US20040235080A1 - Methods and reagents for detecting endotoxin - Google Patents

Methods and reagents for detecting endotoxin Download PDF

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US20040235080A1
US20040235080A1 US10/480,254 US48025404A US2004235080A1 US 20040235080 A1 US20040235080 A1 US 20040235080A1 US 48025404 A US48025404 A US 48025404A US 2004235080 A1 US2004235080 A1 US 2004235080A1
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Lin Chen
Michael Pepe
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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
    • G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS

Definitions

  • the invention relates to reagents and methods for detecting endotoxin.
  • Gram negative bacterial endotoxin is a widespread contaminant of a variety of materials, such as water, food, pharmaceutical products, and parenteral preparations.
  • the most commonly used tests for endotoxin contamination employ amebocyte lysates derived from horseshoe crab hemolymph. As populations of these animals decreases, however, it becomes increasingly important to develop rapid and reliable methods for detecting endotoxin that do not rely on the availability of horseshoe crab hemolymph.
  • the invention provides reagents and methods for detecting endotoxin.
  • One embodiment of the invention is a reagent for detecting endotoxin, comprising a purified horseshoe crab Factor C protein and a surfactant.
  • the surfactant can be an amphoteric surfactant represented by the following formulae:
  • R 1 is an alkylene radical having from 1 to 4 carbon atoms
  • Y and Y′ are each (1) hydrogen, (2) lower alkyl, or (3) hydroxy lower alkyl
  • R 2 and R 3 are each (1) lower alkyl or (2) hydroxy lower alkyl
  • n is 0 or 1, when n is 0, R 4 is alkyl containing from about 8 to about 18 carbon atoms; when n is 1, R 4 is an alkylene radical having from 1 to about 6 carbon atoms;
  • R 5 is an allyl containing from about 8 to about 18 carbon atoms;
  • M is hydrogen, sodium, potassium or ammonium.
  • the surfactant can be an anionic surfactant represented by the following formulae:
  • R 5 , Y, and M have the same meaning as set forth above;
  • R 6 is an alkyl from 8 to 24 carbon atoms;
  • n1 is an integer from 1 to 3;
  • n2 is 1 or 2; and
  • Ar is phenyl or naphthyl.
  • the surfactant can be a cationic surfactant represented by the following formula:
  • the surfactant can be a nonionic surfactant represented by the following formula:
  • the surfactant can be those nonionic surfactants selected from the group consisting of the condensation product of about 10 to 30 moles of ethylene oxide with the monoester of a hexahydric alcohol containing 6 carbon atoms with the ester group containing 10 to 20 carbon atoms.
  • Even another embodiment of the invention is a method of detecting endotoxin in a test sample.
  • a test sample is contacted with (1) a reagent comprising (a) a purified horseshoe crab Factor C protein and (b) a surfactant to form a test sample-reagent mixture and (2) a Factor C substrate to form a contacted test sample. Cleavage of the Factor C substrate generates a detectable signal.
  • the contacted test sample is assayed for the presence or absence of the detectable signal. An amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
  • a further embodiment of the invention is a method of detecting endotoxin in a test sample.
  • a test sample is contacted with (1) a reagent comprising (a) a recombinant Carcinoscorpius rotundicauda Factor C protein and (b) a surfactant and (2) N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin to form a contacted test sample.
  • the Factor C protein is made by the method of culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supernatant.
  • the contacted test sample is assayed for the presence or absence of a fluorescent signal. An amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
  • Another embodiment of the invention is a method of detecting endotoxin in a test sample.
  • a test sample is contacted with a reagent comprising a purified horseshoe crab Factor C protein and a surfactant as described above to form a test sample-reagent mixture.
  • the test sample-reagent mixture is contacted with a Factor C substrate, wherein cleavage of the Factor C substrate generates a detectable signal.
  • the contacted test sample-reagent mixture is assayed for the presence or absence of the detectable signal.
  • An amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
  • Even another embodiment of the invention is a method of detecting endotoxin in a test sample.
  • a test sample is contacted with a reagent comprising a recombinant Carcinoscorpius rotundicauda Factor C protein and a surfactant.
  • the Factor C protein is made by the method of culturing a host cell comprising a vector encoding the Factor C protein in a supernatant under conditions such that the Factor C protein is expressed into the supernatant.
  • the test sample-reagent mixture is contacted with N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin.
  • the contacted test sample-reagent mixture is assayed for the presence or absence of a fluorescent signal.
  • An amount of the fluorescent signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
  • kits for detecting endotoxin comprises a reagent that comprises (a) a purified horseshoe crab Factor C protein and (b) a surfactant as described above, together with instructions for a method of detecting endotoxin in a test sample.
