CA1159754A - Process for preparing limulus lysate - Google Patents
Process for preparing limulus lysateInfo
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- CA1159754A CA1159754A CA000380754A CA380754A CA1159754A CA 1159754 A CA1159754 A CA 1159754A CA 000380754 A CA000380754 A CA 000380754A CA 380754 A CA380754 A CA 380754A CA 1159754 A CA1159754 A CA 1159754A
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- lysate
- carbon atoms
- endotoxin
- methyl
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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
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Abstract
ABSTRACT OF THE DISCLOSURE
This invention relates to a process for treating Limuius Lysate having improved sensitivity to endotoxin, to lysate reagents utilizing such lysate, and to the use of such lysate reagents.
This invention relates to a process for treating Limuius Lysate having improved sensitivity to endotoxin, to lysate reagents utilizing such lysate, and to the use of such lysate reagents.
Description
~ 59754 APPLICATION ~OR
LETTERS PATENT
FOR
IMPROVEMENT IN THE PROCESS FOR
PREPARING LIMULUS LYSATE
-This invention relates to a process for improving the sensitivity of Limulus amebocyte lysate (hereinafter sometimes referred to as LAL or lysate) to endotoxin, to an improved LAL reagent and to the use of sùch LAL reagent.
As is well known, the LAL test for detecting endotoxins i5 perhaps the most practical and sensitive test for determining endotoxins. Commercial assay tests employ amebocyte lysate from Limulus hemolymph obtained from the horseshoe crabs. This lysate is combined with lS appropriate divalent cations, appropriate buffers and other ingredients to form a LAL reayent. This reagent then reacts with endotoxin during the assay to form a gel.
Manufacturers of LAL reagents often experience difficulty in producing lysate of the desired sensitivity for detecting endotoxin. Sensitivity from one preparation to the next is also variable. These problems are attributed at least in part to the presence of an endogenous, undefined endotoxin inhibitor substance in the lysate, hereinafter sometimes referred to as the inhibitor).
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Little is known of the nature of the inhibitor or its in-vivo role in the horseshoe crab. Electro-.. ...
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phoretic studies indicate that the inhibitor is a high molecular weight lipoprotein. It may function in the amebocyte to control the coagulation defense mechanism.
That the inhibitor may be a membrane component freed during cell lysis is also plausible. The uncertainty of ~~ ~ ~~
the role and origin of the inhibitor is compounded by the fact that the mechanism of inhibition is unclear. The inhibitor presumably blocks the enzymatic reaction in some ~ fashion either by association with the enzyme itself, or 10 with the endotoxin or both. Like some other serine pro-teases, the proclotting enzyme is thought to be complexed with calcium and glycerophospholipid. Endotoxin itself is lipoidal, hence, an inhibitor of lipoprotein character would be highly compatible with either component.
That the inhibitor is a lipoprotein is supported by its sensitivity to chloroform. As described in U.S.
Patent No. 4,107,077, the sensitlvlty of LAL is improved substantlally when lysate is treated with an organic solvent such as chloroform to precipitate inhibitor from the lysate. The aqueous phase is then recovered and processed to prepare the LAL reagent.
To date, the above-mentioned solvent extraction procedure is the most rapid means of improving LAL sensi-tivity. Unfortunately, the method has several drawbacks.
Because o~ the absolute requirement that endotoxin-free ~ conditions be maintained throughout the lysate production, ; a cumbersome extraction procedure and subsequent centrifi-cation increases the likelihood of product failure. As noted in the patent, the solvent treatment reduces lysate ~; 30 stability such that the production must be completed ` rapidly in the cold. Also the precipitate removed from ; the lysate by the solvent treatment contains considerable coagulogen, the requlred clotting protein. Malntenance of ~ adequate protein content is a requirement for firm ,, ,,.. , .... - - .
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gelation during endotoxin assay. Obviously, under the latter circumstances, control of reagent sensitivity is difficult. Chloroform, the solvent used most successfully, is well known for its undesirable effects in 5 man. The health and safety of production personnel is therefore a reasonable concern. It is apparent that a process which avoids these pitfalls and yet improves sensitivity to the desired degree would be an improvement in the art.
-It is therefore an ob~ect of the present inven-tion to provide a method for the simple and rapid enhance-ment of the sensitivity of LA~.
In accordance with this invention, there is pro-vided a process for treating under lysate treating ~
15 conditions LAL havlng decreased sensitivity to endotoxin due to the presence of an endogenous inhibitor with an enhancing amount of a lysate sensitivity enhancing agent to neutralize or partially neutralize the lysate inhibitor thereby increasing the LAL sensitivity to endotoxin.
There are certain minimal criteria whlch can delineate LAL sensitivity enhancing characteristics, i.e., the LAL sensitivity enhancing agents useful in the process of this invention should possess (a) the ability to increase the lysate sensitivity to a suitable sensitivity, 25 e.g., by a twofold or greater increase, (b) the ability to withstand depyrogenation, i.e. removal or destruction of endotoxin, by ultrafiltratlon or acid treatment at pH less than 5 or base treatment greater than pH 8, (c) the ability to be sterilized e.g.,by autoclaving e.g. at or 30 above 121C at 15 psi for 15 minutes, (d) the ability to form aqueous solutions of about 2% (w/v) at 25C, (e) the ability to function in the pH range of about 6.0 to about 9, (f) the ability to be compatible with buffers and other ::, ' -- ~ - , .
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' ~ ' ingredients utilized in the LAL reagent and (g) the ability to be compatible with respect to LAL and its reaction with endotoxins.
Such enhancing agents includ~ amphoteric surfac-tants which have both an anionic and cationic group in their structure. Illustrative are the sulfobetaines represented by the following formula (hereinafter Formula A:
' O R2 (R5-C-HN)n-R4 ~ ~ ~ ~Rl SO3 wherein:
Rl is an alkylene radical having from l to about 4 carbon atoms, Y is any non-deleterious, chemically suitable substituen~ including ~l) hydrogen, (2) sub-stituted or unsubstituted lower alkyl, e.g.
containing 1 to 4 carbon atoms such as methyl, ethyl, propyl, or hydroxy etc.;
R2 and R3 are each selected from substituted or unsubstituted lower alkyl containing l to 4 carbon atoms, eg, such as methyl, ethyl, propyl, hydroxy ethyl, hydroxy methyl, hydroxy propyl, etc.
n=0 or 1, when n=0, R4 is substituted or unsubstituted alkyl, e.g. containing about 8 to about 18 carbon atoms, when n=l, R4 is an alkylene radical having from about l to about 6 carbon atoms, R5 is a substituted or unsubstituted alkyl, eg containing about 8 to about 18 carbon atoms;
-. ~ It is to be understood that the term "alkylene"
as it is used herein, encompasses both polymethylene radicals and other divalent saturated aliphatic radlcals ~:
' ~ .~, , . . .. ,, ~ , : ~ :
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li~9'7 and thus there may be branching in the linkage prov~ded ~y the alkylene radical. The item "lower" means a radlcal contalning 1 t~ 4 carbon atoms.
The sulfobetaines which are employed in the ~
compositions Or the present invention are known in the art and have been described as zwitterionic surfactants. The preparation Or such compounds is described, for example, by G. W. Fernley in the JOURNAL OF AMERICAN OIL CHEMISTS
- SOCIETY, January 1978 (Vol. 55), pages 98-103, and by R.
10 Ernst in the U.S. Patent 3,280,179 issued October 18, 1966. , In preferred sulfobetalne surfactants, R2 and R3 in the above structure are methyl. It is also prefered that Rl be propylene. ~ ;
One type of sulfobetaine surfactant which can be employed has the above structure wherein n equals 0 and R4 is an alkyl radical having from about 8 to 18 carbon atoms, preferably a straight chain alkyl radical. For these sulfobetaine surfactants, a convenient source of the 20 R4 component is tallo~ fatty alcohol whlch consists of a mixture Or various chain lengths, with a typical composition being approximately 66 percent Clg, 30 percent C16 and 4 percent C14 and others. Another convenient source is the middle cut Or distilled coconut 25 fatty alcohol, which also consists of a mixture of various chain lengths, with a typical composition being approximately 66 percent C12, 23 percent C14, 9 percent C16 and 2 percent Clo.
, . . . . .
Specific sulfobetaine surfactants of the above 30 structure wherein n equals O are set forth in U.S. Patent -- 3, 539,521 lssued on November 10, 1970 to A. O. Snoddy et al. A surfactant of this type particularly preferred is N-tetradecyl~N,N-dim~thyl-3-ammonio 1-propanesulfonate ...
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commercially available from Calbiochem-Behring Corporation under the trademark ZWITTERGENT 3-14.
Another type of sulfobetaine surfactant which can be employed has the above structure wherein n equals 1 and R4 is an alkylene radical having from about 1 to about 6 carbon atoms. In these sulfobetaines wherein n equals 1, R5 is an alkyl radical having from about 8 to about 18 carbon atoms. It is preferred that R5 be straight chain. As previously discussed, convenient 10 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 the _ 15 previously mentioned U.S. Patent 3,280,179.
Particularly preferred sulfobetaine surfactants for use in compositions of the present invention are 3-(N,N-dimethyl-N-acylamidopropylammonio)-2-hydroxypropane -l-sulfonates wherein the acyl group is derived from 20 tallow fatty alcohol or coconut fatty alcohol, with coconut fatty alcohol preferred. It would 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 25 lengths for the acyl groups would result. As previously discussed, these fatty alcohols contain for the most part carbon chain lengths which will provide acyl groups with ; the desired number of carbon atoms, that is from about 8 to about 18 carbon atoms. Thus, these mixtures obtained 30 from tallow or coconut fatty alcohols are useful in providing thé sulfobetaine surfactant in the compositions of the present invention. A material of this type ~ particularly preferred for u~e in the composition of the .: ..... :
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, present invention is N-cocoamido-propyl-N,N-dimethyl-N-2-hydroxypropyl sulfobetaine, an example of which is lONZAINE CS, commercially available from Lonza, Inc., Fair I,awn, New Jersey, another example of which is VARION CAS
commercially available from Sherex Chemical Company, Inc.
