JPS6364747B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention uses Limulus amebocyte lysate against endotoxin.
(hereinafter sometimes referred to as LAL or lysate), improved LAL reagents and uses of such LAL reagents. As is well known, the LAL reagent, which detects endotoxin (internal toxin in bacteria), is probably the most practical and sensitive test for quantifying endotoxin. Commercial assays use amebocyte lysates from horseshoe crab hemolymph obtained from horseshoe crabs. This lysate is combined with appropriate divalent dissolved ions, appropriate buffers, and other ingredients to form the LAL reagent. This reagent then reacts with the endotoxin during the assay to form a gel. Manufacturers of LAL reagents often experience difficulties when producing lysates with the desired sensitivity for detecting endotoxin. Sensitivity varies from one production to the next. These problems are due, at least in part, to the presence of endogenous, unspecified endotoxin inhibitors (hereinafter sometimes referred to as inhibitors) in the lysate. Little is known about the nature of the inhibitor and its role in horseshoe crabs. Electrophoretic studies indicate that the inhibitor is a high molecular weight lipoprotein. It can function in controlling coagulation defense mechanisms in transformed cells. It is also plausible that the inhibitor could be a component of the membrane that is released during cell lysis. Uncertainty in the role and origin of inhibitors is compounded by the fact that the mechanism of inhibition appears to be unknown. Inhibitors interfere with enzymatic reactions in some manner, either by association with the enzyme itself or with endotoxins, or both. Like certain other serine proteases, the precoagulant enzyme is thought to complex with calcium and glycerophospholipids. Endotoxin itself is fatty, so the inhibitor of lipoprotein properties is highly compatible with both components. That the inhibitor is a lipoprotein is supported by its sensitivity to chloroform. As described in US Pat. No. 4,107,077, the sensitivity of LAL is improved when the lysate is treated with an organic solvent such as chloroform to precipitate the inhibitor from the lysate. The aqueous phase is then collected and processed to prepare the LAL reagent. To date, the solvent extraction method described above is the most rapid means of improving the susceptibility of LAL. Unfortunately, this method has several drawbacks. Since it is imperative that endotoxin-free conditions be maintained throughout lysate preparation, harsh extraction and subsequent centrifugation increases the likelihood of product deficiency. As described in this patent, solvent treatment reduces the stability of the melt since production must be rapidly completed in the cold.
The precipitate removed from the lysate by solvent treatment also contains significant coagulogen, the necessary clotting proteins. Maintaining appropriate protein content is a requirement when forming stiff gels during endotoxin assays. Obviously, in the latter case, control of reagent sensitivity is difficult. It is well known that chloroform, the most effectively used solvent, has undesirable effects on humans. The health and safety of those involved in production is therefore a legitimate concern. A method that avoids these pitfalls and still improves the sensitivity to the desired degree would be an obvious improvement in this technology. Therefore, the aim of the present invention is to provide a method for simply and rapidly increasing the susceptibility of LAL. According to the present invention, under lysate treatment conditions, lysates of horseshoe crab amebocytes with reduced susceptibility to endotoxin due to the presence of endogenous lysate inhibitors can be treated with lysate-sensitivity enhancers having lysate-sensitivity-enhancing properties. A method is provided comprising treating the lysate with an enhancing amount of the agent to neutralize or partially neutralize the lysate inhibitor and thereby increase the susceptibility of the lysate to endotoxin. Minimum criteria exist that can describe the susceptibility-enhancing properties of LAL. That is, LAL susceptibility enhancers useful in the methods of the invention have (a) the ability to increase the susceptibility of a lysate to a moderate sensitivity, e.g., by a factor of 2 or more; (b) an ultraviolet or lower (c) ability to withstand depyrogenic, i.e. endotoxin removal or degradation, by acid treatment at a pH of greater than 8 or by base treatment at a pH greater than 8; ability to draw, (d)25
(e) ability to function in a PH range of about 6.0 to about 9; (f) buffers and other components used in the LAL reagent; (g) Compatibility with respect to reaction with LAL and its endotoxin. Such enhancers include amphoteric surfactants having both anionic and cationic groups in their structure. An example is sulfobetaine, represented by the following formula (hereinafter formula A): where R ₁ is an alkylene group of 1 to about 4 carbon atoms, and Y is (1) hydrogen, (2) optionally substituted lower alkyl, such as methyl, ethyl, propyl, etc. Contains lower alkyl or hydroxy carbon atoms, which have no adverse effects,
It is a chemically suitable substituent, and R ₂ and R ₃ are each optionally substituted 1 to 1.
lower alkyl of 4 carbon atoms, e.g. methyl,
Selected from ethyl, propyl, hydroxyethyl, hydroxymethyl, hydroxypropyl, etc.