  • the method comprises the steps of (1) contacting a test sample with a reagent comprising (a) a purified horseshoe crab Factor C protein and (b) a surfactant as described above to form a test sample-reagent mixture; (2) contacting the test sample-reagent mixture with a Factor C substrate, wherein cleavage of the Factor C substrate generates a detectable signal; and (3) assaying the contacted test sample-reagent mixture for the presence or absence of the detectable signal, wherein an amount of the detectable signal that is increased relative to a control sample that does not contain endotoxin indicates a presence of endotoxin in the test sample.
  • FIG. 1 Graph showing effect of Zwittergent 3-14 and Tween 20 on recombinant Factor C activity.
  • FIG. 2 Graph showing effect of Zwittergent 3-14 and Tween 80 on recombinant Factor C activity.
  • FIG. 3 Graph showing effect of Zwittergent 3-14 and Triton X-114 on recombinant Factor C activity.
  • FIG. 4 Plot showing endotoxin detection using a DPR(N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl)-coumarin substrate.
  • FIG. 5 Plot showing endotoxin detection using a VPR(N-t-BOC-Val-Pro-Arg-7-Amido-4-methyl)-coumarin substrate.
  • FIG. 6 Graph showing Limulus Factor C activity at different Zwittergent concentrations.
  • FIG. 7. Graph showing endotoxin sensitivity in the presence and absence of surfactant.
  • FIG. 8 Graph showing results of a one hour, one-step endotoxin assay, using DPR-coumarin as a substrate.
  • FIG. 9 Graph showing effect of Zwittergent 3-14 and octylthiolglycoside on recombinant Factor C activity.
  • FIG. 10 Graph showing effect of Zwittergent 3-14 and Genapol C-100 on recombinant Factor C activity.
  • FIG. 11 Graph showing effect of Zwittergent 3-14 and IX-100 on recombinant Factor C activity.
  • the invention is a reagent for detecting endotoxin and a method of using the reagent.
  • the reagent comprises a purified horseshoe crab Factor C protein and a surfactant.
  • This reagent can be used in conjunction with a substrate for Factor C that, upon cleavage, generates a detectable signal to detect endotoxin in a test sample.
  • the presence of the surfactant enhances the activation of the purified Factor C by endotoxin by as much as 3-7-fold, permitting more rapid and sensitive measurement of endotoxin levels in a test sample.
  • the reagent preferably contains a recombinant Factor C, thus eliminating the need for a continuous supply of horseshoe crab hemolymph.
  • One component of the reagent of this invention comprises a purified horseshoe crab Factor C protein.
  • Purified native Factor C from any of the four known horseshoe crab species, Limulus polyphemus, Carcinoscorpius rotundicauda, Tachypleudus tridentata , or Tachypleudus gigas , can be used in the practice of the invention.
  • the native Factor C can be purified biochemically or purified Factor C can be produced recombinantly.
  • “Purified Factor C” as used herein means a composition of a Factor C protein as defined hereinafter that contains less than 30% by weight of non-Factor C native amebocyte lysate components from any of the four known horseshoe crab species.
  • the composition specifically includes a cell culture supernatant which comprises recombinant Factor C protein (see below).
  • Methods for purifying horseshoe crab Factor C from its native source are known and are disclosed, for example, in Nakamura et al., Eur. J. Biochem. 154, 511-21, 1986; Navas et al., Biochem. Intl. 21, 805-13, 1990; Tokunaga et al., J. Biochem.
  • the purified Factor C is recombinantly produced.
  • Amino acid sequences for native Factor C from Tachypleus tridentata and Carcinoscorpius rotundicauda are known, as are the naturally occurring coding sequences for these proteins.
  • SEQ ID NOS:1 and 3 provide coding sequences for the Tachypleus tridentata Factor C amino acid sequences shown in SEQ ID NOS:2 and 4, respectively.
  • SEQ ID NOS:5 and 7 provide coding sequences for the Carcinoscorpius rotundicauda Factor C amino acid sequences shown in SEQ ID NOS:6 and 8, respectively. Because of the degeneracy of the genetic code, many other sequences can be envisioned that will encode each of these native Factor C proteins, and the invention specifically contemplates use of any of these coding sequences to produce a purified Factor C.
  • the invention also encompasses use of purified naturally and non-naturally occurring, i.e., recombinantly produced, Factor C variants, provided that the variants retain a Factor C enzyme activity.
  • Factor C enzyme activity can be assessed using any assay for Factor C enzyme activity known in the art. See, e.g., Tokunaga et al., J. Biochem. 109, 150-57 (1991) and Nakamura et al., Eur. J. Biochem. 176, 89-94, 1988.
  • the endotoxin assays described in Examples 1 and 13, below, also can be used.
  • Naturally occurring Factor C variants include, for example, products of Factor C mRNA splice variants or mutated Factor C genes.
  • Non-naturally occurring Factor C variants can be constructed using base substitutions, additions, or deletions to produce proteins having Factor C activity.