Other amphoteric surfactants include, the N-long chain alkyl aminocarboxyli~c acids illustrated by the formula (hereafter Formula B):
~5 - N - Rl - COOM
Y' the N-long chain alkyl iminodicarboxylic acids illustrated by the formula (hereinafter Formula C):
RsN(Rl-cOoM)2 y and the N-long chain alkyl or amido betaines illustrated by the formula (hereinafter Formula D):
(~5 - ~ - HN)n ~ R4 ~ Rl where Rl, R2, R3, R4, Y and n have the same meaning as they have in Formula A, M is hydrogen or a salt-forming metal and Y' has the same meaning as Y in Formula A. Y and Y' may be the same or dif~erent.
Examples of specific amphoteric detergents are N-alkyl-beta-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 ,~ .
* Trade Mark 1~5~?`75~
_ sodlum or other salt forms, which may likewise used in the practice Or this inventlon. Speclfic examples lnclude cocobetaine sold by Wltco Chemical Corporation under the name EMCOL CC 37-18; cocoamldopropyl betaine sold by Lonza Inc. and Sherex Chemical Company under the names LONZAINE
CO and VARION CADG, respectlvely; sodium N-coco-beta-aminopropionate sold by Henkel Corporation under the name DERIPHAT 151; disodium N-lauryl-beta-iminodiproplonate sold by Henkel Corporation 10 under the name DERIPHAT 160, and disodium N-tallow-beta-imlnodipropionate sold by Henkel Corporation under the name of DERIPHAT 154.
Examples of other amphoterlc detergents are the fatty imidazolines such as those made by reacting a long 15 chaln fatty acld (e.g. Or 10 to 20 carbon atoms) with diethylene triamlne and monohalocarboxyllc acids havlng 2 to 6 carbon atoms, e~g. 1-coco-5-hydroxyethyl-5-carboxy-methyllmldazoline.
Specific examples include cocoimidazoline 20 commercially available under the name AMPHOTERGE K-2 from Lonza, Inc., capric dicarboxy imidazoline commercially avallable under the name AMPHOTERG~ KJ2 from Lonza, Inc.
and coco dicarboxy imidazoline blended with sulfated surfactants commercially available under the name 25 AMPHOTERGE 2 WAS MOD from Lonza, Inc.
.
Other e~amples Or enhancing agents include`
anionic synthetic surfactants, generally described as those compounds which contain hydrophillc and lipophilic groups in their molecular structure and ionize in an aqueous medium to give anions contalning both the lipophilic group and hydrophilic group. -The alkyl aryl sulfonates, the alkane sulfates and sulfated oxyethylated alkyl phenols are illustrative Or the anionic type of surface actlve coMpounds.
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* Trade Mark -llS9~S4 The alkyl aryl sulfonates are a class of synthetic anlonic surface active agents represented by the general formula (hereinafter Formula E):
(R6)nl (Y)Ar (so3M)n2 R6 is a straight or branched chain hydrocarbon radical having from abou~ l to about 24 carbon atoms, at least one - R6 having at least 8 carbon atoms; nl is from l to 3;
n2 is from l to 2; Ar is a phenyl or a naphthyl radical and Y and M have the same meaning as in Formula B. R6 10 can be, for example, methyl, ethyl, hexyl, octyl, tetraoctyl, iso-octyl, nonyl, decyl, dodecyl, octadecyl and the like.
Compound illustrative of the alkyl aryl sulfonates include sodium dodecylbenzene sulfonate, 15 sodium decylbenzene sulfonate, ammonium methyl dodecylbenzene sulfonate, ammonium dodecylbenzene ~ulfonate, sodlum octadecylbenzene sulfonate, sodium nonylbenzene sulfonate, sodium dodecylnaphthalene sulfonate, sodium hetadecylbenzene sulfonate, potassium 20 eicososyl naphthalene sulfonate, ethylamine undecylnaphthalene sulfonate and sodium docosylnaphthalene sulfonate.
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The alkyl sulfates are a class of synthetic anionic surface active agents represented by the general 25 formula (hereinafter Formula F):
RsOS03M
where R5 and M have the same meaning as in Formula B.
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Compounds illustrative of alkyl sulfate class of anionic surfactants ~nclude sodium octadecyl sulfate, sodium hexadecyl sulfate, sodium dodecyl sulfate, sodium nonyl sulfate, ammonium decyl sulfate, potassium 5 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 10 represented by the general formula (hereinafter Formula G):
R5 ~ A - [CH2CH20]Z - CH2CH2 - 0S2M
, where A i8 either oxygen, sulfur, a carbonamide group, thiocarbonamlde group, a carboxylic group or 15 thiocarboxylic ester group, z is an integer from 3 to 8 and R5 and M have the same meaning as in Formula B.
Compounds illustrative of the sulfated oxyethylated alkyl phenol class of anionic surfa~tants include ammonium nonylphenoxyl tetraethylenoxy sulfate, 20 sodlum dodecylphenoxy triethyleneoxy sulfate, ethanolamine decylphenoxy tetraethyleneoxy sulfate and potassium octylphenoxy triethyleneoxy sulfate.
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.: ~ Other examples of LAL enhancing agents include . nonionic surface active compounds can be broadly described ~-. 25 as compounds which do not ionize but acquire hydrophilic ~ characteristics from an oxygenated side chain such as .~` polyoxyethylene and the lipophilic part of the molecule ~; mag come from fatty acids, phenol, alcohols, amides or amines. The compounds are usually made by reacting an : 30 alkylene oxide such as ethy~ene oxide, butylene oxlde, ' ' , , l~S~S~
propylene oxide and the llke, with fatty acids, straight or branched chain alcohols containlng one or more hydroxyl groups, phenols, thiophenols, amides and amines to form polyoxyalkylene glycoethers and esters, polyoxyalkylene 5 alkylphenols, polyoxyalkylene thiophenols, polyoxyal~ylene amides and the like. It is generally preferred to react rrom about 3 to about 30, more preferably 10 to 30, moles Or alkylene oxide per mole Or the fatty acids, alcohols, _. phenols, thiophenols, amides or amines.
.
10 Illustrative Or these nonionic surfactants are the products obtained from the reaction Or 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 15 alcohols, the ester group containlng lO 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, dlbu~yl, amyl, octyl, dodecyl, tetradecyl and 20 the like; and with an alkyl amine in which the alkyl group contains between 1 to 8 carbon atoms.
Compounds illustrative of synthetic nonionic surfactants include the products obtained from condensing ethylene oxide or propylene oxide with the 25 following: propylene glycol, ethylene diamine, diethylene glycol,.dodecyl phenol, nonyl phenol, tetradecyl.alcohol, -N-octadecyl diethanolamide, N-dodecyl monoethanolamide, polyoxyethylene (20) sorbitan monooleate sald under the name TWEEN 80 and polyoxyethylene (20) sorbitan - 30 monolaurate sold under the name TWEEN 20.
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~ Trade ~ark ~:lS9'7S4 Other nonionic surfactants include long chaln tertlary amlne oxldes correspondlng to the followlng Keneral formula thereinarter Formula H):
R5R7R8N~o~
s whereln R5 has the same meaning as in Formula A, and R7 and R8 are each methyl or ethyl radlcals. The arrow ln the formula ls a conventional representation of a semi-polar bond. Examples of amine oxldes suitable for use in this invention include dimethyldodecylamine oxide, 10 dimethyloctylamine oxide, dimethyldecylamine o~lde, dimethyltridecylamine oxide, dimethylhexadecylamine oxide.
Catlonic surface active agents may also be employed as LAL enhancing agents. Such agents are those 15 surface acti~e compounds which contain an organic hydrophobic group and a catlonic solubllizlng group.
Typ~cal catlonic solubillzing groups are amine and quaternary groups~ Such ca~ionlc surface actlve agents are represented by the following general formula o (hereinafter Formula I) --r~
~ R5 - C \ ; ~ ~
wherein R5, Y and Y' have the same meaning as in Formula C. An example is QUATERNARY O available from Ciba-Geigy Corporation.
, - Other examples of suitable synthetic cationic 30 surfactants include the diamines such as those of the formula (hereinafter Formula J):
.. . .
R9NHc2H4NH2 . .
y~ ' ' , * Trade Mark . .
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whereln R9 ls an alkyl group of about 12 to 22 carbon atoms, such as N-2-amlnoethyl stearyl amlne and N-?-aminoethyl myristyl amlne; amide-llnked amines such as those of the formula (herelnafter Formula K):
RsCONHC2H4N~3 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 nltrogen atom are alkyl groups which contaln 1 to 3 carbon atoms, 10 including such 1 to 3 carbon alkyl groups bearing inert substltutents, such as phenyl groups and there is present an anlon such as halogen, acetate, methylsulfate, etc.
Typlcal quaternary ammonlum compounds are ethyl-dlmethyl-stearyl ammonlum chlorlde, benzyl-dimethyl-stearyl :~
15 ammonium chloride, benzyldimethyl-stearyl ammonium chlorlde, trimethyl stearyl ammonium chloride, trimethyl-cetyl ammonlum bromlde, dimethylethyl dila~rylammonium chloride, dimethyl-propyl-myristyl ammonium chloride, and the correspondlng methosulfates and acetates.
Another sultable cationic surfactant is : represented by the formula (herelnafter Formula L): -.
/ (cH2cH2o)aH .
Rs - N
( CH2CH20 ) aH
25 wherein Rs has the same meaning as in Formula A and each a is an integer from 1 to 15. An example ls the . : polyethylene glycol amlne of hydrogenated tallow whereln R5 represents the tallow.radical and a+a has an average ~: : . value of 5. It ls avallable from Ciba-Geigy Corporation . 30 under the.trade name BINA COBA 3001.
* Trade Mark ..
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1~5~54 As mentloned, the lysate enhancing agent is used ln enhanclng amounts, l.e. sufflclent to neutralize or partlally neutrallze the endogenous endotoxin inhibitor ln the lysate. Generally, thls ls an amount from about 0.001 to 1.0% (w/v) preferably from about 0.01% to about -'~ ~ ~~~
0.05% (w/v) based on the total volume Or the lysate.