n is 0 or 1; when n is 0, R ₄ is optionally substituted alkyl, such as alkyl of 8 to 18 carbon atoms; when n is 1, R ₄ is 1 to 6 carbon atoms; is an alkylene group, and R ₅ is an optionally substituted alkyl, for example, an alkyl having 8 to 18 carbon atoms. It should be understood that the term "alkylene" as used herein encompasses both polymethylene groups and other divalent saturated aliphatic groups, and that alkylene groups may therefore be branched. be. The term "lower" refers to groups containing 1 to 4 carbon atoms. Sulfobetaines used in the compositions of the present invention are known in the art and have been described as zwitterionic surfactants. Such compounds are described, for example, by GWFernley, the
JOURNAL OF AMERICAN OIL
CHEMISTS SOCIETY, January 1978
(Vol. 55), pages 98-103, and U.S. Pat.
No. 3280176 (R. Ernst). One type of sulfobetaine surfactant that can be used is where n is 0 and R ₄ is about 8-18
It has the above structure, which is an alkyl group of carbon atoms, preferably a straight chain alkyl group. For these sulfobetaine surfactants, a convenient source of the _R4 component is tallow aliphatic alcohol;
It consists of a mixture of various chain lengths, with a typical composition of approximately 66% C ₁₈ , 30% C ₁₆ and 4% C ₁₄
etc. Another convenient source is distilled coconut fatty alcohol center fraction, which also consists of a mixture of various chain lengths, with a typical composition of approximately 66%
_C12 , 23% _C14 , 9% _C16 and 2% _C10 . Certain sulfobetaine surfactants of the above structure where n is 0 are incorporated herein by reference.
U.S. Patent No. 3539521 (AO
Snoddy et al). A particularly preferred surfactant of this type is N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate [trademark ZWITTERGENT3-
Calbiochem Behring Company (Calbiochem)
- commercially available from Behring Corporation]. Another type of sulfobetaine surfactant that can be used is where n is 1 and R ₄ is from about 1 to about 6
It has the above structure which is an alkylene having a carbon atom. In those sulfobetaines where n is 1, R ₅ is an alkyl group of 8 to 18 carbon atoms.
Preferably, R ₅ is linear. As aforementioned,
Sources of alkyl groups having 10 to 18 carbon atoms are tallow aliphatic alcohols and coconut aliphatic alcohols. Certain sulfobetaine surfactants of the above structure where n is 1 are described in the aforementioned US Pat. No. 3,280,179. Particularly preferred sulfobetaine surfactants for use in the compositions of the invention are those in which the acyl group is derived from tallow fatty alcohol or coconut fatty alcohol, preferably coconut fatty alcohol. -dimethyl-N-acylamidopropylammino)-2-hydroxypropane-
1-sulfonate. It is recognized in the art that the conventional preparation of these derivatives of tallow or coconut fatty alcohols results in a mixture of sulfobetaines with varying carbon chain lengths for the acyl group. As mentioned above, these aliphatic alcohols mostly contain a carbon chain length that provides the desired number of carbon atoms, ie, acyl groups having from 8 to 18 carbon atoms. These mixtures obtained from tallow or coconut oil fatty alcohols are thus useful for providing sulfobetaine surfactants in the compositions of the present invention. A particularly preferred substance of this type for use in the compositions of the invention is N-cocoamido-propyl-N,N-dimethyl-N-2-hydroxypropylsulfobetaine, an example of which is available from Lonza, Inc. LONZAINE CS, commercially available from Sherex Chemical Company, Fair Lawn, New Jersey;
VARION CAS, commercially available from Chemical Company, Inc. Other amphoteric surfactants include N-long chain alkylaminocarboxylic acids represented by the following formula (hereinafter Formula B): N-long chain alkylimidodicarboxylic acid represented by the following formula (hereinafter formula C): and N-long chain alkyl or amidobetaine represented by the following formula (hereinafter formula D): wherein R ₁ , R ₂ , R ₃ , R ₄ , Y and n are as defined in formula A, M is hydrogen or a salt-forming metal, and Y' is As defined. Y and
Y′ can be the same or different. Examples of specific amphoteric detergents are N-alkyl-beta-aminopropionic acid, N-alkyl beta-iminodipropionic acid, and N-alkyl-beta-aminopropionic acid.
N,N-dimethylglycine; the alkyl group is, for example, coconut fatty alcohol, lauryl alcohol, myristyl alcohol (or lauryl-myristyl mixture), hydrogenated tallow fatty alcohol, cetyl, stearyl, or such alcohols. It is derived from a blend of Substituted aminopropionic acids and iminopropionic acids are often used in sodium or other forms. These can similarly be used in the practice of this invention. Specific examples include cocoamidopropyl betaine sold under the name EMCOL CC 37-18 by Witco Chemical Corporation;
Inc.) and Sherex Chemical Co.
