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WO2009063307A2 - Procédé de détection de l'activité inflammatoire en utilisant l'il-6 - Google Patents

Procédé de détection de l'activité inflammatoire en utilisant l'il-6 Download PDF

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Publication number
WO2009063307A2
WO2009063307A2 PCT/IB2008/003063 IB2008003063W WO2009063307A2 WO 2009063307 A2 WO2009063307 A2 WO 2009063307A2 IB 2008003063 W IB2008003063 W IB 2008003063W WO 2009063307 A2 WO2009063307 A2 WO 2009063307A2
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inflammatory
test
test sample
animal
inflammatory activity
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WO2009063307A8 (fr
WO2009063307A3 (fr
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Daniel Plaksin
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Bio Technology General Israel Ltd
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Bio Technology General Israel Ltd
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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
    • 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
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6

Definitions

  • the inflammatory response is a defense mechanism to protect the body from infection and/or injury; inflammation increases blood flow to the site of infection or injury, bringing necessary fluids, proteins, and white blood cells (leukocytes) to help in the healing process.
  • one symptom associated with the inflammatory response is an elevation in body temperature or fever, which functions as a defense mechanism to pathogens that cause overheating.
  • the inflammatory response is often, therefore, associated with a variety of "flu-like" symptoms including fever, chilis, fatigue, headaches, loss of appetite, and muscle stiffness.
  • a chemical or biological compound that triggers fever has historically been referred to as a "pyrogen” or a “pyrogenic” compound, referring to the fever response which such compounds may cause.
  • Some chemical or biological compounds are generally proinflammatory and may or may not cause fever as part of the inflammatory response that they cause.
  • an individual can develop life-threatening shock-like symptoms after exposure to an inflammatory agent.
  • Medical products which can be inhaled, injected, or infused and medical devices such as membranes or implanted materials pose a particular risk of pyrogenicity. Even nutrients can represent a risk of pyrogenicity.
  • Inflammatory agents, or pyrogens, contained in medical products and nutrients are referred to as exogenous inflammatory agents (or pyrogens); in contrast, endogenous pyrogens are messenger compounds of the immune system that mediate an individual's inflammatory response to exogenous pyrogens.
  • Pyrogenicity due to contamination of a product can be caused by any one of a diverse group of pyrogens derived from bacteria, viruses, fungi, or even from the host. This problem can persist even if the product is "sterilized” by heat or chemical methods.
  • a commonly encountered pyrogenic compound, bacterial endotoxin (consisting largely of lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria), can remain after the bacteria are killed.
  • LPS lipopolysaccharide
  • Gram-positive bacteria such as Streptococcus, produce proinflammatory cell wall mucopeptides.
  • the rabbit pyrogen test has been historically used to monitor pyrogen contamination in mass-produced pharmaceutical products.
  • the rabbit test is an in-vivo test which consists of injecting a statistically significant number of rabbits with the sample compound intravenously and observing the average rise in body temperature elicited in the test animals.
  • the rabbit test is responsive to a wide spectrum of pyrogenic agents, including non-endotoxin pyrogens, the rabbit test has a relatively low sensitivity (ng endotoxin/ml).
  • the relative insensitivity, poor quantitative and variability of results, as well as ethical issues involved in animal testing have made the rabbit test disfavored in recent years.
  • ROTT rabbit ocular toxicity test
  • owl monkeys Aotus trivigatus
  • the intravitreous injection of owl monkeys has also been used to measure inflammatory activity, most frequently for preparation of bacterially synthesized hyaluronic acid.
  • the owl monkey was used because it was found that, when fully grown, this new world monkey had a liquid vitreous (see Balazs et al. (1959) Comparative Biochemistry. Arch. Blochem Biophvs. 81 :464-79).
  • the presence of a liquid rather than a gel vitreous was a prerequisite for these tests.
  • the exchange of viscous liquid vitreous with the biopolymer solution had to be achieved with minimal surgical trauma in order to evaluate the tolerance of the vitreous substance used.
  • the owl monkey vitreous test was consequently developed as an extremely sensitive method to evaluate the inflammatory reaction in the vitreous after the injection of viscoelastic solutions (see Balazs et al. (1966) "Replacement of the vitreous body of monkeys with reconstituted vitreous and hyaluronic acid" in Modern Problems in Ophthalmology (Surgery of retinal vascular diseases and prophylactic treatment of retinal detachment), Amersfoort 1963 fed. Streiff. E. BX VoI 4 pp. 230-32).
  • the liquid vitreous of the owl monkey can be easily replaced without mechanical damage to the retina and lens.
  • the minimal operational trauma permits a very accurate and sensitive evaluation of the tolerance of the vitreal implant.
  • U.S. Pat. No. 4,141 ,973 describes a modified owl monkey test to test the inflammatory reaction of ultrapure hyaluronic acid.
  • Another primate that has been used to test the inflammatory reaction of ultrapure hyaluronic acid is the rhesus monkey.
  • the total volume of the vitreous of the adult rhesus eye is approximately 24% larger than the owl monkey eye, the volume of the liquid vitreous is smaller (0.8 to 1.2 ml) in the rhesus than the owl monkey eye (1.8-2.2 ml).
  • the volume of vitreous is smaller (0.8 to 1.2 ml) in the rhesus than the owl monkey eye (1.8-2.2 ml).
  • 0.5 ml of vitreous can be removed and replaced.
  • the incidence of eyeball collapse is greatly reduced, and therefore, there is less traumatic injury to the blood aqueous barrier.
  • the rhesus monkey has been shown to have similar inflammatory reactions as the owl monkey, after vitreal injection of HEALONTM.
  • the rhesus monkey can also be used as an extremely sensitive system for detecting small amounts of inflammatory agents (see Denlinger and Balazs, (1980) "Replacement of the Liquid Vitreous with Sodium Hyaluronate in Monkeys" Experimental Eye Research 31 :81-99.)
  • the oxidative burst inflammation test is yet another method of testing for inflammatory activity.
  • the oxidative burst (OB) inflammation test is a standard test for pyrogenic activity in which the activation of an acute phase immune response is measured in an animal ⁇ e.g., mouse) after injection of a test sample by detecting transformation of molecular oxygen into reactive oxygen species (ROS) by leukocytes.
  • the OB method is carried out by measuring the OB of cells harvested from the peritoneum of test sample-injected mice by means of UV spectrophotometric determination of cytochrome C oxidation in the peritoneal exudate cells (i.e., mainly polymorphonuclear leukocytes (PMNs), which are the primary responders to inflammatory material).
  • PMNs polymorphonuclear leukocytes
  • the oxidative burst may be amplified by the addition of phorbol myristate acetate (PMA).
  • the cell- dependent OB response may then be determined by measuring the absorbance of freshly added cytochrome C at 550 nm.