Frequently, amounts ln excess of an enhanclng amount lnterfere wlth the ablllty of the LAL to react wlth endotoxln durlng assay.
LAL may be prepared by those procedures known ln the art, e.g. the procedure, described in British Patent 1,522,127.
, For example, the hemolymph from healthy specimens of Limulus polyphemus ls collected in a saline 15 anticoagulant 'solution generally as described by Levln and Bang -- 'JClottable Proteln ln Llmulus: Its Localization and Klnetics of Its Coagulation by Endotoxin", m romb.
Dlath. Haemorrh. 19: 186 - 197 (1968).-- The amebocytes are collected and washed wlth the sallne antlcoagulant 20 solution with the amebocyte separated from the antlcoagu-lant by centrlfugation.
m e separated amebocytes are suspended in water and the osmotic disruption of the cells is complemented by mechanical agitation. The cellular debris is separated 25 from the lysate by centrifugation and the lysate fractions are pooled and stored at 0-4 C.
To form the LAL reagent, the aforementloned LAL
fractlons are generally buffered to a sultable pH range, e.g. 5.5 to 8.5, preferably 6.5 to 7.5 by means Or a ' - 30 sultable burfer, e.g. trls(hydroxymethyl)amlnomethane, ~' trls(hydroxymethyl)amlnomethane maleate, 1,4-plperazlne-dlethanesulfonlc acld, morphollnopropanesulronlc acld, N-2-hydroxyethylplperazlne-N'-2-ethanesulfon~c acld, .
5~
triethanolamine, imidazole and tris(hydroxymethyl)-imidazole. Then, the LAL reagent can be subdivided into serum vials, e.g. containing 1.2 or 5.2 ml. of solution and lyophilized. Normally, after lyophilization the vials are sealed and refrigerated ~1-5 C.).
Normally, in accordance with this invention the LAL is treated by adding the lysate sensitivity enhancing agent to the LAL after the LAL has been separated from the - amebocyte cellular debris. Usually, it is added prior to or at the time of preparing the LAL reagent, e.g., simultaneously with the buffer and other ingredients.
Sensitivity of the LAL reagent toward endotoxin is further increased by including low concentrations of divalent and monovalent cations. Calcium and manganese ions are the preferred divalent ions, although other alkaline earth ions such as magnesium and strontium ions or other divalent ions may be used. Magnesium and strontium ions are also preferred divalent ions. Sodium ions are the preferred monovalent ions, but other monovalent ions, especially alkali metal ions such as lithium ions may be used. The chlorides (CaC12, NaCl, etc.) are convenient sources of these added ions, although other salts may be used. Preferably these electrolytes are added in endotoxin sensitivity increasing amounts, e.g. for the divalent cation (e.g., Ca+2), the concentration will be in the range of 0.0001 - 0.4 molar ; and for the monovalent cation (e.g., Na~), the concentration will be in the range of .01 - 0.4 molar.
i The LAL reagent may also contain conventional adjuvants such as stabilizers, including lactose. These adjuvants when employed are provided in minor amounts sufficient to impart the intended qualities, but not 1, . 4 ~adverse to, the desired properties of the LAL reagents.
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1~ 5~ t All of the above operations are carried out under lysate treating conditlons, which include insuring that the final product is sterile and free of endotoxin.
Methods of insuring freedom from endotoxins are known to the art. For example, inorganic additives (CaC12,NaCl etc.) may be rendered endotoxin-free by heating the dry salts at 250C. for at least 120 minutes. Organic additives, because of their melting points, etc., must ordinarily be dissolved, rendered acidic (pH<5) or ~ 10 alkaline (pH>9), and the solution autoclaved at 121 C
for 30 - 60 minutes or more to destroy any endotoxins present.
As mentioned, another aspect of the invention is directed to a LAL reagent containing as the essential 15 ingredient an aqueous dispersion of LAL, a LAL sensitivity enhancing agent as described previously in a lysate enhanclng amount and a suitable buffer descrlbed previously ln a bufferlng amount. Optionally, monovalent and divalent catlonæ descrlbed above can be included in 20 lysate sensitivity increasing amounts to further increase the sensitivity of lysate to endotoxin.
Normally, the lysate in the reagent of this ; invention is pre~ent in an endotoxin determining amount, e.g. an amount sufficient to determine endotoxins in a 25 subsequent LAL assay for endotoxins, and generally this is an amount that will detect about 0.007 to about 0.5 ng/ml, ;~ preferably from about 0.007 to about 0.050 ng/ml of FDA
Reference Endotoxins EC-2. The aforementioned LAL reagent can be lyophilized which i~ preferred.
In accordance with this invention the LAL
reagent can be utillzed to determine endotoxin under endotoxin determining conditlons according to the usual procedure, e.g. as described ln British Patent 1,522,127, ~ and hereafter in the Examples.
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m e following examples illustrate the invention;
all parts are by weight/volume unless otherwise stated.
PREPARATION OF LIMULUS LYSATE
Limulus Lysate was prepared by modification of a procedure described originally by Levin and Bang (Thromb.
Diath. Haemorrh. 1~, 186 (1969). Horseshoe crabs, Limulus _ polyphemus, were taken from the Atlantic Ocean in the - vicinity of Beaufort, N.C. Hemolymph (approximately 500 ml) removed by cardiac puncture with a 16-gauge needle was 10 collected in an endotoxin-free one liter glass centrifuge bottle which contained 500 ml of 0.125% N-ethylmaleimide in endotoxin-free 3% saline warmed to 42C. The centrifuge bottle containing hemolymph-anticoagulant solution was warmed to 42 for-8 minutes and then 15 centrifuged at 150 x g for 10 minutes. The plasma supernatant was decanted and the amebocyte pellet was resuspended ln antlcoagulant solutlon. The cells were again pelleted by centrifugation as before. The packed cells were resuspended in 0.9% pyrogen-free saline and 20 transferred to a depyrogenated 50 ml plastic centrifuge tube. The washed cells were centrifuged again at 150 x g.
After decanting the saline, the packed amebocytes were ruptured by addition of pyrogen-free water for inJection in a ratio of 7 ml water to 3 ml packed cells. After 25 mixing on a vortex for 10-15 seconds, the lysed cells were ~`~ , stored for 24 hours at 1-5C. Cell debris was sedimented by centrifugation at 1500 x g for approximately 15 minutes. The lysate was decanted and stored at 0-4C.
; The cell debris was discarded.
~ PREPARATION OF STANDARD ~NDOTOXIN SOLUTIONS
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Standard solutions of Food and Drug Adminlstration (FDA) reference standard endotoxin Lot EC-2 - were prepared in pyrogen-fr~e water for inJection.
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Reconstitution of 1~ g endotoxin supplied in a vial with 10 ml water resulted in an initial concentration of 0.1 ~g/ml. The vial was shaken on a reciprocal shaker for 1 hour. Serial dilutions were prepared to provide the following endotoxin concentrations: 10ng/ml, lng/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.6pg/ml, and 7.8pg/ml. Once prepared, endotoxin solutions were stored up to 48 hours and then discarded.
_ Other endotoxin standards used were solutions of FDA
~ 10 Reference Endotoxin Lot No. 1 from Klebsiella pneumoniae, Escherichia coli endotoxin Lot 071857 (Difco), and a reformulation of reference Lot EC-2 prepared in our laboratory. Endotoxin standard solutions prepared from E.
coli Lot 071857 endotoxin were of the following 15 concentrations: 6.25, 12.5, 50, 75, 100, 150, and 200pg/ml.
LYSATE ASSAY PROCEDURE
Lysate dilutlons of 25 to 70% were prepared in a 0.'M buffer pH 7.0 which was usually tris, i.e., tris (hydroxymethyl)aminomethane. Other buffers used included imidazole, tris imidazole, triethanolamine, tris maleate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 1,4-piperazinediethane sulfonic acid (PIPES), and morpholinopropane sulfonic acid (MOPS).
~, To determine the sensitivity of the lysate, 0.1 ml of each of the endotoxin dilutions was combined with 0.1 ml of lysate in depyrogenated 10x75mm screw capped glass tubes and incubated for 1 hour at 37C.
Results were determined by gently inverting each tube to 180. A clot which remained intact after the inversion indlcated a positive endotoxin test.
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`-`` ~l15~754 PREPARATION AND DEPYROGENATION OF SURFACTANT SOLUTIONS
Stock LAL sensitlvlty enhancing agent solutions were prepared in concentratlons up to 10% active ingre-dlents in aqueous solution (w/v). Most rrequently, the ~~~ ~ ~~
concentration prepared was 1% (w/v). Although the procedure varied in amounts from one agent to the next, enough material was dissolved in 50 ml aqueous solution to yield the desired concentration if diluted to 100 ml. The solution also contained 7.5 ml o~ 0.05M tris(hydroxymethyl)amlnomethane 10 (l.e. TRIZMA BASE, Sigma Chemical Company) and 1 ml Or 2N
NaOH to give a final pH> 11. The agent in alkaline solution was stored for 12 hours or more at 0-4 to insure complete depyrogenation. After ad~usting the solution to approximately pH 8, it was autoclaved at > 121C at ~5 psi 15 for 15 minutes or more. m e pH was ad~usted finally to pH
7.0 + 0.5. The agent concentration was calculated and the dilutlon ad~usted to yleld the rlnal deslred concentratlon.
Alkall-labile agents were depyrogenated by acid treatment ln whlch HCl and tris burrer were substituted for NaOH and 2a TRIZMA BASE, respectlvely.
ADDITION OF AGENTS TO LYSATE
Agent solutions prepared as described above were added to the lysate during dilution with buffer. The amount added varied with the agent used, but the - 25 concentration range for all those tested was 0.001 to 1.0%
(w/v) rinal concentration in the lysate solution. The order Or addition Or components did not alter the resulting lysate sensltivity.
