 20-minute light integrals of tubes A, B and C are used to calculate the amount of LPS in the sample as follows.
- the amount of LPS present in the sample is referred to as "endotoxin Activity” (EA), and calculate from the light integrals as follows:
- the EA is calculated and the decision of whether a patient is endotoxemic or not may be based on a cutoff value of range, i.e. > 35 EA, an indicator of of clinically significant endotoxemia. Further parameters are available from the three-tube assay results as pertains to the stage of sepsis. Responsiveness (R) of the patients white blood cells, a measure of the maximal ability of the white blood cell to bind and respond to opsonized immunocomplexes as defined above, is calculated as follows:
- a measure of the level of white blood cell activation and cell number may be measured as the peak luminometer count rate of tube A during the course of the assay.
- the maximum oxidant production of neutrophils, as measured by CLmax, is a measure of the ability of the white blood cell to respond to programmed opsonic challenge.
Abstract
Description
Claims
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DE69923715T DE69923715T2 (en) | 1999-07-14 | 1999-11-30 | MEASUREMENT OF ANALYTES IN FULL BLOOD |
AU12910/00A AU1291000A (en) | 1999-07-14 | 1999-11-30 | Measurement of analytes in whole blood |
EP99956273A EP1194779B1 (en) | 1999-07-14 | 1999-11-30 | Measurement of analytes in whole blood |
AT99956273T ATE289068T1 (en) | 1999-07-14 | 1999-11-30 | MEASUREMENT OF ANALYTES IN WHOLE BLOOD |
CA2381038A CA2381038C (en) | 1999-07-14 | 1999-11-30 | Measurement of analytes in whole blood |
JP2001509989A JP4323744B2 (en) | 1999-07-14 | 1999-11-30 | Measuring analytes in whole blood |
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US09/353,189 | 1999-07-14 | ||
US09/353,189 US6306614B1 (en) | 1994-06-08 | 1999-07-14 | Measurement of analytes in whole blood |
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AT (1) | ATE289068T1 (en) |
AU (1) | AU1291000A (en) |
CA (1) | CA2381038C (en) |
DE (1) | DE69923715T2 (en) |
ES (1) | ES2237955T3 (en) |
WO (1) | WO2001004630A1 (en) |
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WO2003041737A1 (en) * | 2001-11-13 | 2003-05-22 | The University Of Liverpool | Treatment of inflammatory conditions |
US7632685B2 (en) | 2002-11-12 | 2009-12-15 | Becton, Dickinson And Company | Method of predicting the onset of sepsis in SIRS-positive individuals using mass spectrometry |
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- 1999-11-30 CA CA2381038A patent/CA2381038C/en not_active Expired - Lifetime
- 1999-11-30 DE DE69923715T patent/DE69923715T2/en not_active Expired - Lifetime
- 1999-11-30 ES ES99956273T patent/ES2237955T3/en not_active Expired - Lifetime
- 1999-11-30 EP EP99956273A patent/EP1194779B1/en not_active Expired - Lifetime
- 1999-11-30 AT AT99956273T patent/ATE289068T1/en not_active IP Right Cessation
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US7632685B2 (en) | 2002-11-12 | 2009-12-15 | Becton, Dickinson And Company | Method of predicting the onset of sepsis in SIRS-positive individuals using mass spectrometry |
US7645573B2 (en) | 2002-11-12 | 2010-01-12 | Becton, Dickinson And Company | Diagnosis of sepsis or SIRS using biomarker profiles |
US7645613B2 (en) | 2002-11-12 | 2010-01-12 | Becton, Dickinson And Company | Mass spectrometry techniques for determining the status of sepsis in an individual |
US10443099B2 (en) | 2005-04-15 | 2019-10-15 | Becton, Dickinson And Company | Diagnosis of sepsis |
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US9708661B2 (en) | 2008-04-03 | 2017-07-18 | Becton, Dickinson And Company | Advanced detection of sepsis |
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US10221453B2 (en) | 2008-04-03 | 2019-03-05 | Becton, Dickinson And Company | Advanced detection of sepsis |
Also Published As
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DE69923715D1 (en) | 2005-03-17 |
CA2381038A1 (en) | 2001-01-18 |
AU1291000A (en) | 2001-01-30 |
ATE289068T1 (en) | 2005-02-15 |
DE69923715T2 (en) | 2006-01-05 |
EP1194779B1 (en) | 2005-02-09 |
JP2003524773A (en) | 2003-08-19 |
US20020031786A1 (en) | 2002-03-14 |
JP4323744B2 (en) | 2009-09-02 |
US6306614B1 (en) | 2001-10-23 |
CA2381038C (en) | 2011-01-25 |
EP1194779A1 (en) | 2002-04-10 |
ES2237955T3 (en) | 2005-08-01 |
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