  • a noninflammatory negative control will generally produce an absorbance of cytochrome C at 550 nm (A 550 ) of ⁇ 0.10 at 1 x 10 5 cells/ml, while a sample having inflammatory activity will typically produce an A 550 of 0.15-0.40 at 1 x 10 5 cells/ml.
  • the rabbit ocular, owl monkey, rhesus monkey, OB and other tests are accepted methods for detecting inflammatory activity, they suffer from several inconveniences and inefficiencies.
  • the simian assays are time-consuming, costly and of arguable value in view of ethical considerations.
  • the OB test method has a relatively narrow signal-to-noise ratio, thereby giving rise to a large number of false positive and false negatives and necessitating repeated testing.
  • the OB testing method frequently yields hemolytic samples obtained from animal handling that must be excluded from the test.
  • the OB test for inflammatory activity relies upon a very limited sampling of immune leukocyte cells withdrawn from each individual mouse peritoneum, thereby increasing the potential for testing error. While the limited number of peritoneal cells from individual animals can be pooled, this technique impacts on the statistical analysis of the results and can yield a false positive result in cases where the number of cells from one animal are the majority that are tested for that group.
  • the invention is based, in part, upon the discovery that detection of interleukin-6 (IL-6), or another pro-inflammatory cytokine or other cell-derived mediator of inflammation, provides a particularly sensitive, accurate and convenient method for detecting inflammatory activity as compared to methods based upon detection of the oxidative burst (OB) produced by activated polymorphonuclear leukocytes.
  • IL-6 interleukin-6
  • OB oxidative burst
  • the elevation of IL-6 levels, or other cell-derived mediators of inflammation, in the peritoneal fluid of a test animal may be detected using any available method, such as an IL-6 specific ELISA (Enzyme-Linked Immunosorbent Assay).
  • the method of the invention provides the additional advantage of not requiring the isolation of intact immune cells (e.g., from peritoneal exudate), thereby allowing the use of hemolytic samples that are frequently obtained upon animal handling and which would otherwise have to be excluded when employing an oxidative burst assay.
  • the method of the invention further avoids the additional laborious testing of isolated cells in vitro, and produces more statistically reliable measurements.
  • the invention provides a method of detecting inflammatory activity in a sample by administering a test sample to an animal, and detecting a level of interleukin-6 (IL-6) in the animal.
  • IL-6 interleukin-6
  • the test sample may be a biological sample, a medical device or a biopolymer.
  • exemplary test samples for use in the method of the invention include heteropolysaccharide preparations such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparan sulfate, agar, alginate, carrageenan, gellan, guar gum, locust bean gum and/or xantham gum.
  • test sample is a hyaluronan (or hyaluronic acid or hyaluronate) preparation, e.g., an HA preparation for use in synovial fluid supplementation or as a dermatological filler agent.
  • hyaluronan or hyaluronic acid or hyaluronate
  • the heteropolysaccharide is produced by bacterial fermentation.
  • the heteropolysaccharide is produced by bacterium of the genus Streptococcus, and the inflammatory activity detected may be, e.g., a Streptococcal mucopeptide.
  • the inflammatory activity detected is a bacterial endotoxin, e.g., a lipopolysaccharide.
  • the inflammatory activity detected by the method of the invention is from a Gram-positive bacterium, e.g., a lipoteichoic acid (LTA).
  • LTA lipoteichoic acid
  • test animal used in the method may be, in particular embodiments, a mammal, e.g., a mouse, guinea pig, or rat.
  • the test sample is administered by injecting it into the abdomen of the mammalian test animal.
  • the level of IL-6 may be detected using any method or technique known in the art.
  • the level of IL-6 in the test animal may be detected using an IL-6 antibody; e.g., using an antibody-antigen-antibody sandwich enzyme-linked immunosorbent assay (ELISA).
  • ELISA antibody-antigen-antibody sandwich enzyme-linked immunosorbent assay
  • the level of IL-6 may be detected with a bioassay that is sensitive to IL-6.
  • the level of IL-6 in the animal is determined about 4-16 hours after administering the test sample to the animal.
  • the invention provides a method of detecting an exogenous non-endotoxin pyrogen in a Gram-positive bacterial fermentation product by injecting a mouse with the Gram-positive bacterial fermentation product intra-peritoneally; collecting a peritoneal fluid from the injected mouse at least about 6 hours after injecting the bacterial fermentation product; and measuring the IL-6 concentration in a cell free supernatant of the peritoneal fluid.
  • an exogenous non-endotoxin pyrogen is present in the Gram- positive bacterial fermentation product if the IL-6 concentration is higher than the concentration of IL-6 in a control mouse injected with a non-inflammatory negative control.
  • the exogenous non-endotoxin pyrogen is a Streptococcal mucopeptide.
  • the Gram-positive bacterial fermentation product is hyaluronic acid (HA).
  • the Gram- positive bacterium is a Streptococcus; e.g., a hemolysin mutant strain of Streptococcus zooepidemicus such as HA- 116 (ATCC 39920).
  • the mouse tested in the method of the invention is a Balb/c mouse, a CB6F1 mouse, or the progeny or descendent of a cross between a Balb/c and a CB6F1 mouse.
  • the peritoneal fluid is collected from the mouse approximately 4-16 hours after injecting the Gram-positive bacterial fermentation product.
  • the level of IL-6 may be detected using any method or technique known in the art.
  • the level of IL-6 in the test animal may be detected using an IL-6 antibody; e.g., using an antibody-antigen-antibody sandwich enzyme-linked immunosorbent assay (ELISA).
  • the level of IL-6 may be detected using a bioassay that is sensitive to IL-6.
  • the presence of an exogenous non-endotoxin pyrogen in the bacterial fermentation product is indicated if there is a statistically significant higher concentration of IL-6 concentration in the mouse injected with the Gram-positive bacterial fermentation product than in the control mouse injected with the non-inflammatory negative control.
  • the Gram-positive bacterial fermentation product is an otherwise non-immunogenic preparation of a heteropolysaccharide, e.g., sodium hyaluronate.
  • the invention provides a method of detecting inflammatory activity in a test sample by administering the test sample to an animal; and detecting a level of a pro-inflammatory cytokine or other cell-derived mediator of inflammation in the animal.
  • a pro-inflammatory cytokine or other cell-derived mediator of inflammation in the animal indicates that the test sample has inflammatory activity.
  • the level of one or more particular pro-inflammatory cytokines, such as IL-1 , IL-6, TNF- ⁇ , and/or IFN- ⁇ is detected.
  • the level of one or more particular cell-derived mediators of inflammation is detected.
  • the test sample is an otherwise non-immunogenic preparation of a heteropolysaccharide, e.g., sodium hyaluronate.
  • Figure 1 is a representation of potentially inflammatory bacterial membrane and cell wall components that may contaminate bacterial fermentation preparations including those occurring in Gram-positive bacteria (such as Streptococcus), Gram-negative bacteria, Mycoplasma and Mycobacterium tuberculosis.