  • Non-naturally occurring Factor C variants can differ from naturally occurring Factor C by as much as 50, 75, 80, 85, 90, 95, 97, 98, or 99%, as determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes). Fragments of native and recombinantly produced Factor C that retain Factor C activity also can be used in the practice of the invention.
  • “Factor C” as used herein includes naturally and non-naturally occurring proteins and protein fragments that have the properties described above.
  • Methods of producing proteins recombinantly are well known in the art and generally involve culturing a host cell comprising an expression vector encoding the Factor C protein in a supernatant under conditions such that the protein is expressed.
  • the expressed protein can be recovered or, preferably, the supernatant comprising the expressed protein is used directly as the source of recombinant Factor C.
  • purified Factor C specifically includes a supernatant which comprises recombinant Factor C.
  • Host cells useful for the production of recombinant Factor C include, without limitation, yeast cells and insect cells. Recombinant production of Carcinoscorpius rotuidicauda Factor C in Pichia pastoris and Saccharomyces cerevisiae host cells is specifically disclosed in U.S. Pat. No. 5,985,590.
  • a particularly preferred method of obtaining recombinant Factor C is to produce the protein in a baculovirus system, as described in Examples 2-5. Briefly, a Factor C coding sequence is cloned into pFASTBAC1. The resultant recombinant plasmid is transformed into DH10BAC competent cells that contain a bacmid with a mini-attTn7 target site and a helper plasmid. In the presence of transposition proteins provided by the helper plasmid, the mini-Tn7 transposable element on the pFASTBAC plasmid can transpose to the mini-attTn7 target site on the bacmid.
  • Colonies containing recombinant bacmids are identified by disruption of the lacZa gene.
  • High molecular weight DNA is prepared from selected DH10BAC clones containing the recombinant bacmid. This DNA is then used to transfect S9 insect cells, which will then secrete recombinant Factor C.
  • a particularly surprising discovery of the present invention is that culture medium containing the secreted Factor C, which has been separated from the cultured cells, can be used together with a surfactant (described below) as a reagent in endotoxin assays without further purification.
  • Reagents of the invention also comprise a surfactant.
  • Surfactants useful in the practice of the invention include those described in U.S. Pat. No. 4,322,217, although other surfactants also can be used.
  • Useful surfactants include amphoteric surfactants that contain both an anionic and cationic group in their structure. Illustrative are the sulfobetaines represented by Formula A:
  • R 1 is an alkylene radical having from 1 to about 4 carbon atoms
  • Y is any non-deleterious, chemically suitable substituent including (1) hydrogen, (2) substituted or unsubstituted lower alkyl, e.g., containing 1 to 4 carbon atoms such as methyl, ethyl, propyl, or hydroxy etc.;
  • R 2 and R 3 are each selected from substituted or unsubstituted lower alkyl containing 1 to 4 carbon atoms, e.g., such as methyl, ethyl, propyl, hydroxy ethyl, hydroxy methyl, hydroxy propyl, etc.
  • n 0, or 1
  • R 4 is substituted or unsubstituted alkyl, e.g., containing about 8 to about 18 carbon atoms, and
  • R 4 is an alkylene radical having from about 1 to about 6 carbon atoms
  • R 5 is a substituted or unsubstituted alkyl, e.g., containing about 8 to about 18 carbon atoms.
  • alkylene encompasses both polymethylene radicals and other divalent saturated aliphatic radicals. Thus, there may be branching in the linkage provided by the alkylene radical.
  • lower means a radical containing 1 to 4 carbon atoms.
  • Sulfobetaines that can be used in the reagent of the present invention are known in the art and have been described as zwitterionic surfactants. The preparation of such compounds is described for example, in Fernley, J. Am. Oil Chem. Soc. 55, 98-103 (1978) and U.S. Pat. No. 3,280,179.
  • R 2 and R 3 in the above structure are methyl. It is also preferred that R 1 be propylene.
  • R 4 is an alkyl radical having from about 8 to 18 carbon atoms, preferably a straight chain alkyl radical.
  • R 4 is an alkyl radical having from about 8 to 18 carbon atoms, preferably a straight chain alkyl radical.
  • R 4 component is tallow fatty alcohol, which consists of a mixture of various chain lengths, with a typical composition being approximately 66 percent C 18 , 30 percent C 16 , and 4 percent C 14 and others.
  • Another convenient source is the middle cut of distilled coconut fatty alcohol, which also consists of a mixture of various chain lengths, with a typical composition being approximately 66 percent C 12 , 23 percent C 14 , 9 percent C 16 and 2 percent C 10 .
  • sulfobetaine surfactants of the above structure wherein n equals 0 are set forth in U.S. Pat. No. 3,539,521.
  • a particularly preferred surfactant of this type is N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, which is commercially available from Calbiochem-Behring Corporation under the trademark ZWITTERGENT 3-14.