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* Trade Mark .' , 1:~59754 EXAMPLE I
The LAL sensitlvlty enhancing agent, ZWITTERGENT TM
3-14, N,N-dimethyl-3-ammonio-1-propanesulfonate, a ~ ~ ~ ~~
sulfobetaine sold by Calbiochem-Behring Corporation, was depyrogenated as described above and diluted to a final 1%
concentration (w/v). Lysate lot 9CZC was prepared as a _ 50% dilution with O.lM tris-maleate buffer pH 7Ø
Addition of the enhancing agent ~as carried out to yield final concentration in lysate of 0.005, 0.01, 0.02, 0.05, 10 0.075, and 0.10% (w/v). A control sample contained - pyrogen-free water instead of surfactant. The lysate dilutions were stored overnight at 0-4C and tested the following day with a specially formulated EC endotoxin series of dilutions (designated EC). The results (Table 15 I) indicate both the effective concentration range ~or enhancing agent and the total lncrease in lysate sensitivi.ty to endotoxin as compare to the control lysate.
TABLE I
-Lysate Sample Enhancing Agent (%)Sensitivity*
Control 0 500 ~, LAL plus 0.0051,000.0 ~:~ ! Enhancing Agent 0.01 500.0 0.02 31.2 : ; : 0.0510,000.0 0.07510,000.0 0.1010,000.0 *Expressed as the lowest endotoxin concentration (pg/ml)30 which yields a positlve clot test.
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EXAMPLE II
Standard endotoxin solutions were prepared for E. coli endotoxin 071857, FDA Reference Endotoxin EC-2, and K. ~~ - ~~~
pneumoniae FDA reference Lot No. 1. Lysate was diluted with O.lM tris buffer pH 7.0 and the enhancing agent~
ZWITTERGENTTM 3-14, solution to yield a 30% lysate dilution containing 0.02% ZWITTERGENT 3-14 (w/v). The agent was replaced by water in the control sample. The lysate assay was carried out with each endotoxin dilution series.
TABLE II
-Lysate Endotoxin Sample Sensitivity*
E. coli 071857 Control 75.0 LAL enhancing agent 25.0 15 FDA Reference EC-2 Control 500.0 LAL enhancing agent 62.5 FDA K. pneumonia Control 1,000.0 LAL enhancing agent 125.0 *Expressed as the lowest endotoxin concentration (pg/ml) 20 which yields a positive clot test.
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EXAMPLE III
Twenty-two commercially available LAL enhancing agents were surveyed to determine their effects on lysate sensitivity-~o endotoxin. Solutions of each were prepared and added to lysate Lot 9FI in final concentrations ranging from 0.001 to 0.20% (w/v). Lysate samples were then tested with reformulated FDA EC endotoxin (EC). In Table III, the ._, ~ agents are listed in order of decreasing effectiveness. The most effective concentration tested in lysate and its 10 corresponding lysate sensitivity are shown. The sensitivity of control lysate (50% dilution) containing no agent is also indicated.
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EXAMPLE IV
To determine that the increased sensitivity observed in _ lysate treated with the enhancing agent was maintained during lyophilization, 30% dilution of lysate in 0.05M
tris buffer with and without 0.02% ZWITTERGENTTM 3-14 (final concentration in lysate) were prepared. Lysate - solution (1.2ml) was dispensed into each lO-ml serum vial.
Samples were frozen at -35C and lyophilized under 5Q~
vacuum with a drying time of approximately 32 hours. The 10 vials were sealed with split rubber stoppers and metal caps. The freeze-dried lysate was reconstituted with 1.2ml pyrogen-free water for in~ection and then tested wlth FDA Reference Endotoxin lot EC-2. Control lysate without the enhancing agent had a sensltivity o~
15 62.5pg/ml. In the presence of the enhancing agent, the sensltlvlty of the lysate was lmproved by twofold to 31.2 ; pg/ml.
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Limulus Lysate Day Pool Lot ODH approximately one week old was prepared as a 40% dilution in 0.05M tris(hydroxymethyl) aminomethane maleate buffer, pH 7.0, containing in final concentrations 0.06M CaCl2 and 0.01M MnCl2 and 0.03%
ZWITTERGENTTM 3-14. This solution was dispensed as 1.2 ml aliquots into 8 ml vials and frozen at -45C and _ lyophilized under 50u vacuum with a drying time of approximately 28 hours. The vials were sealed with split - 10 rubber stoppers and capped with plastic screw caps. The freeze dried lysate was reconstituted with 1.2 ml pyrogen-free water for injection and tested with FDA
Reference Endotoxin lot EC-2. Sensitivity of enhancing agent treated lyophilized lysate was 62 pg/ml. A control 40% dilution of LAL without enhancing agent tested before lyophilization had a sensitivity of l ng/ml.
EXAMPLE VI
Limulus Lysate Day Pools of a sensitivity equal to or greater than 500 pg/ml E. coli endotoxin lCF were combined and prepared as a 40% dilution in 0.025M tris(hydroxy-methyl) aminomethane maleate buffer, pH 7.0, containing in final concentrations 0.02M MgC12, 0.01M SrCl2, 0.01M
CaCl2, and 0. 025% ZWITTERGENT~ 3-14. This solution was dispensed in l. 2 ml and 5.2 ml aliquots in 10 ml vials and frozen at -50C. The samples were lyophilized under 100 vacuum with a drying time of approximately 72 hours. The lyophilized product was reconstituted with 1.2 ml or 5.2 ml pyrogen-free Water for Injection depending on the starting volume of lysate. When tested with E. coli endotoxin lCF following lyophilization, the sensitivity of the enhancing agent treated lyophilized lysate for both sample sizes was 25 pg/ml in comparison to the control sensitlvity of 500 pg/ml.
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LETTERS PATENT
FOR
IMPROVEMENT IN THE PROCESS FOR
PREPARING LIMULUS LYSATE
-This invention relates to a process for improving the sensitivity of Limulus amebocyte lysate (hereinafter sometimes referred to as LAL or lysate) to endotoxin, to an improved LAL reagent and to the use of sùch LAL reagent.
As is well known, the LAL test for detecting endotoxins i5 perhaps the most practical and sensitive test for determining endotoxins. Commercial assay tests employ amebocyte lysate from Limulus hemolymph obtained from the horseshoe crabs. This lysate is combined with lS appropriate divalent cations, appropriate buffers and other ingredients to form a LAL reayent. This reagent then reacts with endotoxin during the assay to form a gel.
Manufacturers of LAL reagents often experience difficulty in producing lysate of the desired sensitivity for detecting endotoxin. Sensitivity from one preparation to the next is also variable. These problems are attributed at least in part to the presence of an endogenous, undefined endotoxin inhibitor substance in the lysate, hereinafter sometimes referred to as the inhibitor).
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Little is known of the nature of the inhibitor or its in-vivo role in the horseshoe crab. Electro-.. ...
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phoretic studies indicate that the inhibitor is a high molecular weight lipoprotein. It may function in the amebocyte to control the coagulation defense mechanism.
That the inhibitor may be a membrane component freed during cell lysis is also plausible. The uncertainty of ~~ ~ ~~
the role and origin of the inhibitor is compounded by the fact that the mechanism of inhibition is unclear. The inhibitor presumably blocks the enzymatic reaction in some ~ fashion either by association with the enzyme itself, or 10 with the endotoxin or both. Like some other serine pro-teases, the proclotting enzyme is thought to be complexed with calcium and glycerophospholipid. Endotoxin itself is lipoidal, hence, an inhibitor of lipoprotein character would be highly compatible with either component.
That the inhibitor is a lipoprotein is supported by its sensitivity to chloroform. As described in U.S.
Patent No. 4,107,077, the sensitlvlty of LAL is improved substantlally when lysate is treated with an organic solvent such as chloroform to precipitate inhibitor from the lysate. The aqueous phase is then recovered and processed to prepare the LAL reagent.
To date, the above-mentioned solvent extraction procedure is the most rapid means of improving LAL sensi-tivity. Unfortunately, the method has several drawbacks.
Because o~ the absolute requirement that endotoxin-free ~ conditions be maintained throughout the lysate production, ; a cumbersome extraction procedure and subsequent centrifi-cation increases the likelihood of product failure. As noted in the patent, the solvent treatment reduces lysate ~; 30 stability such that the production must be completed ` rapidly in the cold. Also the precipitate removed from ; the lysate by the solvent treatment contains considerable coagulogen, the requlred clotting protein. Malntenance of ~ adequate protein content is a requirement for firm ,, ,,.. , .... - - .
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gelation during endotoxin assay. Obviously, under the latter circumstances, control of reagent sensitivity is difficult. Chloroform, the solvent used most successfully, is well known for its undesirable effects in 5 man. The health and safety of production personnel is therefore a reasonable concern. It is apparent that a process which avoids these pitfalls and yet improves sensitivity to the desired degree would be an improvement in the art.
-It is therefore an ob~ect of the present inven-tion to provide a method for the simple and rapid enhance-ment of the sensitivity of LA~.
In accordance with this invention, there is pro-vided a process for treating under lysate treating ~
15 conditions LAL havlng decreased sensitivity to endotoxin due to the presence of an endogenous inhibitor with an enhancing amount of a lysate sensitivity enhancing agent to neutralize or partially neutralize the lysate inhibitor thereby increasing the LAL sensitivity to endotoxin.
There are certain minimal criteria whlch can delineate LAL sensitivity enhancing characteristics, i.e., the LAL sensitivity enhancing agents useful in the process of this invention should possess (a) the ability to increase the lysate sensitivity to a suitable sensitivity, 25 e.g., by a twofold or greater increase, (b) the ability to withstand depyrogenation, i.e. removal or destruction of endotoxin, by ultrafiltratlon or acid treatment at pH less than 5 or base treatment greater than pH 8, (c) the ability to be sterilized e.g.,by autoclaving e.g. at or 30 above 121C at 15 psi for 15 minutes, (d) the ability to form aqueous solutions of about 2% (w/v) at 25C, (e) the ability to function in the pH range of about 6.0 to about 9, (f) the ability to be compatible with buffers and other ::, ' -- ~ - , .
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' ~ ' ingredients utilized in the LAL reagent and (g) the ability to be compatible with respect to LAL and its reaction with endotoxins.