  • Gram-positive bacteria such as Streptococcus
  • Gram-negative bacteria such as Mycoplasma
  • Mycobacterium tuberculosis such as Steptococcus
  • Figure 2 is a diagrammatic depiction of the cytochrome c assay for inflammatory activity in a test sample.
  • Figure 3 is a diagrammatic depiction of the IL-6 ELISA assay for inflammatory activity in a test sample.
  • the invention provides materials and methods for the detection of an inflammatory activity in a test sample, e.g., any biomaterial.
  • a test sample e.g., any biomaterial.
  • biomaterial implants can nonetheless often cause adverse reactions, particularly if they are contaminated by even minute amounts of an inflammatory activity (e.g., a bacterial endotoxin, such as LPS, or cell wall mucopeptide, or other gram-positive bacterial inflammatory activity, e.g., lipoteichoic acid).
  • an inflammatory activity e.g., a bacterial endotoxin, such as LPS, or cell wall mucopeptide, or other gram-positive bacterial inflammatory activity, e.g., lipoteichoic acid.
  • hyaluronan a particularly useful and normally non-immunogenic and non-inflammatory biomaterial, hyaluronan, has been shown to possess inflammatory activity when it is degraded down into low molecular weight fragments (see GaIIo et al. (2004) J. Biol. Chem. 279:17079-84). Only small HA fragments of tetra- and hexasaccharide size (sHA), but not intermediate size ⁇ e.g., M.W. 80 - 200 kD) or high molecular weight (e.g., 600 - 1 ,000 kD) were shown to possess such inflammatory activity (Simon et al. (2000) J. Immunol. 165:1863-70). Accordingly the invention also provides a useful means of detecting inflammatory preparations of otherwise pure hyaluronan containing very low molecular weight hyaluronan fragments.
  • sHA tetra- and hexasacchari
  • biomaterials may attract a layer of phagocytic cells (especially, macrophages, monocytes and neutrophils) and fibroblast-like cells.
  • phagocytic cells especially, macrophages, monocytes and neutrophils
  • fibroblast-like cells This process resembles the acute inflammatory response seen with many other types of foreign bodies and, to some extent, with focal bacterial infections.
  • the acute inflammatory response to tissue contact biomaterials is very often followed by chronic inflammation and the appearance of fibrotic tissue surrounding many types of implants.
  • Chronic inflammation and fibrosis are also associated with the degradation and failure of many types of biomaterial implants, including temporomandibular and other joint implants.
  • Such inflammatory responses to inert, nonimmunogenic and nontoxic implanted biomaterials must be minimized. Accordingly, methods of detecting inflammatory activity in a biomaterial preparation are important to guarding against adverse reactions to biomaterials and other implanted matter.
  • the term "animal” is meant to include any eukaryotic metazoan exhibiting an inflammatory response to a foreign agent or tissue damage. Accordingly, the term “animal” includes all mammals such as mice, rats, guinea pigs, rabbits, humans, sheep, elks, deer, mule deer, minks, monkeys, horses, cattle, pigs, goats, dogs and cats. Also included within the scope of the tern animal are birds, including chickens, as well as reptiles, fish, insects and arachnids.
  • antibody refers to intact antibodies including polyclonal antibodies (see, for example Antibodies: A Laboratory Manual, Harlow and Lane (eds), Cold Spring Harbor Press, (1988)), and monoclonal antibodies (see, for example, U.S. Patent Nos. RE 32,011 , 4,902,614, 4,543,439, and U.S. Pat. No. 4,411 ,993, and Monoclonal Antibodies: A New Dimension in Biological Analysis, Plenum Press, Kennett, McKearn and Bechtol (eds.) (1980)).
  • antibody also refers to a fragment of an antibody such as F(ab), F(ab'), F(ab') 2 , Fv, Fc, and single chain antibodies which are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antibody also refers to bispecific or bifunctional antibodies, which are an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. (See Songsivilai et al., Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J,_ Immunol.
  • antibody also refers to chimeric antibodies, that is, antibodies having a human constant antibody immunoglobin domain is coupled to one or more non-human variable antibody immunoglobin domain, or fragments thereof (see, for example, U.S. Pat. No. 5,595,898 and U.S. Pat. No. 5,693,493).
  • Antibodies also refers to "humanized” antibodies (see, for example, U.S. Pat. No.
  • cytokine refers to secreted proteins that are involved in regulation of the immune response including interleukins (ILs), interferons (IFNs), chemokines, tumor necrosis factors (TNFs), and a variety of colony stimulating factors (CSFs).
  • ILs interleukins
  • IFNs interferons
  • chemokines chemokines
  • TNFs tumor necrosis factors
  • CSFs colony stimulating factors
  • IL-6 IL-1 ⁇
  • IL-12p70 IL-10
  • IL-2 granulocyte-macrophage colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • TNF- ⁇ TNF- ⁇
  • “Expression” refers to transcription and translation occurring within a host cell.
  • the level of expression of a DNA molecule in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of DNA molecule encoded protein produced by the host cell (Sambrook et al., 1989, Molecular cloning: A Laboratory Manual, 18.1 -18.88).
  • Genetically engineered refers to any recombinant method used to create a eukaryotic host cell that expresses a protein of interest. Methods and vectors for genetically engineering host cells are well known; for example, various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel etal., eds. (Wiley & Sons, New York, 1988, and quarterly updates). Genetic engineering techniques include, but are not limited to, expression vectors, targeted homologous recombination and gene activation (see, for example, U.S. Pat. No. 5,272,071 to Chappel) and transactivation by engineered transcription factors (see, for example, Segal et al., 1999, Proc Natl Acad Sci USA 96(6):2758- 63).
  • high molecular weight depends upon the particular macromolecular sample (e.g., biopolymer) to be tested but generally refers to a molecular weight greater than 1 x 10 6 daltons.
  • a high molecular weight HA viscoelastic biopolymer is generally in the range of 1 x 10 6 to 1 x 10 7 daltons (more particularly, in the range of 2.5 x 10 6 to 5.0 x 10 6 daltons).
  • Hyaluronic acid and “sodium hyaluronate” are used interchangeably and refer to the acid and/or sodium salt of "hyaluronan.”
  • Hyaluronan is a non-sulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. It is one of the chief components of the extracellular matrix, contributes significantly to cell proliferation and migration, and may also be involved in the progression of some malignant tumors.
  • Hyaluronan is a polymer of disaccharides themselves, composed of D-glucuronic acid and D-N- acetylglucosamine, linked together via alternating ⁇ -1 ,4 and ⁇ -1 ,3 glycosidic bonds.
  • Hyaluronan can be 25,000 disaccharide repeats in length.