  • Another type of useful sulfobetaine surfactant has the above structure wherein n equals 1 and R 4 is an alkylene radical having from about 1 to about 6 carbon atoms.
  • R 5 is an alkyl radical having from about 8 to about 18 carbon atoms. It is preferred that R 5 be straight chain.
  • convenient sources of alkyl radicals having from about 10 to about 18 carbon atoms are tallow fatty alcohol and coconut fatty alcohol.
  • Specific sulfobetaine surfactants of the above structure wherein n equals 1 are set forth in U.S. Pat. No. 3,280,179.
  • Particularly preferred sulfobetaine surfactants are 3-(N,N-dimethyl-N-acylamidopropylammonio)-2-hydroxy-propane-1-sulfonates, wherein the acyl group is derived from tallow fatty alcohol or coconut fatty alcohol, with coconut fatty alcohol preferred. It will be recognized by those skilled in the art that, in the normal preparation of these derivatives of tallow or coconut fatty alcohols, a mixture of sulfobetaines with varying carbon chain lengths for the acyl groups will result.
  • these fatty alcohols contain, for the most part, carbon chain lengths that will provide acyl groups with the desired number of carbon atoms, i.e., from about 8 to about 18 carbon atoms.
  • these mixtures obtained from tallow or coconut fatty alcohols are useful in providing the sulfobetaine surfactant for reagents of the present invention.
  • a material of this type particularly preferred for use in reagents of the present invention is N-cocoamido-propyl-N,N-dimethyl-N-2-hydroxypropyl sulfobetaine.
  • An example of this is LONZAINE CS, which is commercially available from Lonza, Inc., Fair Lawn, N.J.
  • amphoteric surfactants include the N-long chain alkyl aminocarboxylic acids illustrated by Formula B:
  • N-long chain alkyl iminodicarboxylic acids illustrated by Formula C are N-long chain alkyl iminodicarboxylic acids illustrated by Formula C:
  • R 1 , R 2 , R 3 , R 4 , Y, and n have the same meaning as they have in Formula A
  • M is hydrogen or a salt-forming metal
  • Y′ has the same meaning as Y in Formula A.
  • Y and Y′ may be the same or different.
  • amphoteric detergents are N-alkylbeta-aminopropionic acid, N-alkyl-beta-iminodipropionic acid, and N-alkyl-N,N-dimethyl glycine;
  • the alkyl group may be, for example, that derived from coco fatty alcohol, lauryl alcohol, myristyl alcohol (or a lauryl-myristyl mixture), hydrogenated tallow alcohol, cetyl, stearyl, or blends of such alcohols.
  • the substituted aminopropionic and iminodipropionic acids are often supplied in the sodium or other salt forms, which also can be used in reagents of the invention.
  • cocobetaine sold by Witco Chemical Corporation under the name EMCOL CC 37-18
  • cocoamidopropyl betaine sold by Lonza Inc. under the name LONZAINE CO
  • disodium N-tallow-beta-iminodipropionate sold by Henkel Corporation under the name of DERIPHAT 160.
  • amphoteric detergents are the fatty imidazolines such as those made by reacting a long chain fatty acid (e.g., of 10 to 20 carbon atoms) with diethylene triamine and monohalocarboxylic acids having 2 to 6 carbon atoms, e.g. 1-coco-5-hydroxyethyl-5-carboxymethylimidazoline.
  • fatty imidazolines such as those made by reacting a long chain fatty acid (e.g., of 10 to 20 carbon atoms) with diethylene triamine and monohalocarboxylic acids having 2 to 6 carbon atoms, e.g. 1-coco-5-hydroxyethyl-5-carboxymethylimidazoline.
  • Specific examples include cocoimidazoline, which is commercially available under the name AMPHOTERGE K-2 from Lonza, Inc., and capric dicarboxy imidazoline, which is commercially available under the name AMPHOTERGE KJ-2 from Lonza, Inc.
  • useful surfactants include anionic synthetic surfactants, generally described as those compounds that contain hydrophilic and lipophilic groups in their molecular structure and ionize in an aqueous medium to give anions containing both the lipophilic group and hydrophilic group.
  • anionic synthetic surfactants generally described as those compounds that contain hydrophilic and lipophilic groups in their molecular structure and ionize in an aqueous medium to give anions containing both the lipophilic group and hydrophilic group.
  • the alkyl aryl sulfonates, the alkane sulfates, and sulfated oxyethylated alkyl phenols are illustrative of the anionic type of surface active compounds.
  • alkyl aryl sulfonates are a class of synthetic anionic surface active agents represented by Formula E:
  • R 6 is a straight or branched chain hydrocarbon radical having from about 1 to about 24 carbon atoms, at least one R 6 having at least 8 carbon atoms; n1 is from 1 to 3; n2 is from 1 to 2; Ar is a phenyl or a naphthyl radical, and Y and M have the same meaning as in Formula B.