Such enhancing agents includ~ amphoteric surfac-tants which have both an anionic and cationic group in their structure. Illustrative are the sulfobetaines represented by the following formula (hereinafter Formula A:
' O R2 (R5-C-HN)n-R4 ~ ~ ~ ~Rl SO3 wherein:
Rl is an alkylene radical having from l to about 4 carbon atoms, Y is any non-deleterious, chemically suitable substituen~ including ~l) hydrogen, (2) sub-stituted or unsubstituted lower alkyl, e.g.
containing 1 to 4 carbon atoms such as methyl, ethyl, propyl, or hydroxy etc.;
R2 and R3 are each selected from substituted or unsubstituted lower alkyl containing l to 4 carbon atoms, eg, such as methyl, ethyl, propyl, hydroxy ethyl, hydroxy methyl, hydroxy propyl, etc.
n=0 or 1, when n=0, R4 is substituted or unsubstituted alkyl, e.g. containing about 8 to about 18 carbon atoms, when n=l, R4 is an alkylene radical having from about l to about 6 carbon atoms, R5 is a substituted or unsubstituted alkyl, eg containing about 8 to about 18 carbon atoms;
-. ~ It is to be understood that the term "alkylene"
as it is used herein, encompasses both polymethylene radicals and other divalent saturated aliphatic radlcals ~:
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li~9'7 and thus there may be branching in the linkage prov~ded ~y the alkylene radical. The item "lower" means a radlcal contalning 1 t~ 4 carbon atoms.
The sulfobetaines which are employed in the ~
compositions Or the present invention are known in the art and have been described as zwitterionic surfactants. The preparation Or such compounds is described, for example, by G. W. Fernley in the JOURNAL OF AMERICAN OIL CHEMISTS
- SOCIETY, January 1978 (Vol. 55), pages 98-103, and by R.
10 Ernst in the U.S. Patent 3,280,179 issued October 18, 1966. , In preferred sulfobetalne surfactants, R2 and R3 in the above structure are methyl. It is also prefered that Rl be propylene. ~ ;
One type of sulfobetaine surfactant which can be employed has the above structure wherein n equals 0 and R4 is an alkyl radical having from about 8 to 18 carbon atoms, preferably a straight chain alkyl radical. For these sulfobetaine surfactants, a convenient source of the 20 R4 component is tallo~ fatty alcohol whlch consists of a mixture Or various chain lengths, with a typical composition being approximately 66 percent Clg, 30 percent C16 and 4 percent C14 and others. Another convenient source is the middle cut Or distilled coconut 25 fatty alcohol, which also consists of a mixture of various chain lengths, with a typical composition being approximately 66 percent C12, 23 percent C14, 9 percent C16 and 2 percent Clo.
, . . . . .
Specific sulfobetaine surfactants of the above 30 structure wherein n equals O are set forth in U.S. Patent -- 3, 539,521 lssued on November 10, 1970 to A. O. Snoddy et al. A surfactant of this type particularly preferred is N-tetradecyl~N,N-dim~thyl-3-ammonio 1-propanesulfonate ...
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commercially available from Calbiochem-Behring Corporation under the trademark ZWITTERGENT 3-14.
Another type of sulfobetaine surfactant which can be employed has the above structure wherein n equals 1 and R4 is an alkylene radical having from about 1 to about 6 carbon atoms. In these sulfobetaines wherein n equals 1, R5 is an alkyl radical having from about 8 to about 18 carbon atoms. It is preferred that R5 be straight chain. As previously discussed, convenient 10 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 the _ 15 previously mentioned U.S. Patent 3,280,179.
Particularly preferred sulfobetaine surfactants for use in compositions of the present invention are 3-(N,N-dimethyl-N-acylamidopropylammonio)-2-hydroxypropane -l-sulfonates wherein the acyl group is derived from 20 tallow fatty alcohol or coconut fatty alcohol, with coconut fatty alcohol preferred. It would 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 25 lengths for the acyl groups would result. As previously discussed, these fatty alcohols contain for the most part carbon chain lengths which will provide acyl groups with ; the desired number of carbon atoms, that is from about 8 to about 18 carbon atoms. Thus, these mixtures obtained 30 from tallow or coconut fatty alcohols are useful in providing thé sulfobetaine surfactant in the compositions of the present invention. A material of this type ~ particularly preferred for u~e in the composition of the .: ..... :
11S~7S~
, present invention is N-cocoamido-propyl-N,N-dimethyl-N-2-hydroxypropyl sulfobetaine, an example of which is lONZAINE CS, commercially available from Lonza, Inc., Fair I,awn, New Jersey, another example of which is VARION CAS
commercially available from Sherex Chemical Company, Inc.
Other amphoteric surfactants include, the N-long chain alkyl aminocarboxyli~c acids illustrated by the formula (hereafter Formula B):
~5 - N - Rl - COOM
Y' the N-long chain alkyl iminodicarboxylic acids illustrated by the formula (hereinafter Formula C):
RsN(Rl-cOoM)2 y and the N-long chain alkyl or amido betaines illustrated by the formula (hereinafter Formula D):
(~5 - ~ - HN)n ~ R4 ~ Rl where Rl, R2, R3, R4, Y and n have the same meaning as they have in Formula A, M is hydrogen or a salt-forming metal and Y' has the same meaning as Y in Formula A. Y and Y' may be the same or dif~erent.
Examples of specific amphoteric detergents are N-alkyl-beta-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 ,~ .
* Trade Mark 1~5~?`75~
_ sodlum or other salt forms, which may likewise used in the practice Or this inventlon. Speclfic examples lnclude cocobetaine sold by Wltco Chemical Corporation under the name EMCOL CC 37-18; cocoamldopropyl betaine sold by Lonza Inc. and Sherex Chemical Company under the names LONZAINE
CO and VARION CADG, respectlvely; sodium N-coco-beta-aminopropionate sold by Henkel Corporation under the name DERIPHAT 151; disodium N-lauryl-beta-iminodiproplonate sold by Henkel Corporation 10 under the name DERIPHAT 160, and disodium N-tallow-beta-imlnodipropionate sold by Henkel Corporation under the name of DERIPHAT 154.
Examples of other amphoterlc detergents are the fatty imidazolines such as those made by reacting a long 15 chaln fatty acld (e.g. Or 10 to 20 carbon atoms) with diethylene triamlne and monohalocarboxyllc acids havlng 2 to 6 carbon atoms, e~g. 1-coco-5-hydroxyethyl-5-carboxy-methyllmldazoline.
Specific examples include cocoimidazoline 20 commercially available under the name AMPHOTERGE K-2 from Lonza, Inc., capric dicarboxy imidazoline commercially avallable under the name AMPHOTERG~ KJ2 from Lonza, Inc.
and coco dicarboxy imidazoline blended with sulfated surfactants commercially available under the name 25 AMPHOTERGE 2 WAS MOD from Lonza, Inc.
.
Other e~amples Or enhancing agents include`
anionic synthetic surfactants, generally described as those compounds which contain hydrophillc and lipophilic groups in their molecular structure and ionize in an aqueous medium to give anions contalning both the lipophilic group and hydrophilic group. -The alkyl aryl sulfonates, the alkane sulfates and sulfated oxyethylated alkyl phenols are illustrative Or the anionic type of surface actlve coMpounds.
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* Trade Mark -llS9~S4 The alkyl aryl sulfonates are a class of synthetic anlonic surface active agents represented by the general formula (hereinafter Formula E):
(R6)nl (Y)Ar (so3M)n2 R6 is a straight or branched chain hydrocarbon radical having from abou~ l to about 24 carbon atoms, at least one - R6 having at least 8 carbon atoms; nl is from l to 3;
n2 is from l to 2; Ar is a phenyl or a naphthyl radical and Y and M have the same meaning as in Formula B. R6 10 can be, for example, methyl, ethyl, hexyl, octyl, tetraoctyl, iso-octyl, nonyl, decyl, dodecyl, octadecyl and the like.
Compound illustrative of the alkyl aryl sulfonates include sodium dodecylbenzene sulfonate, 15 sodium decylbenzene sulfonate, ammonium methyl dodecylbenzene sulfonate, ammonium dodecylbenzene ~ulfonate, sodlum octadecylbenzene sulfonate, sodium nonylbenzene sulfonate, sodium dodecylnaphthalene sulfonate, sodium hetadecylbenzene sulfonate, potassium 20 eicososyl naphthalene sulfonate, ethylamine undecylnaphthalene sulfonate and sodium docosylnaphthalene sulfonate.
~: .
The alkyl sulfates are a class of synthetic anionic surface active agents represented by the general 25 formula (hereinafter Formula F):
RsOS03M
where R5 and M have the same meaning as in Formula B.
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Compounds illustrative of alkyl sulfate class of anionic surfactants ~nclude sodium octadecyl sulfate, sodium hexadecyl sulfate, sodium dodecyl sulfate, sodium nonyl sulfate, ammonium decyl sulfate, potassium 5 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 10 represented by the general formula (hereinafter Formula G):
R5 ~ A - [CH2CH20]Z - CH2CH2 - 0S2M
, where A i8 either oxygen, sulfur, a carbonamide group, thiocarbonamlde group, a carboxylic group or 15 thiocarboxylic ester group, z is an integer from 3 to 8 and R5 and M have the same meaning as in Formula B.
Compounds illustrative of the sulfated oxyethylated alkyl phenol class of anionic surfa~tants include ammonium nonylphenoxyl tetraethylenoxy sulfate, 20 sodlum dodecylphenoxy triethyleneoxy sulfate, ethanolamine decylphenoxy tetraethyleneoxy sulfate and potassium octylphenoxy triethyleneoxy sulfate.