  • Polymers of hyaluronan can range in size from 5,000 to 20,000,000 Da in vivo. The average molecular weight in human synovial fluid is 3-4 million Da, and hyaluronan purified from human umbilical cord is approximately 3,140,000 Da.
  • inflammatory activity refers to any agent that causes, or tends to cause, inflammation in an animal subject to which it is administered. Inflammation is a local response to cellular injury or noxious agent that is marked by capillary dilation, leukocytic infiltration, redness, heat, pain, and swelling.
  • isolated refers to a polynucleotide or polypeptide that has been separated from at least one contaminant (polynucleotide or polypeptide) with which it is normally associated.
  • contaminant polynucleotide or polypeptide
  • an isolated antibody or an isolated polynucleotide is in a context or in a form that is different from that in which it is found in nature.
  • Polynucleotide refers to a sequence of nucleotides.
  • the nucleotides are either a sequence of polyribonucleotides or polydeoxyribonucleotides, or a mixture of both.
  • Examples of polynucleotides in the context of the present invention include single and double stranded DNA, single and double stranded RNA, and hybrid molecules that have both mixtures of single and double stranded DNA and RNA.
  • the polynucleotides of the present invention can include one or more modified nucleotide.
  • protein and “polypeptide” are used interchangeably and is considered to be any chain of at least ten amino acids linked by peptide bonds.
  • Purification of a protein from contaminating proteins can be accomplished through any number of known techniques, including, ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.
  • Various protein purification techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates).
  • the percent identity of one polynucleotide, polypeptide or protein in relation to another may be determined by visual inspection and mathematical calculation, or by a comparison of two sequences using various computer programs used by those of skill in the art.
  • the percent identity of two sequences can be determined using the GAP computer program, based on the algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, (available from the University of Wisconsin Genetics Computer Group (UWGCG), University Research Park, Madison, Wis.).
  • the preferred default parameters for the GAP program include: (1) a scoring matrix, blosum62, as described by Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915 ); a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for end gaps.
  • Other programs used by those skilled in the art of sequence comparison may also be used.
  • Vector refers to a first polynucleotide molecule, usually double-stranded, which can have inserted into it a second polynucleotide molecule, for example a heterologous polynucleotide.
  • a heterologous polynucleotide may or may not be naturally found in the host cell, and can be one or more additional copy of a nucleic acid sequence naturally present in the host genome.
  • the vector transports the foreign polynucleotide into a suitable host cell. Once in the host cell, the vector can be capable of integrating into the host cell chromosomes.
  • the vector can also contain the necessary elements to select cells containing the integrated polynucleotide, as well as elements to promote transcription of mRNA from the transfected polynucleotide.
  • vectors that may be adapted for use in certain embodiments of the present invention include, but are not limited to, plasmids, bacteriophages, cosmids, retroviruses, and artificial chromosomes.
  • the presence of a pyrogen or other inflammatory activity in a test sample stimulates blood monocytes, as well as other leukocytes, to produce and release these endogenous pyrogenic mediators of the inflammatory response.
  • the invention provides methods for detecting one or more inflammatory activities, or pyrogens, in a sample by measuring the production and release of endogenous pyrogenic mediators of the inflammatory response, including cytokines (e.g., IL-6, TNF- ⁇ and/or IL-1 ⁇ ).
  • the pro-inflammatory cytokine IL-6 is particularly useful in the method of the invention, however any endogenous mediator of the inflammatory response that is detectable may be used as the basis of the pyrogen test of the present invention.
  • a cytokine or endothelin marker is employed because they are easy to detect (e.g., by ELISA).
  • cytokines including, but not limited to pro-inflammatory cytokines (IL-6, as well as TNF- ⁇ , IL-1 , IL-6), anti-inflammatory cytokines (IL-4, IL- 10, IL-13, IL-1 ra, TGF), Th1 (IL-2, IFN, IL-12), Th2 (IL-4, IL-5, IL-6, IL-I O, IL-13), IL-I ⁇ , IL-1 ra, IL-8, and PGE 2 .
  • pro-inflammatory cytokines IL-6
  • anti-inflammatory cytokines IL-4, IL- 10, IL-13, IL-1 ra, TGF
  • Th1 IL-2, IFN, IL-12
  • Th2 IL-4, IL-5, IL-6, IL-I O, IL-13
  • IL-I ⁇ , IL-1 ra, IL-8 particularly useful cytokine markers for use in the invention include IL-6, TNF- ⁇ , IL-1 ⁇
  • IL-6 is a particularly useful cytokine marker to assay in the method of the invention.
  • IL-6 is produced in detectable amounts with a relatively short latency period.
  • Immunoreactive IL-6 unlike immunoreactive IL-1 ⁇ and TNF- ⁇ , is secreted in relatively large quantities, permitting a more complete and accurate estimation of its level in the body of the test animal, as reflective of the relative amount of inflammatory activity in the test sample.
  • TNF- ⁇ or IL-1 ⁇ couid also be used as a cytokine marker in the present invention.
  • TNF- ⁇ is produced earlier than IL-6 in the monocyte pyrogen response.
  • an embodiment of the invention which assays TNF- ⁇ would use a shorter incubation time (about 1 to 2 hours) than embodiments which assay IL-6.
  • Different pyrogenic contaminants may elicit different cytokine responses in the cell culture. Therefore, the invention may be tailored to detect the formation of particular cytokines when contamination with a particular pyrogen which causes secretion of those cytokines is probable for a pharmaceutical product.
  • cytokine or other cell-derived mediator of inflammation
  • a specific method for detecting that cytokine is selected for use in the method, of invention. Any of a number of different detection techniques may be adapted to the method of the invention.
  • Particularly useful detection methods employ one or more antibodies specific to the analyte to be detected (e.g., the pro-inflammatory cytokine or other cell-derived mediator of inflammation of interest).
  • an antibody to that analyte may be obtained commercially, or by routine experimentation, for use in the method of the invention.
  • polyclonal antibodies purified under stringent conditions ⁇ e.g., as described in Example 1 of U.S. Pat. No. 6,696,261 work well in the pyrogen test.
  • the animal blood from which the polyclonal antibodies are isolated is naturally pyrogen-free (if taken from healthy animals), one must simply prevent contamination of the raw materials with pyrogens during purification to obtain a pyrogen-free product.
  • Pyrogen-free buffers and solid phases are used in affinity chromatography columns to obtain pyrogen-free polyclonal antibodies are known in the art.
  • monoclonal antibodies from hybridoma cultures can be used. However, when using monoclonal antibodies, reasonable care must be taken to isolate the antibodies free of contaminating pyrogens which may be present in the hybridoma cell culture, particularly the pyrogen to which the assay is to be directed.
  • the antibody to the cytokine may be applied to a surface in an assay system.