  • R 6 can be, for example, methyl, ethyl, hexyl, octyl, tetraoctyl, iso-octyl, nonyl, decyl, dodecyl, octadecyl, and the like.
  • Compounds illustrative of the alkyl aryl sulfonates include sodium dodecylbenzene sulfonate, sodium decylbenzene sulfonate, ammonium methyl dodecylbenzene sulfonate, ammonium dodecylbenzene sulfonate, sodium octadecylbenzene sulfonate, sodium nonylbenzene sulfonate, sodium dodecylnaphthalene sulfonate, sodium hetadecylbenzene sulfonate, potassium eicososyl naphthalene sulfonate, ethylamine undecylnaphthalene sulfonate and sodium docosylnaphthalene sulfonate.
  • alkyl sulfates are a class of synthetic anionic surface active agents represented by Formula F:
  • R 5 and M have the same meaning as in Formula B.
  • Compounds illustrative of alkyl sulfate class of anionic surfactants include sodium octadecyl sulfate, sodium hexadecyl sulfate, sodium dodecyl sulfate, sodium nonyl sulfate, ammonium decyl sulfate, potassium tetradecyl sulfate, diethanolamino octyl sulfate, triethanolamine octadecyl sulfate, and ammonium nonyl sulfate.
  • the sulfated oxyethylated alkylphenols are a class of synthetic anionic surface active agents represented by Formula G:
  • A is either oxygen, sulfur, a carbonamide group, a thiocarbonamide group, a carboxylic group, or a thiocarboxylic ester group
  • z is an integer from 3 to 8
  • R 5 and M have the same meaning as in Formula B.
  • Compounds illustrative of the sulfated oxyethylated alkyl phenol class of anionic surfactants include ammonium nonylphenoxyl tetraethylenoxy sulfate, sodium dodecylphenoxy triethyleneoxy sulfate, ethanolamine decylphenoxy tetraethyleneoxy sulfate, and potassium octylphenoxy triethyleneoxy sulfate.
  • nonionic surface active compounds which can be broadly described as compounds that do not ionize but acquire hydrophilic characteristics from an oxygenated side chain, such as polyoxyethylene; the lipophilic part of the molecule may come from fatty acids, phenol, alcohols, amides, or amines.
  • the compounds are usually made by reacting an alkylene oxide, such as ethylene oxide, butylene oxide, propylene oxide and the like, with fatty acids, straight or branched chain alcohols containing one or more hydroxyl groups, phenols, thiophenols, amides, or amines to form polyoxyalkylene glycoethers and esters, polyoxyalkylene alkylphenols, polyoxyalkylene thiophenols, polyoxyalkylene amides and the like. It is generally preferred to react from about 3 to about 30, more preferably 10 to 30, moles of alkylene oxide per mole of the fatty acids, alcohols, phenols, thiophenols, amides, or amines.
  • an alkylene oxide such as ethylene oxide, butylene oxide, propylene oxide and the like
  • fatty acids straight or branched chain alcohols containing one or more hydroxyl groups, phenols, thiophenols, amides, or amines to form polyoxyalkylene glycoethers
  • nonionic surfactants are the products obtained from the reaction of alkylene oxide with an aliphatic alcohol having from 8 to 18 carbon atoms, such as octyl, nonyl, decyl, octadecyl, dodecyl, tetradecyl and the like, with monoesters of hexahydric alcohols, the ester group containing 10 to 20 carbon atoms such as sorbitan monolaureate, sorbitan monooleate and sorbitan monopalmitate, with an alkyl phenol in which the alkyl group contains between 4 and 20 carbon atoms, such as butyl, dibutyl, amyl, octyl, dodecyl, tetradecyl, and the like, or with an alkyl amine in which the alkyl group contains between 1 to 8 carbon atoms.
  • an aliphatic alcohol having from 8 to 18 carbon atoms such as octyl, nonyl
  • Compounds illustrative of synthetic nonionic surfactants include the products obtained from condensing ethylene oxide or propylene oxide with the following: propylene glycol, ethylene diamine, diethylene glycol, dodecyl phenol, nonyl phenol, tetradecyl alcohol, N-octadecyl diethanolamide, N-dodecyl monoethanolamide, polyoxyethylene (20) sorbitan monooleate sold under the name TWEEN 80 and polyoxyethylene (20) sorbitan monolaurate sold under the name TWEEN 20.
  • nonionic surfactants include long chain tertiary amine oxides corresponding to Formula H:
  • R 5 has the same meaning as in Formula A
  • R 7 and R 8 are each methyl or ethyl radicals.
  • the arrow in the formula is a conventional representation of a semi-polar bond.