Chemical Company)
Cocoamidopropyl betaine sold by LONZAINE CO and VARION CADG; name from Henkel Corporation
Sodium N sold at DERIPHAT 151
- Coco-beta-aminopropionate: disodium N-lauryl-beta-iminodipropionate, sold under the name DERIPHAT 160 by Henkel;
DERIPHAT 154 disodium N-tallow-beta-iminodipropionate. Examples of other amphoteric detergents include aliphatic imidazolines made by reacting long chain fatty acids (e.g., 10 to 20 carbon atoms) with diethylenetriamine and monohalocarboxylic acids of 2 to 6 carbon atoms, e.g. 1-coco-5-hydroxyethyl-5-carboxymethylimidazoline. Specific examples include cocoimidazoline, commercially available from Lonza, Inc. under the name AMPHOTERGE K-2;
Caprylic dicarpoxyimidazoline, commercially available under the name AMPHOTERGE KJ2, and cocodicarboximidazoline blended with a sulfated surfactant, commercially available from Lonza under the designation AMPHOTERGE 2 WAS MOD. Other examples of enhancers are commonly described as compounds that contain hydrophilic and lipophilic groups in their molecular structure and that produce anions containing both lipophilic and hydrophilic groups in aqueous media. Includes anionic synthetic surfactants. Alkylaryl sulfonates, alkanesulfonates, and sulfated oxyethylated alkylphenols are examples of anionic types of surface-active compounds. Alkylaryl sulfonates are a class of synthetic anionic _surface active compounds represented by the following general formula ₍ hereinafter _Formula E ₎ ; is a linear or molecular chain hydrocarbon group of 1 to 24 carbon atoms, at least one R ₆ has at least 8 carbon atoms; n ₁ is 1 to 3;
n ₂ is 1-2; Ar is a phenyl or naphthyl group, and Y and M are as defined in formula B. R ₆ is, for example, methyl,
It can be ethyl, hexyl, octyl, tetratecyl, isooctyl, nonyl, decyl, dodecyl, octadecyl, and the like. Compounds exemplifying alkylaryl sulfonates are sodium dodecylpenzene sulfonate, sodium decylbenzene sulfonate, ammonium methyldodecylbenzene sulfonate, ammonium dodecylbenzene sulfonate, sodium octadecylbenzene sulfonate, sodium nonylbenzene sulfonate, sodium dodecylnaphthalene sulfonate, and sodium heptadecylbenzene. sulfonates, including potassium eiconsylnaphthalene sulfonate, ethylamine undecylnaphthalene sulfonate and sodium docosylnaphthalene sulfonate. Alkyl sulfates are a class of synthetic anionic surfactants represented by the following general formula (hereinafter Formula F): R ₅ OSO ₃ M where R ₅ and M are as defined in Formula B. Compounds that exemplify the 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, Includes diethanolaminooctyl sulfate, triethanolamine octadecyl sulfate and ammonium nonyl sulfate. Sulfated oxyethylated alkylphenols are
It is a class of synthetic anionic surfactants represented by the following general formula (hereinafter Formula G): where A is oxygen, sulfur, a carbonamide group, a thiocarbonamide group, a carboxyl group or a thiocarboxylic acid ester group, z is an integer from 3 to 8, and R ₅ and M are as defined in formula B. be. Compounds that exemplify the sulfated oxyethylated alkylphenol class of anionic surfactants include ammonium nonyl phenoxytetraethylene oxysulfate, sodium dodecyl phenoxy triethylene oxysulfate, and ethanolamine decyl phenoxy tetraethylene. oxysulfate and potassium octylphenoxytriethylene oxysulfate. Other examples of LAL enhancers include nonionic surface-active compounds, which can be broadly described as compounds that do not ionize but acquire hydrophilic properties from oxygenated side chains such as polyoxyethylene, and this The lipophilic portion of the molecule can be derived from fatty acids, phenols, alcohols, amides or amines. These compounds are prepared by reacting alkylene oxides such as ethylene oxide, butylene oxide, propylene oxide, etc. with fatty acids, straight or branched chain alcohols containing one or more hydroxyl groups, phenols, thiophenols, amides and amines. , polyoxyalkylene glycoethers and esters,
It is usually produced by producing polyoxyalkylenealkylphenols, polyoxyalkylenethiophenols, polyoxyalkyleneamides, etc. Examples of these nonionic surfactants are alkylene oxides, aliphatic alcohols of 8 to 18 carbon atoms, such as octyl, nonyl, dodecyl, octadecyl, dodecyl, tetradecyl;
Monoesters in which the ester group of the hexahydric alcohol contains 10 to 20 carbon atoms, such as sorbitan monolaurate, sorbitan monooleate and sorbitan monopalmitate; in which the alkyl group contains 4 to 20 carbon atoms, e.g. butyl,