  • Methods, such as coating, for binding antibodies onto a surface in an assay system, such as a microtiter well are well known in the biochemical arts. Many assay systems are available commercially, and the manufacturer usually provides materials and instructions for coating antibodies onto a surface of the system. Because of their ease of reading and the small sample volume required, microtiter wells in which a portion of the interior surface of the well is coated by the antibody are used in a preferred embodiment of the present invention.
  • the microtiter well be part of a microtiter plate, which is a planar array of similar wells, situated so that the array of wells may be read with automated immunoassay plate reading equipment (see, e.g., U.S. Pat. No. 5,281 ,540, hereby incorporated by reference).
  • Automated equipment such as ELISA plate readers (e.g., the Ultramark Microplate Reader, available from Bio- Rad Laboratories, Inc.), automate the assay evaluation process and greatly decrease the per-test cost.
  • microtiter well plates can be rendered pyrogen-free (if not already supplied as such) by extensive washing with pyrogen- free buffer.
  • anti- IL-6 polyclonal antibodies are coated onto the wells of an ELISA plate.
  • other immuno-diagnostic test formats e.g., in which the antibody is coated on a dipstick or bead
  • the antibody is coated on a dipstick or bead
  • a labeled detection antibody such as a biotinylated or enzyme-labeled antibody
  • the level of cytokine or other cell-derived inflammatory mediator may be measured using any of a broad number of techniques available to the person of ordinary skill in the art, e.g., direct physical measurements (e.g., mass spectrometry) or binding assays (e.g., immunoassays, agglutination assays, and immunochromatographic assays).
  • direct physical measurements e.g., mass spectrometry
  • binding assays e.g., immunoassays, agglutination assays, and immunochromatographic assays.
  • the method may also comprise measuring a signal that results from a chemical reaction, e.g., a change in optical absorbance, a change in fluorescence, the generation of chemiluminescence or electrochemi- luminescence, a change in reflectivity, refractive index or light scattering, the accumulation or release of detectable labels from the surface, the oxidation or reduction or redox species, an electrical current or potential, changes in magnetic fields, etc.
  • a chemical reaction e.g., a change in optical absorbance, a change in fluorescence, the generation of chemiluminescence or electrochemi- luminescence, a change in reflectivity, refractive index or light scattering, the accumulation or release of detectable labels from the surface, the oxidation or reduction or redox species, an electrical current or potential, changes in magnetic fields, etc.
  • Suitable detection techniques may detect binding events by measuring the participation of labeled binding reagents through the measurement of the labels via their photoluminescence ⁇ e.g., via measurement of fluorescence, time- resolved fluorescence, evanescent wave fluorescence, up-converting phosphors, multi-photon fluorescence, etc.), chemiluminescence, electrochemiluminescence, light scattering, optical absorbance, radioactivity, magnetic fields, enzymatic activity ⁇ e.g., by measuring enzyme activity through enzymatic reactions that cause changes in optical absorbance or fluorescence or cause the emission of chemiluminescence).
  • Binding assays for measuring cytokine levels may use solid phase or homogenous formats. Suitable assay methods include sandwich or competitive binding assays. Examples of sandwich immunoassays are described in U.S. Pat. No. 4,168,146 and U.S. Pat. No. 4,366,241. Examples of competitive immunoassays include those disclosed in U.S. Pat. No. 4,235,601 , U.S. Pat. No. 4,442,204, and U.S. Pat. No. 5,208,535.
  • Multiple cytokines may be measured using a multiplexed assay format, e.g., multiplexing through the use of binding reagent arrays, multiplexing using spectral discrimination of labels, multiplexing by flow cytometric analysis of binding assays carried out on particles (e.g., using the Luminex system).
  • Suitable multiplexing methods include array based binding assays using patterned arrays of immobilized antibodies directed against the cytokines of interest.
  • Various approaches for conducting multiplexed assays have been described. For example, multiplexed testing is described in U.S. Pat. Publ. Nos. 20040022677 and US20050052646, as well as, U.S. Pat. Publ. No.
  • Also available for use in the methods of the invention is a multiplex binding assay in which 64 different bead sets of microparticles are employed, each having a uniform and distinct proportion of two dyes (see Vignali, (2000) J. Immunol. Meth. 243: 243-255). Similar approaches include a method involving a set of 15 different beads of differing size and fluorescence have been disclosed as useful for simultaneous typing of multiple pneumococcal serotypes (see Park, et al. (2000) Clin Diaqn Lab Immunol. 7: 486- 9); and a multiplex sandwich assay for simultaneous quantification of six human cytokines (see Bishop, et al. (1999) Clin Chem. 45:1693-1694).
  • the test method involves testing one or more samples, e.g., body fluid samples, particularly an intraperitoneal body fluid sample, from the test animal.
  • the test methods of the invention may be conducted on a single sample from the test animal including, but not limited to, peritoneal fluid, blood, serum, plasma, hair, sweat, urine, feces, tissue, biopsies, saliva, skin, mucosa, CNS fluid, bone marrow, tissue extracts, cells, cell extracts, cell culture supernatants, and lymphatic fluids.
  • peritoneal fluid blood, blood serum, blood plasma, or biopsy tissue.
  • multiple samples from the test animal may be tested for one or more inflammatory cytokines and/or other cell-derived mediators of inflammation using any of the preceding detection methods.
  • the invention provides methods of detecting an inflammatory activity in a test sample, particularly a test sample primarily composed of a nonimmunogenic, noninflammatory agent such as hyalronan or other biopolymer.
  • Suitable test samples including preparations of polysaccharides, including homopolysaccharides and heteropolysaccharide for use in biological applications such as in medical implants or as biologic agents.
  • An exemplary heteropolysaccharide containing test sample for use in the method of the invention is a glycosaminoglycan (GAG) test sample.
  • GAG glycosaminoglycan
  • Glycosaminoglycans especially suitable for use in the present invention are hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratan sulfae, heparin and heparan sulfate.
  • the biopolymer may be obtained from a biological source, or it may be a product of in vitro enzymatic or chemical synthesis, or combinations thereof.
  • a biological source may be a bacterial, yeast, plant, amphibian, avian or mammalian organism.
  • HA may be isolated from any of fermented Streptococcal cultures, human umbilical cords, bovine cartilage or rooster combs.
  • Enzymatic in vitro enzymatic synthesis of polysaccharides including HA, cellulose, polymannuronic acid and chitin is described in WO 95/24497.
  • the use of a recombinantly produced hyaluronan synthase enzyme for in vitro synthesis of HA is described in U.S. Pat. No. 6,602,693.
  • HA obtained from any of the aforementioned sources may be subsequently chemically modified or derivatized as described for example in U.S. Pat. No. 4,851 ,521 , U.S. Pat. No. 4,713,448, U.S. Pat. No. 5,336,767 and U.S. Pat. No. 5,099,013.