  • amine oxides suitable for use in this invention include dimethyldodecylamine oxide, dimethyloctylamine oxide, dimethyldecylamine oxide, dimethyltridecylamine oxide, and dimethylhexadecylamine oxide.
  • Cationic surface active agents can also be used as surfactants. Such agents are those surface-active compounds which contain an organic hydrophobic group and a cationic solubilizing group. Typical cationic solubilizing groups are amine and quaternary groups. Such cationic surface-active agents are represented by Formula I:
  • R 5 , Y, and Y′ have the same meaning as in Formula C.
  • Suitable synthetic cationic surfactants include the diamines such as those of Formula J:
  • R is an alkyl group of about 12 to 22 carbon atoms, such as N-2-aminoethyl stearyl amine and N-2-aminoethyl myristyl amine; amide-linked amines such as those of Formula K:
  • N-2-amino ethylstearyl amide and N-amino ethyl myristyl amide such as N-2-amino ethylstearyl amide and N-amino ethyl myristyl amide; quaternary ammonium compounds wherein typically one of the groups linked to the nitrogen atom are alkyl groups which contain 1 to 3 carbon atoms, including such 1 to 3 carbon alkyl groups bearing inert substituents, such as phenyl groups and there is present an anion such as halogen, acetate, methylsulfate, etc.
  • Typical quaternary ammonium compounds are ethyl-dimethylstearyl ammonium chloride, benzyl-dimethyl-stearyl ammonium chloride, benzyldimethyl-stearyl ammonium chloride, trimethyl stearyl ammonium chloride, trimethylcetyl ammonium bromide, dimethylethyl dilaurylammonium chloride, dimethyl-propyl-myristyl ammonium chloride, and the corresponding methosulfates and acetates.
  • Another suitable cationic surfactant is represented by Formula L:
  • R 5 has the same meaning as in Formula A and each a is an integer from 1 to 15.
  • An example is the polyethylene glycol amine of hydrogenated tallow wherein R 5 represents the tallow radical and a+a has an average value of 5.
  • Factor C-surfactant reagent examples include Triton X-100, Triton X-114, octyl-beta-D-thioglucoside (OTG, Amresco #J575), Genapol C-100 (an alkyl polyoxyethylene C12E10; Calbiochem #345794), Tween 20, and Tween 80.
  • purified Factor C and a surfactant are combined in an aqueous solution.
  • a suitable buffer solution for this purpose contains 30 mM Tris, pH 8.0, 30 mM NaCl, and 0.3% lactose.
  • the concentration of purified Factor C in a reagent of the invention preferably ranges from 0.03-3 ⁇ g/ml.
  • the concentration of surfactant in the reagent of the invention preferably ranges from 0.001-0.003%.
  • Optimum concentrations of purified Factor C and of the surfactant will vary, depending, for example, on the purity of the Factor C and the particular surfactant. Optimum concentrations of purified Factor C and of surfactant can be determined using routine testing. In the assay exemplified in Examples 1 and 13, the optimal concentration of the surfactant ZWITTERGENT 3-14 is 0.0025%.
  • the reagent described above can be used in an assay to detect endotoxin in a test sample.
  • the test sample can be any sample in which it would be useful to detect endotoxin, including water, aqueous solutions such as buffers, pharmaceutical preparations (e.g., vaccines, intravenous fluids, drug preparations), biomedical imaging reagents (e.g., dyes, radioactive solutions), enzyme preparations (e.g., collagenase), tissue culture media, beverages, blood, urine, cerebrospinal fluid, lymph, serum, and solutions formed by incubating water or an aqueous solution with a solid sample, such as a foodstuff or a surgical glove.
  • pharmaceutical preparations e.g., vaccines, intravenous fluids, drug preparations
  • biomedical imaging reagents e.g., dyes, radioactive solutions
  • enzyme preparations e.g., collagenase
  • tissue culture media beverages, blood, urine, cerebrospinal fluid, lymph,
  • Either a two-step or a one-step assay can be performed.
  • a test sample is incubated with a reagent containing purified Factor C and a surfactant, then a Factor C substrate that will generate a detectable signal upon cleavage is added.
  • a Factor C substrate that will generate a detectable signal upon cleavage is added.
  • a one-step assay Example 13
  • endotoxin, the reagent containing purified Factor C and a surfactant, and a substrate are added together.
  • Substrates that will generate a fluorescent signal upon cleavage are particularly preferred, such as N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin (“DPR,” Bachem 1-1560.0050) and N-t-BOC-Val-Pro-Arg-7-Amido-4-methyl coumarin (“VPR,” Bachem 1-1120.0050).
  • DPR N-t-BOC-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin
  • VPR N-t-BOC-Val-Pro-Arg-7-Amido-4-methyl coumarin
  • Concentrations of DPR generally range from about 10-100 mM, 25-100 mM, 5-100 mM, 75-100 mM, 25-75 mM, 50-75 mM, 25-50 mM, or 50-75 mM.