It is a product obtained by reacting with an alkylphenol such as dibutyl, amyl, octyl, dodecyl, tetradecyl, etc.; and an alkylamine in which the alkyl group contains 1 to 8 carbon atoms. Compounds illustrating synthetic nonionic surfactants include those obtained by condensing ethylene oxide or propylene oxide with the following compounds: propylene glycol, ethylene diamine,
Diethylene glycol, dodecylphenol, nonylphenol, tetradecyl alcohol, N-
Octadecyl diethanolamide, N-dodecyl monoethanolamide, polyoxyethylene 20 sorbitan monooleate (sold under the name TWEEN80) and polyoxyethylene 20 sorbitan monolaurate (sold under the name TWEEN20) other non-ionic The surfactants include long chain tertiary amine oxides corresponding to the following general formula (hereinafter Formula H): R ₅ R ₇ R ₈ N→O where R ₅ is as defined in Formula A. ,
and R ₇ and R ₈ are each a methyl or ethyl group. The arrow in the formula is the usual representation of a semipolar bond. Examples of amine oxides suitable for use in the present invention include dimethyldodecylamine oxide,
These are dimethyloctylamine oxide, dimethyldecylamine oxide, dimethyltridecylamine oxide, and dimethylhexadecylamine oxide. Cationic surfactants can also be used as LAL enhancers. Such enhancers are surface-active compounds containing organic hydrophobic groups and cationic solubilizing groups. Typical cationic solubilizing groups are amine groups and quaternary groups. Such cationic surfactants are represented by the following general formula (hereinafter formula): In the formula, R ₅ , Y and Y' are as defined in formula C. An example is Ciba-Geigy.
Available from Geigy Corporation
It is QUATERNARYO. Other examples of suitable synthetic cationic surfactants include:
Formula (hereinafter Formula J) R ₉ NHC ₂ H ₄ NH ₂ where R ₉ is about 12 to 22 carbon atoms. diamines such as those of the formula (hereinafter formula K) R ₅ CONHC ₂ H ₄ NH _{2 ,} such as N-2-aminoethylstearylamine and N-2-aminoethyl myristylamine; N
-2-aminoethylsterylamide and N-aminoethylmyristylamide; typically one of the groups attached to the nitrogen atom is an alkyl group of 1 to 3 carbon atoms and an inert substituent, e.g. quaternary ammonium compounds containing alkyl groups of 1 to 3 carbon atoms and in which anions such as halogen, acetic acid, methyl sulfate, etc. are present. Typical quaternary ammonium compounds are ethyl-dimethylstearylammonium chloride, benzyl-dimethyl-stearylammonium chloride, benzyldimethyl-stearylammonium chloride, trimethylstearylammonium chloride, trimethylcetylammonium bromide, dimethylethyl dilauryl ammonium chloride, dimethyl- Propyl-myristyl ammonium chloride and the corresponding methosulfate and acetate. Other suitable cationic surfactants are represented by the following formula (hereinafter Formula L): In the formula, R ₅ is as defined in formula A,
And each a is an integer from 1 to 15. An example is a hydrogenated tallow polyethylene glycolamine in which R represents a tallow group and a+a has an average value of 5. It is named BINA from Ciba-Geigy.
Available in COBA 3001. As mentioned above, the lysate enhancer is used in an enhancing amount, ie, an amount sufficient to neutralize or partially neutralize the endogenous endotoxin inhibitor in the lysate. Generally, this will be about 0.001 to 1.0% (W/V) based on the total volume of melt, preferably about 0.01
% to about 0.05% (W/V). often,
Excess amounts interfere with the ability of LAL to react with the endotoxin in the assay. LAL shall be made in a manner known in the art, e.g.
It can be produced by the method described in No. 1522127. For example, hemolymph from a healthy specimen of Limulus polyphemus is commonly used by Levin and Bang ^--
“Clottable Protein in Limulus: Its
Localization and Kinetics of Its Coagulation
by Endotoxin”, Thromb.Diath.Haemorrh. 19 :
186-197 (1968), in a salt anticoagulant solution. The amebocytes are collected, washed with salt anticoagulant solution, and the amebocytes are separated from the anticoagulant by centrifugation. The separated deformed cells are suspended in water, and the osmotic rupture of the cells is supplemented by mechanical stirring. Cell debris is centrifuged from the lysate and the lysate portion is collected and stored at 0-4°C. To form the LAL reagent, the aforementioned LAL moiety is generally adjusted to a suitable PH range, e.g. 5.5-8.5, preferably 6.5-7.5, with a suitable buffer, e.g. tris(hydroxymethyl)aminomethane, tris(hydroxymethyl) ) aminomethane maleate,
Buffer with 1.4-piperazinediethanesulfonic acid, morpholinopropanesulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, triethanolamine, imidazole and tris(hydroxymethyl)imidazole.