  • HA is a naturally occurring biopolymer consisting of repeating disaccharide units of D-glucuronic acid in ⁇ -(1-3) linkage with N-acetyL-D- glucosamine, wherein each disaccharide unit is connected to its adjoining neighbors by ⁇ -(1 -4) linkages.
  • the salt sodium hyaluronate (NaHA) is found at physiological pH in human and vertebrate joint synovial fluid, connective tissue, vitreous humor of the eye and healthy skin tissue, and is an extracellular secretion product of several bacterial species, particularly of the genus Streptococcus.
  • the method of the invention is particularly suitable for HA. NaHA has been isolated and purified from microorganisms and vertebrates.
  • Bacterially-derived NaHA may be derived from fermentation of Streptococcus zooepidemicus, e.g. particularly a hemolysin negative mutant such as HA-116 (ATCC 239920) as described in further detail in U.S. Patent Nos. 4,780,414 and 4,784,990.
  • hyaluronic acid means hyaluronic acid, salts thereof, such as sodium, potassium, magnesium, calcium, lysine, ammonium, triethanolamine and propanolamine hyaluronates, metal salts thereof, such as cobalt, zinc, copper, iron, manganese and lithium hyaluronate, and chemically modified and derivatized forms thereof, as disclosed for example in U.S. Pat. No. 4,851 ,521 , U.S. Pat. No. 5,099,013, U.S. Pat. No. 5,336,767 and U.S. Pat. No. 6,017,901.
  • Major medical applications of manufactured NaHA products include ophthalmic surgery for cataracts and intraocular lens implantation, and in viscoelastic supplementation for the treatment of osteoarthritis in humans and large mammals. Viscoelastic supplementation, particularly in the knee, is aimed at restoring the normal rheological homeostasis of the joint network and for providing immediate protection, lubrication, shock absorption, hydrodynamic resistance and a mechanochemical barrier against stress. Intra-articular injections of NaHA have been shown to improve function and mobility and decrease pain. More recent medical applications of manufactured NaHA include medical device coatings, surgical adhesion prevention products, drug delivery vehicles, bone replacement materials and wound healing materials.
  • Additional viscoelastic biopolymers which are the products of bacterial fermentation include for example curdlan gum ( ⁇ -1 -3-D-glucan), produced by Alcaligenes faecalis, gellan gum (tetrasaccharide backbone of L- rhamnose and D-glucose with glyceryl and acetyl substituents) produced by Sphingomonas elodea and xanthum gum ( ⁇ -1-4-D- glucan with mannose and glucuronic acid side chains), produced by Xanthomonas campedis.
  • curdlan gum ⁇ -1 -3-D-glucan
  • Alcaligenes faecalis Alcaligenes faecalis
  • gellan gum tetrasaccharide backbone of L- rhamnose and D-glucose with glyceryl and acetyl substituents
  • Sphingomonas elodea and xanthum gum ⁇
  • the viscoelastic biopolymer suitable for the method of the invention may be a homopolysaccharide i.e. assembled from a single type of monosaccharide, or a heteropolysaccharide Le assembled from two or more different types of monosaccharides.
  • homopolysaccharides include carboxymethylcellulose, chitin, polymannuronic acid, curdlan gum, scleroglucan and dextran.
  • heteropolysaccharides include glycosaminoglycans, alginates, carageenans, guar gum, pectins, locust bean gum and xanthum gum.
  • glycosaminoglycans also known as acid mucopolysaccharides, which are composed of repeating disaccharide units in which one of the two monosaccharides is always either N- acetylglucosamine or N-acetylgalactosamine.
  • glycosaminoglycans include HA, chondroitin, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, dermatan sulfate, keratan sulfate, heparin and heparan sulfate. Both homo- and heteropolysaccharides may be linear or branched structures.
  • the component monosaccharides of a biopolymer may be released by acid hydrolysis and detected by analytical techniques such as thin layer chromatography and/ or high pressure liquid chromatography.
  • Suitable viscoelastic biopolymers may be within a wide range of molecular weight (e.g., 1 x 10 4 to 1 x 10 7 daltons).
  • a bulk manufactured and purified viscoelastic biopolymer is generally sterilized, e.g. by filter sterilization, before it is tested for inflammatory activity by the method of the invention.
  • Example 1 Cell-Dependent Oxidative Burst Inflammatory Test
  • a mouse-based cell-dependent oxidative burst test has been used successfully to assay for biologically active inflammatory impurities in hyaluronic acid and other heteropolysaccharides and implanted materials.
  • an acute inflammatory response is initiated by intraperitoneal (ip) injection of unpurified HA into the mouse, but is not initiated by injection of highly purified HA.
  • a positive control group and a negative control group three experimental groups are used: a positive control group and a negative control group, and a test sample group. Since the local inflammatory response is complex and varies as a function of different stimulants, partially purified hyaluronate was used as the inflammatory reference material (positive control) in the assay.
  • the negative control NaHa reference standard dissolved in saline served to confirm proper execution of the test procedure and the quality of the test animals.
  • the test NaHA sample was prepared as a 1% solution in saline. The NaHA reference standard was used as the negative control.
  • mice of the (BALB/c x C57B1) Fi inbred strain were obtained from Jackson Laboratories (Bar Harbor, Maine). Male mice 1-2 months of age (20-22g) were used for the test and were kept for 5 days prior to being used in the assay to insure adequate adjustment to their new environment.
  • the following assay reagents were prepared in advance: Ferricytochrome c type III (Sigma, cat. no. C-2506); tissue culture grade dimethylsulf oxide (DMSO) (Sigma, Cat. No. D2650); phorbol myristate acetate (PMA, or TPA, Sigma, cat. no. P-8139) dissolved to a concentration of 2 mM and serially diluted to 0.2 mM, 200 nM, and 2 nM stocks.
  • DMSO tissue culture grade dimethylsulf oxide
  • PMA phorbol myristate acetate
  • PMA phorbol myristate acetate
  • the test consists of three groups: group I, the negative control, consisting of 10 mice injected with NaHA reference standard dissolved in sterile saline; group II, the positive control, consisting of 3 mice injected with inflammatory (unrefined) NaHA; and group III, the experimental group, consisting of 15 mice injected with test samples.
  • a 1% NaHA solution of the test samples, negative control sample and positive control (inflammatory reference) sample were prepared by dissolving bulk NaHA at a concentration of 11.5 mg/ml saline (to compensate for water and salt in the sample). The same saline solution should be used for dissolution of the negative control and the test sample.
  • test samples negative control and positive control were loaded into 5 ml syringes and sterile 23G needles were attached to the syringes. Mice were injected at about the center of the lower left quadrant of the abdomen by holding the syringe at an -20 degree angle to the skin, towards the chest. Each mouse was injected with 1.0 ml of sample while being careful not to damage internal organs.