  • Concentration of VPR generally range from about 50-200 mM, 50-150 mM, 50-100
  • the optimum concentration for a particular substrate varies.
  • the optimum concentration for DPR is 50 mM
  • the optimum concentration for VPR is 100 mM.
  • the particular substrate can be chosen depending on the endotoxin levels expected to be present in the test sample.
  • the VPR substrate has a better linear range at higher endotoxin concentrations
  • the DPR substrate has a better linear range at lower endotoxin concentrations.
  • DPR can be used in assays in which higher sensitivity is desired, whereas VPR is the preferred substrate when higher endotoxin levels are expected to be present and a lower sensitivity is required.
  • Fluorescence can be measured using any means known in the art. If a fluorimeter is used with the DPR or VPR substrates described above, the excitation is set at 360, 380, 390, or 395 nm (slit width of 540 nm) and the emission is measured at 440 or 460 nm (slit width of 2.5-40 nm). Measurement can be either qualitative or quantitative, by reference to a standard endotoxin concentration curve.
  • the assay can be carried out in a clear vessel, such as a glass or polystyrene tissue culture plate, or can be carried out in a black vessel (e.g., a black 96-well microplate). If desired, the assay can be adapted for high-throughput screening of multiple samples using, for example, 96-well microtiter plates.
  • the invention also provides a kit for use in detecting endotoxin.
  • the kit comprises a purified horseshoe crab Factor C protein and a surfactant, as described above. Instructions for using the reagent to detect endotoxin can be included. Kits also can include a Factor C substrate for use in the assay.
  • Endotoxin was pre-incubated with culture medium containing recombinant Carcinoscorpius rotundicauda Factor C obtained from recombinant Factor C-producing Sf9 cells for 1 hour in assay buffer (30 mM Tris, pH 8.0, 30 mM NaCl, 0.3% lactose, and 0.0025% Zwittergent 3-14).
  • assay buffer (30 mM Tris, pH 8.0, 30 mM NaCl, 0.3% lactose, and 0.0025% Zwittergent 3-14.
  • the substrate N-t-Boc-Asp(Obzl)-Pro-Arg-7-Amido-4-methyl coumarin (DPR-coumarin) was added to the mixture to a final concentration of 50 mM.
  • the fluorescence generated from the cleavage of the substrate by activated Factor C was measured after 15-20 minutes. The results are shown in FIG. 4. Results of a similar assay carried out using the substrate N-t-Bo
  • Sf9 cells are seeded at a density of 5 ⁇ 10 6 per 35 mm well in a 6-well tissue culture plate in insect cell culture medium (ICCM) containing 50 U/ml penicillin and 50 mg/ml streptomycin.
  • ICCM insect cell culture medium
  • Insect-Xpress BioWhittaker Cat. #04-10270
  • Sf-900 SFM are suitable media; however, any comparable medium in which SF9 cells grow can be used.
  • the plates are incubated at 27° C. for 1 hour to allow the cells to settle.
  • the adhered Sf9 cells are washed gently with 2 ml ICCM (without antibiotics).
  • One ml of CELLFECTIN-DNA complex is added to each well and incubated at 27° C. for 5 hours.
  • the transfection mixtures are removed completely, and 2 ml of ICCM containing antibiotics is added.
  • the cell culture supernatant is harvested.
  • the supernatant is clarified by centrifugation at 5000 rpm for 10 minutes in a SIGMA 3K10 swing-out rotor, Nr. 11133.
  • the supernatant, containing the recombinant baculovirus is stored at 4° C.
  • Virus amplification can be done with Sf9 cells either in a monolayer or in suspension.
  • the medium is decanted from a culture of 80% confluent one day-old Sf9 cells in a 75 cm 2 tissue culture flask.
  • the cell monolayer is infected with 1 ml of virus (Example 2) using an MOI of 0.1-1.
  • the virus stock is sterile-filtered using a Millipore GV millex filter (yellow; low-protein binding).
  • volume ⁇ ⁇ of ⁇ ⁇ inoculum MOI ⁇ total ⁇ ⁇ cell ⁇ ⁇ number virus ⁇ ⁇ titer ⁇ ⁇ in ⁇ ⁇ pfu ⁇ / ⁇ ml
  • the volume of the inoculum is 0.5 ml.
  • the volume of the inoculum is adjusted to 1 ml with ICCM before it is introduced to the cells.
  • the flask is rocked several times to ensure that the cell monolayer is completely covered by the 1 ml viral inoculum.
  • the flask is then incubated at 27° C. for one hour without rocking. After the incubation, the flask is placed upright, and 14 ml of fresh ICCM is added. The flask is then incubated for 3 days at 27° C.
  • the culture supernatant is harvested into sterile pyrogen-free tubes and centrifuged at 2000 rpm for 10 minutes at 4° C. using a Sigma 3K10 swing out rotor.