`: `
.: ~ Other examples of LAL enhancing agents include . nonionic surface active compounds can be broadly described ~-. 25 as compounds which do not ionize but acquire hydrophilic ~ characteristics from an oxygenated side chain such as .~` polyoxyethylene and the lipophilic part of the molecule ~; mag come from fatty acids, phenol, alcohols, amides or amines. The compounds are usually made by reacting an : 30 alkylene oxide such as ethy~ene oxide, butylene oxlde, ' ' , , l~S~S~
propylene oxide and the llke, with fatty acids, straight or branched chain alcohols containlng one or more hydroxyl groups, phenols, thiophenols, amides and amines to form polyoxyalkylene glycoethers and esters, polyoxyalkylene 5 alkylphenols, polyoxyalkylene thiophenols, polyoxyal~ylene amides and the like. It is generally preferred to react rrom about 3 to about 30, more preferably 10 to 30, moles Or alkylene oxide per mole Or the fatty acids, alcohols, _. phenols, thiophenols, amides or amines.
.
10 Illustrative Or these nonionic surfactants are the products obtained from the reaction Or 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 15 alcohols, the ester group containlng lO 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, dlbu~yl, amyl, octyl, dodecyl, tetradecyl and 20 the like; and with an alkyl amine in which the alkyl group contains between 1 to 8 carbon atoms.
Compounds illustrative of synthetic nonionic surfactants include the products obtained from condensing ethylene oxide or propylene oxide with the 25 following: propylene glycol, ethylene diamine, diethylene glycol,.dodecyl phenol, nonyl phenol, tetradecyl.alcohol, -N-octadecyl diethanolamide, N-dodecyl monoethanolamide, polyoxyethylene (20) sorbitan monooleate sald under the name TWEEN 80 and polyoxyethylene (20) sorbitan - 30 monolaurate sold under the name TWEEN 20.
.
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~ Trade ~ark ~:lS9'7S4 Other nonionic surfactants include long chaln tertlary amlne oxldes correspondlng to the followlng Keneral formula thereinarter Formula H):
R5R7R8N~o~
s whereln R5 has the same meaning as in Formula A, and R7 and R8 are each methyl or ethyl radlcals. The arrow ln the formula ls a conventional representation of a semi-polar bond. Examples of amine oxldes suitable for use in this invention include dimethyldodecylamine oxide, 10 dimethyloctylamine oxide, dimethyldecylamine o~lde, dimethyltridecylamine oxide, dimethylhexadecylamine oxide.
Catlonic surface active agents may also be employed as LAL enhancing agents. Such agents are those 15 surface acti~e compounds which contain an organic hydrophobic group and a catlonic solubllizlng group.
Typ~cal catlonic solubillzing groups are amine and quaternary groups~ Such ca~ionlc surface actlve agents are represented by the following general formula o (hereinafter Formula I) --r~
~ R5 - C \ ; ~ ~
wherein R5, Y and Y' have the same meaning as in Formula C. An example is QUATERNARY O available from Ciba-Geigy Corporation.
, - Other examples of suitable synthetic cationic 30 surfactants include the diamines such as those of the formula (hereinafter Formula J):
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R9NHc2H4NH2 . .
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whereln R9 ls an alkyl group of about 12 to 22 carbon atoms, such as N-2-amlnoethyl stearyl amlne and N-?-aminoethyl myristyl amlne; amide-llnked amines such as those of the formula (herelnafter Formula K):
RsCONHC2H4N~3 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 nltrogen atom are alkyl groups which contaln 1 to 3 carbon atoms, 10 including such 1 to 3 carbon alkyl groups bearing inert substltutents, such as phenyl groups and there is present an anlon such as halogen, acetate, methylsulfate, etc.
Typlcal quaternary ammonlum compounds are ethyl-dlmethyl-stearyl ammonlum chlorlde, benzyl-dimethyl-stearyl :~
15 ammonium chloride, benzyldimethyl-stearyl ammonium chlorlde, trimethyl stearyl ammonium chloride, trimethyl-cetyl ammonlum bromlde, dimethylethyl dila~rylammonium chloride, dimethyl-propyl-myristyl ammonium chloride, and the correspondlng methosulfates and acetates.
Another sultable cationic surfactant is : represented by the formula (herelnafter Formula L): -.
/ (cH2cH2o)aH .
Rs - N
( CH2CH20 ) aH
25 wherein Rs has the same meaning as in Formula A and each a is an integer from 1 to 15. An example ls the . : polyethylene glycol amlne of hydrogenated tallow whereln R5 represents the tallow.radical and a+a has an average ~: : . value of 5. It ls avallable from Ciba-Geigy Corporation . 30 under the.trade name BINA COBA 3001.
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1~5~54 As mentloned, the lysate enhancing agent is used ln enhanclng amounts, l.e. sufflclent to neutralize or partlally neutrallze the endogenous endotoxin inhibitor ln the lysate. Generally, thls ls an amount from about 0.001 to 1.0% (w/v) preferably from about 0.01% to about -'~ ~ ~~~
0.05% (w/v) based on the total volume Or the lysate.
Frequently, amounts ln excess of an enhanclng amount lnterfere wlth the ablllty of the LAL to react wlth endotoxln durlng assay.
LAL may be prepared by those procedures known ln the art, e.g. the procedure, described in British Patent 1,522,127.
, For example, the hemolymph from healthy specimens of Limulus polyphemus ls collected in a saline 15 anticoagulant 'solution generally as described by Levln and Bang -- 'JClottable Proteln ln Llmulus: Its Localization and Klnetics of Its Coagulation by Endotoxin", m romb.
Dlath. Haemorrh. 19: 186 - 197 (1968).-- The amebocytes are collected and washed wlth the sallne antlcoagulant 20 solution with the amebocyte separated from the antlcoagu-lant by centrlfugation.
m e separated amebocytes are suspended in water and the osmotic disruption of the cells is complemented by mechanical agitation. The cellular debris is separated 25 from the lysate by centrifugation and the lysate fractions are pooled and stored at 0-4 C.
To form the LAL reagent, the aforementloned LAL
fractlons are generally buffered to a sultable pH range, e.g. 5.5 to 8.5, preferably 6.5 to 7.5 by means Or a ' - 30 sultable burfer, e.g. trls(hydroxymethyl)amlnomethane, ~' trls(hydroxymethyl)amlnomethane maleate, 1,4-plperazlne-dlethanesulfonlc acld, morphollnopropanesulronlc acld, N-2-hydroxyethylplperazlne-N'-2-ethanesulfon~c acld, .
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triethanolamine, imidazole and tris(hydroxymethyl)-imidazole. Then, the LAL reagent can be subdivided into serum vials, e.g. containing 1.2 or 5.2 ml. of solution and lyophilized. Normally, after lyophilization the vials are sealed and refrigerated ~1-5 C.).
Normally, in accordance with this invention the LAL is treated by adding the lysate sensitivity enhancing agent to the LAL after the LAL has been separated from the - amebocyte cellular debris. Usually, it is added prior to or at the time of preparing the LAL reagent, e.g., simultaneously with the buffer and other ingredients.
Sensitivity of the LAL reagent toward endotoxin is further increased by including low concentrations of divalent and monovalent cations. Calcium and manganese ions are the preferred divalent ions, although other alkaline earth ions such as magnesium and strontium ions or other divalent ions may be used. Magnesium and strontium ions are also preferred divalent ions. Sodium ions are the preferred monovalent ions, but other monovalent ions, especially alkali metal ions such as lithium ions may be used. The chlorides (CaC12, NaCl, etc.) are convenient sources of these added ions, although other salts may be used. Preferably these electrolytes are added in endotoxin sensitivity increasing amounts, e.g. for the divalent cation (e.g., Ca+2), the concentration will be in the range of 0.0001 - 0.4 molar ; and for the monovalent cation (e.g., Na~), the concentration will be in the range of .01 - 0.4 molar.
i The LAL reagent may also contain conventional adjuvants such as stabilizers, including lactose. These adjuvants when employed are provided in minor amounts sufficient to impart the intended qualities, but not 1, . 4 ~adverse to, the desired properties of the LAL reagents.
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1~ 5~ t All of the above operations are carried out under lysate treating conditlons, which include insuring that the final product is sterile and free of endotoxin.
Methods of insuring freedom from endotoxins are known to the art. For example, inorganic additives (CaC12,NaCl etc.) may be rendered endotoxin-free by heating the dry salts at 250C. for at least 120 minutes. Organic additives, because of their melting points, etc., must ordinarily be dissolved, rendered acidic (pH<5) or ~ 10 alkaline (pH>9), and the solution autoclaved at 121 C
for 30 - 60 minutes or more to destroy any endotoxins present.
As mentioned, another aspect of the invention is directed to a LAL reagent containing as the essential 15 ingredient an aqueous dispersion of LAL, a LAL sensitivity enhancing agent as described previously in a lysate enhanclng amount and a suitable buffer descrlbed previously ln a bufferlng amount. Optionally, monovalent and divalent catlonæ descrlbed above can be included in 20 lysate sensitivity increasing amounts to further increase the sensitivity of lysate to endotoxin.
Normally, the lysate in the reagent of this ; invention is pre~ent in an endotoxin determining amount, e.g. an amount sufficient to determine endotoxins in a 25 subsequent LAL assay for endotoxins, and generally this is an amount that will detect about 0.007 to about 0.5 ng/ml, ;~ preferably from about 0.007 to about 0.050 ng/ml of FDA
Reference Endotoxins EC-2. The aforementioned LAL reagent can be lyophilized which i~ preferred.
In accordance with this invention the LAL
reagent can be utillzed to determine endotoxin under endotoxin determining conditlons according to the usual procedure, e.g. as described ln British Patent 1,522,127, ~ and hereafter in the Examples.
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m e following examples illustrate the invention;
all parts are by weight/volume unless otherwise stated.
PREPARATION OF LIMULUS LYSATE
Limulus Lysate was prepared by modification of a procedure described originally by Levin and Bang (Thromb.
Diath. Haemorrh. 1~, 186 (1969). Horseshoe crabs, Limulus _ polyphemus, were taken from the Atlantic Ocean in the - vicinity of Beaufort, N.C. Hemolymph (approximately 500 ml) removed by cardiac puncture with a 16-gauge needle was 10 collected in an endotoxin-free one liter glass centrifuge bottle which contained 500 ml of 0.125% N-ethylmaleimide in endotoxin-free 3% saline warmed to 42C. The centrifuge bottle containing hemolymph-anticoagulant solution was warmed to 42 for-8 minutes and then 15 centrifuged at 150 x g for 10 minutes. The plasma supernatant was decanted and the amebocyte pellet was resuspended ln antlcoagulant solutlon. The cells were again pelleted by centrifugation as before. The packed cells were resuspended in 0.9% pyrogen-free saline and 20 transferred to a depyrogenated 50 ml plastic centrifuge tube. The washed cells were centrifuged again at 150 x g.