  • mice were sacrificed by cervical dislocation, and the peritoneal exudate cells (PEC) by rinsing the peritoneal cavity with 15 ml of ice-cold Hank's balanced salt solution, and collecting the wash in 50 ml centrifuge tubes. The peritoneal cavity was then washed with an additional 15 ml of Hank's solution, which was added to the first wash. The tubes were numbered in the order that the cells were harvested in, and kept on ice at all times.
  • PEC peritoneal exudate cells
  • the exudate was centrifuged at 10°C at 1400 rpm for 10 minutes and the supernatant was discarded.
  • the cell pellets were visually inspected and hemolytic (bloody pellets) were discarded and replaced with PEC from one or more of the reserved mice.
  • the cell pellets were divided from each test group according to size (i.e., tubes containing large cell pellets and small cell pellets). The tubes were further divided into four subgroups (4 mice each), two subgroups of four mice each for the negative control and three subgroups of four mice each for the test samples.
  • the cell pellets were resuspended in 1 ml Hank's salt solution and the cells of each subgroup were pooled in one test tube.
  • the cell pellets of the positive control groups can be pooled together without subdividing.
  • THe samples were centrifuged and gently resuspended in 1 ml of Hank's solution.
  • the resuspended cell pellets were diluted in duplicate to yield samples between 10 4 and 10 5 cells/ml (this required diluting the PEC of the negative control and the test sample between 1 :3 to 1 :10 and those of the positive control between 1 :50 to 1 :200).
  • Each dilution was counted in duplicate with a hemocytometer by counting at least two different fields, with each field containing 16 subdivisions, of the hemocytometer and calculating the cell number per ml by multiplying the mean result by the dilution factor and by 10 4 .
  • ferricytochrome C solution was dissolved to a final concentration of 160 ⁇ M (2 mg/m!) in Hank's buffer.
  • the cells were suspended in Hank's containing 80 ⁇ M freshly dissolved ferricytochrome C at a concentration of 1 x 10 5 cells/ml, and 1 ml aliquots were dispensed, 6 wells per group, into 24-well microtiter plates.
  • PMA was added at concentrations of 0 nM, 2 nM and 200 nM, in a volume of 10 ml per well, using duplicate wells for each concentration.
  • the stock solution of 2 mN PMA in DMSO serves for the serial dilutions and DMSO only is also applied to the wells as a control.
  • the 24-well plates were then placed at 37°C (5% CO 2 /air) incubator for 60 minutes.
  • the reduction of cytochrome C was quantified by measuring the absorbance of each well solution at 550 nM against a blank of Hank's solution containing 80 ⁇ M ferricytochrome C, but without PEC. The results were graphed by plotting the oxidative burst (A 550 n m) as a function of PMA concentration in each culture. The weighted average oxidative burst for each subgroup was calculated by multiplying the oxidative burst at 200 nM PMA measured for each individual test subgroup by the number of mice in that subgroup; the sum of the values was calculated and divided by the total number of mice for all of the subgroups in the group.
  • An acceptable test sample (Ae., one without significant inflammatory activity) met the following requirements: the plots obtained showed the typical shape, i.e., an increase in the As 50 in samples containing PMA; the negative control group had an oxidative burst of ⁇ 0.10 (A 550 ), and the positive control had an oxidative burst of 0.15-0.40; while the oxidative burst of the test sample was ⁇ 0.10 (A 550 ).
  • Example 2 IL-6 Inflammatory Test of Sodium Hvaluronate (NaHA) Bacterial Fermentation Product
  • IL-6 interleukin-6
  • This example summarizes the validation of an ELISA method to determine the absence of inflammation triggering response in NaHA by quantitating interleukin-6 (IL-6) concentrations in cell-free peritoneal fluid supernatant.
  • IL-6 interleukin-6
  • High molecular weight NaHA is reported to be a non-pyrogenic noninflammatory material. Nonetheless, international safety standards for use of NaHA as a medical device or biologic requires demonstration that the NaHA produced is free of inflammatory triggering components.
  • the example demonstrates successful inflammatory activity testing of NaHA prepared by extensive chemical purification of a batch fermentation product of Streptococcus zooepidemicus (see U.S. Pat. Nos. 4,780,414 and 4,784,990).
  • mice Male Balb/c mice, 34-36 days old weighing 20-22 Grams were obtained from Jackson Laboratories (Bar Harbor, Maine). Following receipt, the animals were kept for five days to ensure adequate adjustment to their new environment prior to being used in an inflammatory assay.
  • Group I the negative control, consisted of 10 mice injected with NaHA reference standard dissolved in sterile saline.
  • Group II the positive control consisted of 3 mice injected with a previously characterized inflammatory NaHA preparation.
  • Group III the experimental group, consisted of 15 mice injected with a test lot of purified NaHA product.
  • a 1% solution of NaHA solution of the test sample, negative control sample and positive control (inflammatory reference) sample was prepared by dissolving bulk NaHA at a concentration of 11.5 mg/ml (w/w) in saline by agitating on a shaker for 2-3 days at 4°C. The same saline solution was used for dissolution of the negative control, the positive control and the test samples. The final concentration, 10 mg/ml, was confirmed by measuring a 1 :10 dilution of the final NaHA preparation polarimetrically
  • the positive control an inflammatory NaHA preparation
  • the negative control non-inflammatory NaHA preparation was a preparation that had been previously tested and confirmed free of inflammatory activity by both the oxidative burst (OB) and IL-6 induction assays. All samples were loaded into 5 ml syringes and sterile 23G needles were attached.
  • mice were injected with 1.0 ml of the test sample, positive control or negative control directly into the abdominal cavity, while being careful not to damage internal organs. At six hours post-injection the mice were sacrificed by cervical dislocation or exposure to CO 2 . A lavage from the peritoneal cavity was prepared by flushing with 3 ml of PBS, and the resulting exudate was centrifuged at 1400 rpm for 10 minutes at 10 0 C to remove cells and debris. The supernatants were transferred to new tubes and kept overnight at 2-8°C before measuring IL-6 levels by ELISA.
  • the negative control and test sample supernatants were diluted 10:1 in PBS x10 - BSA 10% (9 parts supernatant with 1 part of PBS x10 - BSA 10%).
  • the positive control supernatant was diluted 1 :2 in PBS followed by dilution 10:1 in PBS x10 - BSA 10% (9 parts supernatant with 1 part of PBS x10 - BSA 10%).
  • IL-6 ELISA's were performed using a commercial kit under conditions prescribed by the manufacturer (BD OptEIA mouse IL-6 ELISA kit, Becton Dickinson Biosciences, San Jose, CA). The wells of a 96-well microtiter plate coated with an IL-6 monoclonal capture antibody were washed with 1x PBS + 0.05% Tween-20 (PBST) and 200 ⁇ l of the test, negative or positive control sample was added to each well. In addition a standard curve was established using 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 pg/ml dilutions of IL-6. The microtiter plate was sealed and incubated for 2 hours at room temperature.