The LAL reagent is then divided into serum vials, eg each vial containing 1.2 ml or 5.2 ml of solution, and these are lyophilized. After freeze-drying, the vial is usually sealed and refrigerated (1-5°C). Typically, in accordance with the present invention, LAL is treated by adding a lysate susceptibility enhancer to the LAL after separating it from cell debris of amebocytes. usually,
It is added prior to or during the manufacture of the LAL reagent, eg, simultaneously with buffers and other ingredients. The sensitivity of the LAL reagent to endotoxin is
It is further increased by including low concentrations of divalent cations and monovalent cations. Calcium and magnesium ions are the preferred divalent ions, but other alkaline earth metal ions,
For example, magnesium and strontium ions or other divalent ions can be used. Magnesium and strontium ions are also preferred ions. Sodium is the preferred monovalent ion, but other monovalent ions can be used, especially alkali metal ions, such as lithium ion. Chloride (CaCl ₂ , NaCl, etc.) is a preferred source of these added ions, but other salts can be used. Preferably these electrolytes are in endotoxin susceptibility increasing amounts, e.g. for divalent cations (e.g. Ca ⁺² ), concentrations between 0.0001 and
0.4 moles and monovalent ions (e.g.
For Na ⁺ ), the concentration will range from 0.01 to 0.4 molar. LAL reagents can also contain common adjuvants such as stabilizers, including lactose. These adjuvants, when used, are supplied in small amounts that impart the intended quality but do not adversely affect the desired properties of the LAL reagent. All of the above operations are performed under lysate processing conditions, including ensuring that the final product is sterile and free of endotoxins. Methods of ensuring endotoxin freedom are known in the art. For example, inorganic additives (CaCl ₂ ,
(e.g. NaCl), dry the salt at least 120°C to 250°C.
By heating for minutes, it can be made endotoxin-free. Because of this melting point, organic additives are usually dissolved, made acidic (PH<5) or basic (PH>9), and the solution heated at 121°C for 30-60°C.
Autoclave for at least a minute to destroy any endotoxin that may be present. As mentioned above, another aspect of the invention relates to a LAL reagent containing as essential ingredients an aqueous dispersion of LAL, a lysate-enhancing amount of the aforementioned LAL susceptibility enhancer, and a buffering amount of the aforementioned suitable buffer. . If desired, the aforementioned monovalent and divalent cations can be included in lysate susceptibility increasing amounts to further increase the susceptibility of the lysate to endotoxin. Typically, the lysate present in the reagents of the invention will be present in an amount sufficient to quantify endotoxin in a subsequent LAL assay for endotoxin, and generally this will be within the FDA reference endotoxin EC-2.
about 0.007 to about 0.5 ng/ml, preferably about 0.007 to about
This is the amount that can be detected at 0.050ng/ml. The aforementioned LAL reagents can be lyophilized, which is preferred. The following examples illustrate the invention. All parts are weight/volume unless otherwise specified. Preparation of horseshoe crab lysates Horseshoe crab lysates were prepared by Levin and Bang.
It was prepared by a modification of the procedure originally described by Thromb. Diath. Haemorrh. 19 , 186 (1969). horseshoe crab
Limulus polyphemus was collected near Beaufort, NC in the Atlantic Ocean. Hemolymph (approximately 500 ml) was removed by cardiac puncture with a 16-gauge needle and heated to 42°C in a 3% endotoxin-free solution.
500ml of 0.125% N-ethylmaleimide in saline solution
was collected in an endotoxin-free 1-volume glass centrifuge bottle containing . The centrifuge bottle containing the hemolymph anticoagulant solution was warmed to 42° C. for 8 minutes and then centrifuged at 150×g for 10 minutes. The plasma supernatant was decanted and the amebocyte pellet was resuspended in anticoagulation solution. Cells were again pelleted by centrifugation as described above. The clogged cells were resuspended in 0.9% pyrogen-free saline and transferred to depyrogenated 50 ml plastic centrifuge tubes. The washed cells were centrifuged again at 150 xg. After decanting the saline, the clogged dysmorphic cells were diluted with 7 ml of pyrogen-free water for injection.
