Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
877-232
~ 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).
.~ . .
Little is known of the nature of the inhibitor
or its in-vivo role in the horseshoe crab. Electro-
.. ...
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1~ , ,; ' '' '
:t' ~
, ~ .
'~ :'` : ' , ' ' : ' ' .
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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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SA
-- 3 --
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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--
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-- 4 --
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
R3 Y
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
~:
'
~ .~, , . . .. ,, ~ ,
: ~ :
, .
li~9'7
-- 5 --
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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,
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'
~`` 115g7S~
-- 6 --
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):
O R2
(~5 - ~ - HN)n ~ R4 ~ Rl
3 Y
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.
'' .
* 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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'~ ~
I_ ,~,, ,~,, ,, :
:: , ~ : :
-~ llS975~
-- 10 --
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):
.. . .
R9NHc2H4NH2
. .
y~ ' ' ,
* Trade Mark
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.. .
.. . . . .
. .
1~5975~
.
- 13 -
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
..
- , . . : . - , .
1~5~54
4 --
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~
- 15 -
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.
" ~ .
;, ~ ,, ,
.
1~ 5~ t
- 16 -
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.
: ,~
,
.
11597S4
-- 17 --
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).
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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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-- 19 --
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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- 20 -
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
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Lysate
Sample Enhancing Agent (%)Sensitivity*
Control 0 500
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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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- 21 -
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
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~ 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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EXAMPLE V
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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