  • the viral stock is stored at 4° C.
  • the viral stock is placed at ⁇ 80° C.
  • Virus titer (Plaque Forming Units (PFU) per ml) can be determined with plaque assay, end-point dilution, or other viral titer kits (e.g., BacPAK Baculovirus Rapid Titer Kit by Clontech #K1599-1). Plaque assay is performed in immobilized monolayer culture. Plaque assays to be are performed are determined as follows:
  • Plaque assay number Number of viral dilution ⁇ 4 (quadruplicates)+2 positive and 2 negative controls.
  • 10 5 , 10 6 , 10 7 , 10 8 for viral dilution each dilution in quadruplicate wells, would be a 16-well plaque assay. Include two positive controls and two negative controls in the plaque assay, for a total of 20 wells.
  • [0104] Prepare 10-fold serial viral dilutions using 4.5 ml media and 0.5 ml of viral stock in sterile 15-ml tubes. Aspirate the medium from each well. Add viral dilution 1 ml per well and incubate at 27° C. for 2 hours.
  • Virus titer is calculated using the following formula:
  • High viability Sf9 cells are used (e.g., viability of 95-100% in serum-free conditions).
  • Culture log-phase Sf9 cells to a cell density of between 1.5 ⁇ 10 6 and 2.5 ⁇ 10 6 cells per ml in suspension culture.
  • Recombinant factor C production also can be carried out by infecting Sf9 cells grown in monolayer culture. Determine cell density and viability. Dilute Sf9 cells with fresh InsectXPRESS to 1.5 ⁇ 10 6 cells per ml. Add recombinant baculovirus high titer stock to the Sf9 culture, at an MOI of 1 (virus/cell), as follows: Vol .
  • ⁇ of ⁇ ⁇ inoculum ⁇ ⁇ HTS ⁇ ⁇ ( ml ) ( Total ⁇ ⁇ cells ⁇ / ⁇ ml ) ⁇ ( Total ⁇ ⁇ Vol . ⁇ of ⁇ ⁇ culture ) ⁇ ( MOI ) ( Virus ⁇ ⁇ Titer ⁇ ⁇ in ⁇ ⁇ pfu ⁇ / ⁇ ml )
  • Sf9 cells grow at 27° C. without CO 2 . Maximal aeration is preferred. If spinner culture bottles are used, loosen the side screw-caps to increase aeration. Sf9 cells adapted to grow in serum-free medium should be subcultured at 4-5 days intervals on a routine basis.
  • the recombinant Factor C (rFC) assay was carried out using 20 ⁇ l of each detergent ( ⁇ 10 ⁇ of final concentration), 150 ⁇ l of 1 EU/ml EC-6, 10 ⁇ l of rFC supernatant 031901I, 20 ⁇ l of 300 mM Tris, pH 8.0, and 20 ⁇ l of 0.55 mM DPR-coumarin substrate.
  • the results of the assay were read at 38° C. using a Cytofluor reader set at 390/440 nm, 5 minutes per cycle, for 1 hour. The data were graphed after 30 minutes.
  • Table 1 summarizes the results obtained by testing nine detergents. These detergents can be divided into three groups: detergents that have a narrow concentration range for enhancing recombinant Factor C activity, detergents that have wide concentration range for enhancing recombinant Factor C activity, and detergents that inhibit recombinant Factor C activity.
  • Endotoxin O55:B5, 0.01, 0.1, 1, and 10 EU/ml were used for the standard curve.
  • Blank and endotoxin standards (100 ⁇ l each) were added to a 96-well plate.
  • Recombinant Factor C supernatant, assay buffer (150 mM NaCl, 150 mM Tris, pH 8.0, and 1.5% lactose, with or without 0.0125% Zwittergent) and substrate solution (0.2 mM in water) were mixed at 1:4:5 ratios. This mixture was added into each blank and endotoxin standard.
  • the fluorescence was recorded at time zero and time 1 hour.
  • the difference in fluorescences (delta fluorescence) were normalized with the blank. Normalized delta fluorescence was then graphed against endotoxin concentration in a log-log scale. Each data point is the result of duplicate assays.
  • a one-step endotoxin assay can be carried out in a 96-well plate, using 100 ⁇ l each of blank and endotoxin standards.
  • One hundred microliters of a mixture of recombinant Factor C supernatant (Baculovirus-infected Sf9 cell culture medium), buffer (150 mM Tri, pH 8.0, 150 mM NaCl, 1.5% beta-lactose, and 0.0125% Zwittergent 3-14) and fluorogenic substrate (DPR-coumarin, 0.2 mM) at a ratio of 1:4:5 is added to wells of the plate.
  • the plate is incubated at 37° C. for 1 hour. Excitation and emission are read at 390 and 440 nm, respectively, in a fluorescence microplate reader.

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