Rupture was achieved by adding a ratio of 3 ml of packed cells. After vortexing for 10-15 seconds, the lysed cells were stored at 1-5°C for 24 hours. Cell debris was sedimented by centrifugation at 1500 xg for approximately 15 minutes. Decant the lysate and
Stored at 4°C. Cell debris was discarded. Preparation of Standard Endotoxin Solutions Standard Solutions of Foods and Drugs Administration (FDA) Reference Standard Endotoxin Lots
EC-2 was prepared in a pyrogen-free solution for injection. Reconstitution by supplying 1 μg of endotoxin in a vial containing 10 ml of water resulted in an initial concentration of 0.1 μg/ml. 1 small bottle with a reciprocating shaker
Shake for an hour. A series of dilutions was prepared to obtain the following endotoxin concentrations: 10 ng/ml, 1 ng/ml.
ml, 500pg/ml, 250pg/ml, 125pg/ml,
62.5pg/ml, 31.25pg/ml, 15.6pg/ml, and
7.8pg/ml. Once prepared, the endotoxin solution was stored for up to 48 hours and then discarded. Other endotoxin standards used were Klebsiella
Pneumoniae, Escherichia coli endotoxin lot 071857 (Difco) was a reformulation solution of FDA reference endotoxin lot No. 1 and reference lot EC-2 prepared in our laboratory. Endotoxin standard solutions prepared from E. coli lot 071857 endotoxin were at the following concentrations: 6.25, 12.5, 50, 75, 100, 150, and
200pg/ml. Lysate assay procedure A 25-70% lysate solution was prepared in a 0.1 molar buffer pH 7.0, usually Tris, i.e., tris(hydroxymethyl)aminomethane. Other buffers used were imidazole, trisimidazole, triethanolamine, trismaleate, N-2-hydroxyethylpiperazine-N-
They were 2-ethanesulfonic acid (HEPES), 1,4-piperazinediethanesulfonic acid (PIPES), and morpholinopropanesulfonic acid (MOPS). To determine the susceptibility of the lysates, 0.1 ml of each endotoxin dilution was added to 0.1 ml of the lysate in a depyrogenated 10 x 75 mm threaded cap tube and 37
It was kept warm at ℃ for 1 hour. Results were determined by gently inverting each tube 180°. Clots remaining intact after inversion showed a positive endotoxin test. Preparation of surfactant solutions and depyrogen removal Raw LAL susceptibility enhancement solutions were prepared at concentrations of active ingredient up to 10% (W/V) in aqueous solutions. The most frequently prepared concentration was 1% (W/V). The procedure varied the amounts from one reagent to the next, but when diluting to 100 ml, sufficient material was dissolved in 50 ml of aqueous solution to obtain the desired concentration. The solution also contains 7.5 ml of 0.05 mol tris(hydroxymethyl)aminomethane (i.e.
TRIZMA BASE, Sigma Chemical Company)
and 1 ml of 2N NaOH to give a final pH ≧11. Reagents in alkaline solution for more than 12 hours 0-4
Stored at °C to protect complete depyrogenation. After adjusting the solution to approximately PH8, ≧121℃ and 15psi
autoclaved for over 15 minutes. The pH was finally adjusted to PH7.0±0.5. Calculate the reagent concentration,
The dilution was adjusted to the final desired concentration. Alkali-labile reagents were depyrogenized by acid treatment, in which HCl and Tris buffer were used in place of NaOH and TRIZMA BASE, respectively. Addition of Enhancer to the Lysate The solution of enhancer prepared as above was added to the lysate during the buffer addition. The amount added varied with the enhancer used, but the concentration range for all tested was 0.001-1.0% in the lysate solution.
It was (W/V). The order of addition of ingredients did not adversely affect the sensitivity of the resulting lysate. Example LAL susceptibility enhancer, ZWITTERGENT ^TM 3-
14,N,N-dimethyl-3-ammonio-1-propanesulfonate (sulfobetaine, available from Calbiochem-Behring Corporation) was depyrogenated as described above to a final concentration of 1% ( W/V). Lysate lot 9CZC was prepared as a 50% dilution in 0.1 molar tris-maleate buffer pH 7.0.
The addition of the enhancer was carried out at a final concentration of 0.005, 0.01,
Tests were performed to yield 0.02, 0.05, 0.075, and 0.10% (W/V). Control samples contained pyrogen-free water instead of surfactant. Dilutions of the lysate were stored overnight at 0-4°C and tested the next day using a specially formulated EC endotoxin dilution series (labeled EC). The results (table) show the effective concentration range for the enhancer and the total increase in lysate sensitivity to endotoxin compared to the control lysate.