After decanting the saline, the packed amebocytes were ruptured by addition of pyrogen-free water for inJection in a ratio of 7 ml water to 3 ml packed cells. After 25 mixing on a vortex for 10-15 seconds, the lysed cells were ~`~ , stored for 24 hours at 1-5C. Cell debris was sedimented by centrifugation at 1500 x g for approximately 15 minutes. The lysate was decanted and stored at 0-4C.
; The cell debris was discarded.
~ PREPARATION OF STANDARD ~NDOTOXIN SOLUTIONS
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Standard solutions of Food and Drug Adminlstration (FDA) reference standard endotoxin Lot EC-2 - were prepared in pyrogen-fr~e water for inJection.
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Reconstitution of 1~ g endotoxin supplied in a vial with 10 ml water resulted in an initial concentration of 0.1 ~g/ml. The vial was shaken on a reciprocal shaker for 1 hour. Serial dilutions were prepared to provide the following endotoxin concentrations: 10ng/ml, lng/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.6pg/ml, and 7.8pg/ml. Once prepared, endotoxin solutions were stored up to 48 hours and then discarded.
_ Other endotoxin standards used were solutions of FDA
~ 10 Reference Endotoxin Lot No. 1 from Klebsiella pneumoniae, Escherichia coli endotoxin Lot 071857 (Difco), and a reformulation of reference Lot EC-2 prepared in our laboratory. Endotoxin standard solutions prepared from E.
coli Lot 071857 endotoxin were of the following 15 concentrations: 6.25, 12.5, 50, 75, 100, 150, and 200pg/ml.
LYSATE ASSAY PROCEDURE
Lysate dilutlons of 25 to 70% were prepared in a 0.'M buffer pH 7.0 which was usually tris, i.e., tris (hydroxymethyl)aminomethane. Other buffers used included imidazole, tris imidazole, triethanolamine, tris maleate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 1,4-piperazinediethane sulfonic acid (PIPES), and morpholinopropane sulfonic acid (MOPS).
~, To determine the sensitivity of the lysate, 0.1 ml of each of the endotoxin dilutions was combined with 0.1 ml of lysate in depyrogenated 10x75mm screw capped glass tubes and incubated for 1 hour at 37C.
Results were determined by gently inverting each tube to 180. A clot which remained intact after the inversion indlcated a positive endotoxin test.
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`-`` ~l15~754 PREPARATION AND DEPYROGENATION OF SURFACTANT SOLUTIONS
Stock LAL sensitlvlty enhancing agent solutions were prepared in concentratlons up to 10% active ingre-dlents in aqueous solution (w/v). Most rrequently, the ~~~ ~ ~~
concentration prepared was 1% (w/v). Although the procedure varied in amounts from one agent to the next, enough material was dissolved in 50 ml aqueous solution to yield the desired concentration if diluted to 100 ml. The solution also contained 7.5 ml o~ 0.05M tris(hydroxymethyl)amlnomethane 10 (l.e. TRIZMA BASE, Sigma Chemical Company) and 1 ml Or 2N
NaOH to give a final pH> 11. The agent in alkaline solution was stored for 12 hours or more at 0-4 to insure complete depyrogenation. After ad~usting the solution to approximately pH 8, it was autoclaved at > 121C at ~5 psi 15 for 15 minutes or more. m e pH was ad~usted finally to pH
7.0 + 0.5. The agent concentration was calculated and the dilutlon ad~usted to yleld the rlnal deslred concentratlon.
Alkall-labile agents were depyrogenated by acid treatment ln whlch HCl and tris burrer were substituted for NaOH and 2a TRIZMA BASE, respectlvely.
ADDITION OF AGENTS TO LYSATE
Agent solutions prepared as described above were added to the lysate during dilution with buffer. The amount added varied with the agent used, but the - 25 concentration range for all those tested was 0.001 to 1.0%
(w/v) rinal concentration in the lysate solution. The order Or addition Or components did not alter the resulting lysate sensltivity.
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* Trade Mark .' , 1:~59754 EXAMPLE I
The LAL sensitlvlty enhancing agent, ZWITTERGENT TM
3-14, N,N-dimethyl-3-ammonio-1-propanesulfonate, a ~ ~ ~ ~~
sulfobetaine sold by Calbiochem-Behring Corporation, was depyrogenated as described above and diluted to a final 1%
concentration (w/v). Lysate lot 9CZC was prepared as a _ 50% dilution with O.lM tris-maleate buffer pH 7Ø
Addition of the enhancing agent ~as carried out to yield final concentration in lysate of 0.005, 0.01, 0.02, 0.05, 10 0.075, and 0.10% (w/v). A control sample contained - pyrogen-free water instead of surfactant. The lysate dilutions were stored overnight at 0-4C and tested the following day with a specially formulated EC endotoxin series of dilutions (designated EC). The results (Table 15 I) indicate both the effective concentration range ~or enhancing agent and the total lncrease in lysate sensitivi.ty to endotoxin as compare to the control lysate.
TABLE I
-Lysate Sample Enhancing Agent (%)Sensitivity*
Control 0 500 ~, LAL plus 0.0051,000.0 ~:~ ! Enhancing Agent 0.01 500.0 0.02 31.2 : ; : 0.0510,000.0 0.07510,000.0 0.1010,000.0 *Expressed as the lowest endotoxin concentration (pg/ml)30 which yields a positlve clot test.
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EXAMPLE II
Standard endotoxin solutions were prepared for E. coli endotoxin 071857, FDA Reference Endotoxin EC-2, and K. ~~ - ~~~
pneumoniae FDA reference Lot No. 1. Lysate was diluted with O.lM tris buffer pH 7.0 and the enhancing agent~
ZWITTERGENTTM 3-14, solution to yield a 30% lysate dilution containing 0.02% ZWITTERGENT 3-14 (w/v). The agent was replaced by water in the control sample. The lysate assay was carried out with each endotoxin dilution series.
TABLE II
-Lysate Endotoxin Sample Sensitivity*
E. coli 071857 Control 75.0 LAL enhancing agent 25.0 15 FDA Reference EC-2 Control 500.0 LAL enhancing agent 62.5 FDA K. pneumonia Control 1,000.0 LAL enhancing agent 125.0 *Expressed as the lowest endotoxin concentration (pg/ml) 20 which yields a positive clot test.
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EXAMPLE III
Twenty-two commercially available LAL enhancing agents were surveyed to determine their effects on lysate sensitivity-~o endotoxin. Solutions of each were prepared and added to lysate Lot 9FI in final concentrations ranging from 0.001 to 0.20% (w/v). Lysate samples were then tested with reformulated FDA EC endotoxin (EC). In Table III, the ._, ~ agents are listed in order of decreasing effectiveness. The most effective concentration tested in lysate and its 10 corresponding lysate sensitivity are shown. The sensitivity of control lysate (50% dilution) containing no agent is also indicated.
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EXAMPLE IV
To determine that the increased sensitivity observed in _ lysate treated with the enhancing agent was maintained during lyophilization, 30% dilution of lysate in 0.05M
tris buffer with and without 0.02% ZWITTERGENTTM 3-14 (final concentration in lysate) were prepared. Lysate - solution (1.2ml) was dispensed into each lO-ml serum vial.
Samples were frozen at -35C and lyophilized under 5Q~
vacuum with a drying time of approximately 32 hours. The 10 vials were sealed with split rubber stoppers and metal caps. The freeze-dried lysate was reconstituted with 1.2ml pyrogen-free water for in~ection and then tested wlth FDA Reference Endotoxin lot EC-2. Control lysate without the enhancing agent had a sensltivity o~
15 62.5pg/ml. In the presence of the enhancing agent, the sensltlvlty of the lysate was lmproved by twofold to 31.2 ; pg/ml.
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Limulus Lysate Day Pool Lot ODH approximately one week old was prepared as a 40% dilution in 0.05M tris(hydroxymethyl) aminomethane maleate buffer, pH 7.0, containing in final concentrations 0.06M CaCl2 and 0.01M MnCl2 and 0.03%
ZWITTERGENTTM 3-14. This solution was dispensed as 1.2 ml aliquots into 8 ml vials and frozen at -45C and _ lyophilized under 50u vacuum with a drying time of approximately 28 hours. The vials were sealed with split - 10 rubber stoppers and capped with plastic screw caps. The freeze dried lysate was reconstituted with 1.2 ml pyrogen-free water for injection and tested with FDA
Reference Endotoxin lot EC-2. Sensitivity of enhancing agent treated lyophilized lysate was 62 pg/ml. A control 40% dilution of LAL without enhancing agent tested before lyophilization had a sensitivity of l ng/ml.
EXAMPLE VI
Limulus Lysate Day Pools of a sensitivity equal to or greater than 500 pg/ml E. coli endotoxin lCF were combined and prepared as a 40% dilution in 0.025M tris(hydroxy-methyl) aminomethane maleate buffer, pH 7.0, containing in final concentrations 0.02M MgC12, 0.01M SrCl2, 0.01M
CaCl2, and 0. 025% ZWITTERGENT~ 3-14. This solution was dispensed in l. 2 ml and 5.2 ml aliquots in 10 ml vials and frozen at -50C. The samples were lyophilized under 100 vacuum with a drying time of approximately 72 hours. The lyophilized product was reconstituted with 1.2 ml or 5.2 ml pyrogen-free Water for Injection depending on the starting volume of lysate. When tested with E. coli endotoxin lCF following lyophilization, the sensitivity of the enhancing agent treated lyophilized lysate for both sample sizes was 25 pg/ml in comparison to the control sensitlvity of 500 pg/ml.