  • PBST 0.05% Tween-20
  • the samples were removed by aspiration, the wells were washed and the amount of IL-6 captured on the surface of the plate was quantitated by adding a streptavidin-horseradish peroxidase (Avidin HRP) conjugate mixed with biotinylated anti-mouse IL-6 antibody to produce antibody-(IL-6 antigen)-antibody "sandwich”.
  • the microtiter wells were washed 7 times before adding 3, 3', 5, 5'- tetramethylbenzidine (TMB) colorimetric indicator in buffered saline to each well.
  • TMB 3, 3', 5, 5'- tetramethylbenzidine
  • the reaction was stopped by adding 100 ml of HRP stop solution per well.
  • the absorbance of the wells was measured using the Multicalc protocol for the detection of IL-6 using BD's kit.
  • a nonparametric Mann-Whitney test was used to statistically analyze the results. Prior to statistical analysis, the mean of IL-6 concentration values was determined and all IL-6 concentration values derived from the ELISA were transformed to In (natural log). The negative control and positive control groups were compared by the Mann-Whitney non-parametric test. A p ⁇ 0.05 value indicates a significant difference between the positive control and the negative control. Next, the positive control and the test sample were compared using the Mann-Whitney nonparametric test. A p ⁇ 0.05 value indicates a significant difference between the positive control and the test sample. Finally, the negative control and the test sample groups were compared by the Mann-Whitney nonparametric test.
  • a p > 0.05 indicates no significant difference between the negative control and the test sample.
  • the test samples were determined to be acceptable based upon the following criteria: the positive control versus the test sample gave a p value ⁇ 0.05, indicating a significant difference between the positive control and the test samples; and both the negative control and the test sample groups are uniformly homogenous and the same ELISA result is obtained, or the difference between the negative control and the test sample provided a p value of > 0.05, which indicates that the difference is not statistically significant.
  • OB test The cell-dependent oxidative burst (OB) test of cells harvested from the peritoneum of NaHA-injected mice was validated against the owl-monkey inflammatory assay.
  • a review of historical test results shows that the OB test method has a narrow signal-to-noise ratio, a tendency to yield hemolytic samples (obtained frequently upon animal handling and which have to be excluded from the test) and is limited to the number of cells withdrawn from individual mouse peritoneum available for OB testing.
  • IL-6 interleukin-6
  • Test results were subjected to statistical analysis by nonparametric Mann-Whitney test; results were tested for statistically significant differences to the positive controls. Animal handling, NaHA administration, and peritoneal fluid withdrawal remained unchanged in the modified test procedure. This example describes how comparability between the two test methods was determined in order to support the replacement of the OB method by the IL-6 ELISA method for determination of the inflammation response in NaHA batches.
  • Tables 1 and 2 summarize the modified inflammation assay (IL-6 ELISA method) performance and results.
  • Table 1 shows the values of IL-6 concentrations (transformed to the natural logarithm (In)) in peritoneal fluid of each mouse. Most of the negative control and test sample values were below the minimal detection concentration (MDC) and therefore are noted as the MDC value.
  • Table 2 shows the statistical analysis of the comparison between the positive control (PC) and negative control (NC) and test samples (TS), and NC vs. TS. Significant differences were obtained between the PC and NC and between the PC and TS, while no statistically significant differences were obtained between the PC and NC and between the PC and TS. No statistically significant differences were observed between the NC vs. TS samples. All test batches met the IL-6 ELISA method requirements.
  • a standard operating procedure for testing bacterially fermented batches of purified HA for use in synovial fluid supplementation utilizes 8 animals for the negative control, 8 animals for the test sample and 5 animals for the positive control.

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Abstract

Une activité inflammatoire contaminant un dispositif médical ou un agent biologique peut grandement affecter à la fois sa sécurité et son efficacité. La présente invention concerne des procédés de détection de l'activité inflammatoire dans un échantillon test en administrant l'échantillon test à un animal et en détectant le niveau d'une cytokine pro-inflammatoire ou d'un autre médiateur de l'inflammation dérivé de cellules. Des niveaux élevés de cytokines pro-inflammatoires telles que l'IL-I, l'IL-6, la TNF-α, ou l'IFN-γ, ou d'autres médiateurs de l'inflammation dérivés de cellules tels que des granules de lysosomes, l'histamine, l'IL-S, le leukotriène B4, l'oxyde nitrique, ou la prostaglandine E2 (PGE2), indiquent que l'échantillon test possède une activité inflammatoire. L'activité inflammatoire peut être due à un quelconque facteur pro-inflammatoire, tel qu'une endotoxine bactérienne (par exemple, un lipopolysaccharide) ou un pyrogène sans endotoxine (par exemple, un mucopeptide de streptocoque).
PCT/IB2008/003063 2007-11-14 2008-11-14 Procédé de détection de l'activité inflammatoire en utilisant l'il-6 Ceased WO2009063307A2 (fr)

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CN103969438A (zh) * 2014-04-29 2014-08-06 北京普恩光德生物科技开发有限公司 白介素6检测试剂盒
CN110672857A (zh) * 2019-10-10 2020-01-10 四川大学华西第二医院 一种TNF-α的高敏Elisa检测试剂盒及其使用方法与应用
WO2020160040A1 (fr) * 2019-01-31 2020-08-06 Fresenius Medical Care Holdings, Inc. Diagnostic rapide de péritonite chez des patients en dialyse péritonéale
WO2022253173A1 (fr) * 2021-05-31 2022-12-08 Fresenius Medical Care Deutschland Gmbh H-ngal pour la détection de la péritonite

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EP1234181A4 (fr) * 1999-12-03 2005-01-26 Baxter Int Essai de pyrogenicite a utiliser avec des systemes de dosage immunologique automatises
WO2002066978A2 (fr) * 2000-12-29 2002-08-29 Reddy Us Therapeutics, Inc. Methodes et compositions de detection de composes modulateurs de reponses inflammatoires
EP1537423B1 (fr) * 2002-09-09 2006-05-31 Medifact-Publishing GmbH Procede de detection locale de mediateurs d'inflammation et d'allergie
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WO2020160040A1 (fr) * 2019-01-31 2020-08-06 Fresenius Medical Care Holdings, Inc. Diagnostic rapide de péritonite chez des patients en dialyse péritonéale
CN113383234A (zh) * 2019-01-31 2021-09-10 费森尤斯医疗保健控股公司 在腹膜透析患者中快速诊断腹膜炎
US11280788B2 (en) 2019-01-31 2022-03-22 Fresenius Medical Care Holdings, Inc. Rapid diagnosis of peritonitis in peritoneal dialysis patients
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WO2022253173A1 (fr) * 2021-05-31 2022-12-08 Fresenius Medical Care Deutschland Gmbh H-ngal pour la détection de la péritonite

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