[Table] Wasu.
Examples Standard endotoxin solutions include E. coli endotoxin 071857, FDA reference endotoxin EC-2,
and K. pneumoniae FDA Reference Lot No. 1. The lysate was buffered with 0.1 molar Tris buffer PH7.0 and the enhancer ZWITTERGENT ^TM 3-
ZWITTERGENT 0.02% diluted with a solution of 14
A 30% dilution of the lysate was made containing 3-14 (W/V). Water was used in place of this enhancer in control samples. Lysate assays were performed using each endotoxin dilution series.

[Table] Wasu.
The commercially available LAL enhancer of Example 22 is studied to determine its effect on the susceptibility of the lysate to endotoxin. Each solution was prepared and added to lysate lot 9F1 at a final concentration of 0.001-0.20% (W/V). A sample of the lysate was then tested with reformulated FDA EC endotoxin (EC). In the table, the enhancers are listed in order of decreasing effectiveness. The most effective concentrations tested in lysates and their corresponding lysate sensitivities are shown. The sensitivity of a control lysate containing enhancer (50% dilution) is also shown.

Table Example: To determine that the increased sensitivity observed in enhancer-treated lysates is maintained during lyophilization, 0.02% in 0.05 molar Tris buffer
A 30% dilution of the lysate was prepared in the presence or absence of ZWITTERGENT ^™ 3-14 (final concentration in the lysate). The lysate solution (1.2 ml) was dispensed into each 10 ml serum vial. The samples were then frozen at -35°C and lyophilized under 50μ vacuum for approximately 32 hours. The vial was sealed with a split rubber stopper and a metal cap. Lyophilized lysate
Reconstituted with 1.2 ml of pyrogen-free water for injection and then tested using the EDA reference endotoxin lot EC-2. The control lysate without enhancer was
It had a sensitivity of 62.5 pg/ml. In the presence of enhancers,
Lysate sensitivity was improved two-fold to 31.2 pg/ml. EXAMPLE A Day Pool lot of approximately one week old horseshoe crab lysate ODH was added to a final concentration of 0.06 M CaCl ₂ , 0.01 M MnCl ₂ and 0.03%
It was prepared as a 40% solution in 0.05M tris(hydroxymethyl)aminomethane maleate buffer, pH 7.0, containing ZWITTERGENT ^™ 3-14. This solution was dispensed in 1.2 ml aliquots into 8 ml vials, frozen at -45°C, and lyophilized under 50μ vacuum for approximately 28 hours. The vial was sealed with a split rubber stopper and capped with a plastic screw cap. Lyophilized lysate
Reconstitute with 1.2 ml pyrogen-free water for injection;
Tested with FDA reference endotoxin lot EC-2. The sensitivity of the enhancer-treated, lyophilized lysate was 62 pg/ml. Lyophilize as before,
A control 40% solution of LAL without enhancer tested had a sensitivity of 1 ng/ml. EXAMPLE A horseshoe crab lysate day pool with susceptibility equal to or greater than 500 pg/ml E. coli endotoxin ICF was combined with a final concentration of 0.02M MgCl ₂ , 0.01M SrCl ₂ , 0.01M CaCl ₂
and 0.025 mole tris(hydroxymethyl) containing 0.025% ZWITTERGENT ^TM 3-14
Aminomethane maleate buffer, PH7.0, medium 40
% solution. Dispense this solution into 10 ml vials with 1.2 ml and 5.2 ml Alcoat and -
Frozen at 50°C. These samples were lyophilized under 100μ vacuum for approximately 72 hours. Add the lyophilized product to 1.2 ml or
Reconstituted with 5.2 ml of pyrogen-free water for injection.
When tested with the E. coli endotoxin ICF after lyophilization, the sensitivity of the enhancer-treated, lyophilized lysate was 25 pg/ml for both sample series, compared to a sensitivity of 500 pg/ml for the control. .

Claims (1)

[Scope of Claims] 1. A buffered aqueous dispersion of a lysate of horseshoe crab amebocytes having improved sensitivity to endotoxin and a lysate susceptibility enhancer having lysate susceptibility enhancing properties; is the following formula In the formula, R ₁ is an alkylene group of 1 to 4 carbon atoms, Y is (1) hydrogen, (2) alkyl of 1 to 4 carbon atoms, or (3) hydroxyalkyl, and R ₂ and R ₃ are each ( 1) alkyl of 1 to 4 carbon atoms or (2) hydroxyalkyl, n is 0 or 1, and when n is 0,
R ₄ is alkyl of 8 to 18 carbon atoms, and n is 1
an amphoteric surfactant having: , R ₄ is an alkylene group containing 1 to 6 carbon atoms, and R ₅ is an alkyl group containing 8 to 18 carbon atoms. Limulus amebocyte lysate reagent for quantifying endotoxin. 2 The cations Na ⁺ , Mn ⁺⁺ and Ca ⁺⁺ are further present in lysate susceptibility increasing amounts.