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Claims (28)
1. A process for treating under lysate treating con-ditions Limuius amebocyte lysate having decreased sensi-tivity to endotoxin due to the presence of an endogenous lysate inhibitor with an enhancing amount of a lysate sensi-tivity enhancing agent having lysate sensitivity enhancing characteristics to neutralize or partially neutralize said lysate inhibitor thereby increasing the lysate sensitivity to endotoxin, said lysate enhancing agent being selected from the group consisting of (I) amphoteric surfactants having the following formulae:
(A) (B) (C) (D) wherein R1 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;
R2 and R3 are each (1) lower alkyl or (2) hydroxy lower alkyl;
n is 0 or 1, when n is 0, R4 is alkyl containing from about 8 to about 18 carbon atoms;
when n is 1, R4 is an alkylene radical having from 1 to about 6 carbon atoms;
R5 is an alkyl containing from about 8 to about 18 carbon atoms;
M is hydrogen, sodium, potassium or ammonium;
(II) anionic surfactants have the following formulae:
(E) (R6)n1?(Y)Ar?(SO3M)n2 (F) R5OSO3M
wherein R5,Y and M have the same meaning as set forth above R6 is an alkyl from 8 to 24 carbon atoms n1 is an integer from 1 to 3 n2 is 1 or 2 Ar is phenyl or naphthyl;
(III) cationic surfactants having the following formula:
wherein R5, Y and Y' have the same meaning as set forth above; and (IV) nonionic surfactants having the following formula:
(H) R5R7R8N?O
wherein R5 has the same meaning as set forth above R7 and R8 are each methyl or ethyl; and those nonionic surfactants selected from the group consisting 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.
(A) (B) (C) (D) wherein R1 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;
R2 and R3 are each (1) lower alkyl or (2) hydroxy lower alkyl;
n is 0 or 1, when n is 0, R4 is alkyl containing from about 8 to about 18 carbon atoms;
when n is 1, R4 is an alkylene radical having from 1 to about 6 carbon atoms;
R5 is an alkyl containing from about 8 to about 18 carbon atoms;
M is hydrogen, sodium, potassium or ammonium;
(II) anionic surfactants have the following formulae:
(E) (R6)n1?(Y)Ar?(SO3M)n2 (F) R5OSO3M
wherein R5,Y and M have the same meaning as set forth above R6 is an alkyl from 8 to 24 carbon atoms n1 is an integer from 1 to 3 n2 is 1 or 2 Ar is phenyl or naphthyl;
(III) cationic surfactants having the following formula:
wherein R5, Y and Y' have the same meaning as set forth above; and (IV) nonionic surfactants having the following formula:
(H) R5R7R8N?O
wherein R5 has the same meaning as set forth above R7 and R8 are each methyl or ethyl; and those nonionic surfactants selected from the group consisting 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.
2. A process according to Claim 1, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula (A) of Claim 1.
3. A process according to Claim 2, wherein the enhancing agent is present in an amount of from about 0.01 to about 0.05% (w/v).
4. A process according to Claim 3, wherein n is 0, R4 is tetradecyl, R2 and R3 are each methyl, and is trimethylene.
5. A process according to Claim 3, wherein n is 1, R4 is trimethylene, R2 and R3 are each methyl and is
6. A process according to Claim 1, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula (D) of Claim 1.
7. A process according to Claim 6, wherein the enhancing agent is present in an amount of from about 0.01 to about 0.05% (w/v).
8. A process according to Claim 7, wherein n is 1, R4 is propylene, R2 and R3 are each methyl and is methylene.
9. A process according to Claim 7, wherein n is 0, R2 and R3 are each methyl and is methylene.
10. A Limuius ambeocyte lysate reagent for determining endotoxin comprising a buffered aqueous dispersion of Limuius amebocyte lysate present in an endotoxin determining amount having improved sensitivity to endotoxin and an enhancing amount of a lysate sensitivity enhancing agent having lysate sensitivity enhancing characteristics, said lysate enhancing agent being selected from the group consisting of (I) amphoteric surfactants having the following formulae:
(A) (B) (C) (D) wherein R1 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;
R2 and R3 are each (1) lower alkyl or (2) hydroxy lower alkyl;
n is 0 or 1, when n is 0, R4 is alkyl containing from about 8 to about 18 carbon atoms;
when n is 1, R4 is an alkylene radical having from 1 to about 6 carbon atoms;
R5 is an alkyl containing from about 8 to about 18 -carbon atoms;
M is hydrogen, sodium, potassium or ammonium;
(II) anionic surfactants have the following formulae:
(E) (R6)n1?(Y)Ar?(SO3M)n2 (F) R5OSO3M
wherein R5,Y and M have the same meaning as set forth above R6 is an alkyl from 8 to 24 carbon atoms n1 is an integer from 1 to 3 n2 is 1 or 2 Ar is phenyl or naphthyl;
(III) cationic surfactants having the following formula:
(G) wherein R5, Y and Y' have the same meaning as set forth above; and (IV) nonionic surfactants having the following formula:
(H) R5R7R8N?O
wherein R5 has the same meaning as set forth above R7 and R8 are each methyl or ethyl; and the 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.
(A) (B) (C) (D) wherein R1 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;
R2 and R3 are each (1) lower alkyl or (2) hydroxy lower alkyl;
n is 0 or 1, when n is 0, R4 is alkyl containing from about 8 to about 18 carbon atoms;
when n is 1, R4 is an alkylene radical having from 1 to about 6 carbon atoms;
R5 is an alkyl containing from about 8 to about 18 -carbon atoms;
M is hydrogen, sodium, potassium or ammonium;
(II) anionic surfactants have the following formulae:
(E) (R6)n1?(Y)Ar?(SO3M)n2 (F) R5OSO3M
wherein R5,Y and M have the same meaning as set forth above R6 is an alkyl from 8 to 24 carbon atoms n1 is an integer from 1 to 3 n2 is 1 or 2 Ar is phenyl or naphthyl;
(III) cationic surfactants having the following formula:
(G) wherein R5, Y and Y' have the same meaning as set forth above; and (IV) nonionic surfactants having the following formula:
(H) R5R7R8N?O
wherein R5 has the same meaning as set forth above R7 and R8 are each methyl or ethyl; and the 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.
11. A reagent according to Claim 10, wherein additionally are present the cations, Na+, Mn++ and Ca++ in lysate sensitivity increasing amounts.
12. A reagent according to Claim 11, wherein the Lysate is present in an amount to detect from about 0.007 to about 0.050 ng/ml of FDA reference endotoxin EC-2.
13. A reagent according to Claim 12, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula A of Claim 10.
14. A reagent according to Claim 13, wherein n is 0, R4 is tetradecyl, R2 and R3 are each methyl and is trimethylene.
15. A reagent according to Claim 13, wherein n is 1, R4 is trimethylene, R2 and R3 are each methyl and is
16. A reagent according to Claim 12, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula D of Claim 10.
17. A reagent according to Claim 16, wherein n is 1, R4 is propylene, R2 and R3 are each methyl and is methylene.
18. A reagent according to Claim 16, wherein n is 0, R2 and R3 are each methyl and is methylene.
19. A reagent according to Claim 10 which is lyophilized.
20. In a method for determining endotoxin under endotoxin determining conditions wherein the endotoxin is reacted with a Limuius amebocyte lysate reagent, the improvement comprising utilizing the Limuius amebocyte lysate reagent of Claim 10.
21. A reagent according to Claim 10, wherein additionally are present the cations, Na+, Sr++, Ca++
and Mg++ in lysate sensitivity increasing amounts.
and Mg++ in lysate sensitivity increasing amounts.
22. A reagent according to Claim 21, wherein the Lysate is present in an amount to detect from about 0.007 to about 0.050 ng/ml of FDA reference endotoxin EC-2.
23. A reagent according to Claim 22, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by forumla A of Claim 10.
24. A reagent according to Claim 23, wherein n is O, R4 is tetradecyl, R2 and R3 are each methyl and ?1 is trimethylene.
25. A reagent according to Claim 23, wherein n is 1, R4 is trimethylene, R2 and R3 are each methyl and ?1 is
26. A reagent according to Claim 21, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula D of Claim 10.
27. A reagent according to Claim 26, wherein n is 1, R4 is propylene, R2 and R3 are each methyl and R1 is methylene.
28. A reagent according to Claim 26, wherein n is O, R2 and R3 are each methyl and ?1 is methylene.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US16345680A | 1980-06-27 | 1980-06-27 | |
| US163,456 | 1980-06-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1159754A true CA1159754A (en) | 1984-01-03 |
Family
ID=22590085
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000380754A Expired CA1159754A (en) | 1980-06-27 | 1981-06-26 | Process for preparing limulus lysate |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPS5742851A (en) |
| BE (1) | BE889407A (en) |
| CA (1) | CA1159754A (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0226914Y2 (en) * | 1987-04-02 | 1990-07-20 | ||
| DE8910715U1 (en) * | 1989-09-08 | 1989-10-19 | Ruhrkohle Ag, 4300 Essen | Coke oven chamber door |
| JP2957251B2 (en) * | 1990-09-28 | 1999-10-04 | 生化学工業株式会社 | Endotoxin assay |
| FR2812884B1 (en) | 2000-08-08 | 2002-11-08 | Aventis Pharma Sa | MODIFIED YEASTS AND USES, ESPECIALLY FOR THE PRODUCTION OF STEROID DERIVATIVES |
| MXPA03012032A (en) * | 2001-06-28 | 2005-07-01 | Cambrex Bio Science Walkersvil | Methods and reagents for detecting endotoxin. |
| JP5437660B2 (en) * | 2009-02-19 | 2014-03-12 | 興和株式会社 | Coagulogen raw material, method for producing the same, method and apparatus for measuring biologically active substances derived from organisms using the same |
-
1981
- 1981-06-26 CA CA000380754A patent/CA1159754A/en not_active Expired
- 1981-06-26 BE BE0/205235A patent/BE889407A/en not_active IP Right Cessation
- 1981-06-27 JP JP56100372A patent/JPS5742851A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6364747B2 (en) | 1988-12-13 |
| BE889407A (en) | 1981-10-16 |
| JPS5742851A (en) | 1982-03-10 |
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