Reagents listed in section. 3 Furthermore, cations Na ⁺ , Sr ⁺⁺ , Ca ⁺⁺ and
2. The reagent of claim 1, wherein Mg ⁺⁺ is further present in a lysate sensitivity increasing amount. 4. The enhancer has the formula according to claim 1.
The reagent according to claim 2 or 3, which is selected from the group of amphoteric surfactants represented by (A). 5 R ₂ and R ₃ are each methyl, n is 0 or 1, when n is 0, R ₄ is tetradecyl and [Formula] is trimethylene, and when n is 1, R ₄ is trimethylene and The reagent according to claim 1, wherein [Formula] is [Formula]. 6 n is 1, R ₄ is propylene, R ₂
and R ₃ are each methyl, and [Formula] is methylene. 7. The reagent according to claim 5, wherein n is 0, R ₂ and R ₃ are each methyl, and [Formula] is methylene. 8 consisting of a buffered aqueous dispersion of a lysate of horseshoe crab amebocytes with improved sensitivity to endotoxin and a lysate susceptibility enhancer having lysate susceptibility enhancing properties, said lysate susceptibility enhancer having the following formula: In the formula, R ₁ is an alkylene group of 1 to 4 carbon atoms, Y and Y' are each (1) hydrogen, (2) alkyl of 1 to 4 carbon atoms, or (3) hydroxyalkyl, and R ₅ is 8 An amphoteric surfactant for determining endotoxin, characterized in that it is an alkyl group containing ~18 carbon atoms, and M is hydrogen, sodium, potassium or ammonium. Amebocyte lysate reagent. 9 consisting of a buffered aqueous dispersion of a lysate of horseshoe crab amebocytes with improved sensitivity to endotoxin and a lysate susceptibility enhancer having lysate susceptibility enhancing properties, said lysate susceptibility enhancer having the following formula ( E) (R ₆ ) _o1・(Y)Ar・(SO ₃ M) _o2 In the formula, R ₆ is alkyl of 8 to 24 carbon atoms,
Y is (1) hydrogen, (2) alkyl of 1 to 4 carbon atoms, or
(3) is hydroxyalkyl, M is hydrogen, sodium, potassium or ammonium, n ₁ is an integer from 1 to 3, n ₂ is 1 or 2, and Ar is phenyl or naphthyl. A horseshoe crab amebocyte lysate reagent for quantifying endotoxin, characterized in that it is an anionic surfactant selected from the group consisting of: 10 consisting of a buffered aqueous dispersion of a lysate of horseshoe crab amebocytes with improved susceptibility to endotoxin and a lysate susceptibility enhancer having lysate susceptibility enhancing properties, said lysate susceptibility enhancer having the following formula ( F) an anionic surfactant from the group consisting of R ₅ OSO ₃ M in which R ₅ is an alkyl group containing 8 to 18 carbon atoms and M is hydrogen, sodium, potassium or ammonium; A horseshoe crab amebocyte lysate reagent for quantifying endotoxin, characterized in that: 11 consisting of a buffered aqueous dispersion of a lysate of horseshoe crab amebocytes with improved susceptibility to endotoxin and a lysate susceptibility enhancer having lysate susceptibility enhancing properties, said lysate susceptibility enhancer having the following formula: where R ₅ is an alkyl group containing 8 to 18 carbon atoms, and Y and Y' are each (1) hydrogen, (2) alkyl of 1 to 4 carbon atoms, or (3) hydroxyalkyl. A horseshoe crab amebocyte lysate reagent for quantifying endotoxin, characterized in that it is a cationic surfactant selected from the group consisting of: 12 consisting of a buffered aqueous dispersion of a lysate of horseshoe crab amebocytes with improved sensitivity to endotoxin and a lysate susceptibility enhancer having lysate susceptibility enhancing properties, said lysate susceptibility enhancer having the following formula ( H) a nonionic surfactant having R ₅ R ₇ R ₈ N→O in which R ₅ is an alkyl group containing 8 to 18 carbon atoms, and R ₇ and R ₈ are each methyl or ethyl; And 10~
a nonionic surfactant selected from the group consisting of condensation products of 30 moles of ethylene oxide and monoesters of hexahydric alcohols containing 6 carbon atoms and containing 10 to 20 carbon atoms; A horseshoe crab amebocyte lysate reagent for quantifying endotoxin, characterized in that the reagent is selected from: