Methods and compositions for the detection of bacterial endotoxins

The invention provides methods and compositions for the detection and/or quantification of bacterial endotoxins. In particular, provided herein is an inexpensive and reproducible method for producing an improved amebocyte lysate preparation having reduced Factor G activity. Provided also is an endotoxin-specific amebocyte lysate preparation produced by such a method. In addition, the invention provides methods and compositions for enhancing the sensitivity to endotoxins of amebocyte lysate preparations having reducing Factor G activity. In particular, the sensitivity of such amebocyte lysate preparations to endotoxins can be enhanced by the addition of exogenous (1.fwdarw.3) .beta.-D-glucan.

FIELD OF THE INVENTION
 This invention relates generally to an amebocyte lysate preparation for use
 in the detection and/or quantification of a bacterial endotoxin in a
 sample, and more particularly to an endotoxin-specific amebocyte lysate
 preparation having reduced Factor G activity for use in the detection
 and/or quantification of a bacterial endotoxin in a sample.
 BACKGROUND OF THE INVENTION
 Bacterial endotoxins, also known as pyrogens, are the fever-producing
 byproducts of Gram negative bacteria and can be dangerous or even deadly
 to humans. Symptoms of infection may range from fever, in mild cases, to
 death. In order to promptly initiate proper medical treatment, it is
 important to identify, as early as possible, the presence of an endotoxin
 and, if possible, the concentration of the endotoxin in the subject of
 interest. Similarly, the U.S. Food and Drug Administration (USFDA)
 requires certain manufacturers to establish that their products, for
 example, parenteral drugs and medical devices, are free of detectable
 levels of Gram negative bacterial endotoxin.
 To this end, a variety of methods have been developed for use in the
 detection of bacterial endotoxins. A currently preferred method involves
 the use of amebocyte lysate (AL) produced from the hemolymph of a
 horseshoe crab, for example, a horseshoe crab selected from the group
 consisting of Limulus polyphemus, Tachpleus gigas, Tachypleus tridentatus,
 and Carcinoscorpius rotundicauda. Amebocyte lysates produced from Limulus,
 Tachpleus, and Carcinoscorpius maybe referred to as LAL, TAL, and CAL,
 respectively.
 Presently, LAL is employed in bacterial endotoxin assays of choice because
 of its sensitivity, specificity and relative ease for avoiding
 interference by other components that may be present in a sample of
 interest. LAL, when combined with a sample containing bacterial endotoxin,
 reacts with the endotoxin to produce a product, for example, a gel or
 chromogenic product, that can be detected, for example, either visually or
 by the use of an optical detector.
 The endotoxin-mediated activation of LAL is well understood and has been
 thoroughly documented in the art. See, for example, Levin et al. (1968)
 Thromb. Diath. Haemorrh. 19: 186, Nakamura et al. (1986) Eur. J. Biochem.
 154: 511, Muta et al. (1987) J. Biochem. 101: 1321, and Ho et al. (1993)
 Biochem. & Mol. Biol. Int. 29: 687. When bacterial endotoxin is contacted
 with LAL, the endotoxin initiates a series of enzymatic reactions,
 referred to in the art as the Factor C pathway, that involve at least
 three serine protease zymogens called Factor C, Factor B and pro-clotting
 enzyme (see FIG. 1). Briefly, upon exposure to endotoxin, the
 endotoxin-sensitive factor, Factor C is activated. Activated Factor C
 thereafter hydrolyses and activates Factor B, whereupon activated Factor B
 activates proclotting enzyme to produce clotting enzyme. The clotting
 enzyme thereafter hydrolyzes specific sites, for example, Arg.sup.18
 -Thr.sup.19 and Arg.sup.46 -Gly.sup.47 of coagulogen, an invertebrate,
 fibrinogen-like clottable protein, to produce a coagulin gel. See, for
 example, U.S. Pat. No. 5,605,806.
 Although the clotting cascade of LAL initially was considered specific for
 endotoxin, it was later discovered that (1.fwdarw.3)-B-D glucans also
 activate the clotting cascade of LAL through a unique enzymatic pathway,
 referred to in the art as the Factor G pathway (see FIG. 1). Upon exposure
 to (1.fwdarw.3)-B-D glucan, Factor G is activated to produce activated
 Factor G. Activated Factor G thereafter converts the proclotting enzyme
 into clotting enzyme, whereupon the clotting enzyme converts coagulogen
 into coagulin, similar to the case with endotoxin. Accordingly, the
 coagulation system of LAL, like the mammalian blood coagulation system,
 consists of at least two coagulation cascades which include an
 endotoxin-mediated pathway (the Factor C pathway), and a (1.fwdarw.3)-B-D
 glucan-mediated pathway (the Factor G pathway). See, for example, Morita
 et al. (1981) FEBS Lett. 129: 318-321 and Iwanaga et aL (1986) J. Protein
 Chem. 5: 255-268.
 In view of the Factor C and Factor G pathways of LAL, the detection of
 bacterial endotoxin in a sample can, under certain circumstances, become
 ambiguous. As a result, attempts have been made to increase the
 specificity of LAL for endotoxin, i.e., to produce an endotoxin-specific
 amebocyte lysate preparation.
 In one approach, polysaccharide based Factor G inhibitors are combined with
 amebocyte lysate to reduce or eliminate clotting induced by
 (1.fwdarw.3)-B-D glucan present in the biological sample, i.e., inhibit
 the Factor G cascade. See, for example, U.S. Pat. Nos.: 5,155,032;
 5,179,006; 5,318,893; 5,474,984; and 5,641,643.
 In an alternative approach, several groups have attempted to remove Factor
 G from LAL thereby to produce a Factor G depleted amebocyte lysate that is
 insensitive to (1.fwdarw.3)-B-D glucan. For example, Obayashi et al.
 (1985) Clin. Chim. Acta 149:55-65 disclose a method for fractionating
 coagulation enzymes in LAL and then recombining only those factors
 involved in the endotoxin induced coagulation cascade (i.e., the Factor C
 cascade) to produce a Factor G depleted amebocyte lysate. The resulting
 lysate, however, may not only lack Factor G but also other components
 required for a complete Factor C cascade. The reconstituted lysate
 produced by this procedure, apparently does not produce a natural coagulin
 type clot and can be used only with synthetic chromogenic substrates.
 U.S. Pat. No. 5,401,647 discloses a method for removing Factor G from LAL
 by combining LAL with (1.fwdarw.3)-B-D glucan immobilized on an insoluble
 carrier. Once bound to the carrier via the (1.fwdarw.3)-B-D glucan moiety,
 the Factor G can thereafter be removed from the LAL to produce a Factor G
 depleted lysate. Similarly, U.S. Pat. No. 5,605,806 discloses an
 immunoaffinity based method using a Factor G specific antibody to remove
 Factor G from LAL thereby to produce a Factor G depleted amebocyte lysate.
 There still exists, however, a demand for an endotoxin-specific amebocyte
 lysate that can be produced economically in commercial quantities. A
 method for producing such an amebocyte lysate should be rapid,
 reproducible, inexpensive, simple to conduct, and preferably should result
 in an amebocyte lysate that can be used in a reliable, and quantitative
 determination of endotoxin in a sample of interest.
 SUMMARY OF THE INVENTION
 The invention features improved amebocyte lysate preparations having
 reduced Factor G activity, methods of making such lysate preparations, and
 methods of using such lysate preparations in the detection and/or
 quantitation of one or more bacterial endotoxins in a sample of interest.
 In one aspect, the invention provides a method of producing an
 endotoxin-specific amebocyte lysate preparation for use in the detection
 of bacterial endotoxins in a sample. The amebocyte lysate preparation is
 rendered endotoxin-specific by the reduction and/or elimination of Factor
 G activity in the preparation. The amebocyte lysate preparation of the
 invention is produced by (a) admixing crude amebocyte lysate, i.e.,
 amebocyte lysate reactive with both endotoxin and (1.fwdarw.3)-B-D glucan,
 with a surfactant in an amount sufficient to produce a solution containing
 a precipitate; and (b) separating the precipitate from the solution
 thereby to produce an amebocyte lysate preparation which is less reactive
 with a (1.fwdarw.3)-.beta.-D glucan than is the crude amebocyte lysate.
 The precipitate produced by addition of surfactant to crude lysate may
 contain any component necessary for a complete Factor G cascade, however,
 the production of a precipitate actually containing Factor G is preferred.
 The amebocyte lysate preparation produced by the methodologies described
 herein comprises all the components necessary for a complete Factor C
 cascade, i.e., is still capable of producing a coagulin gel via the
 endotoxin-mediated pathway. Accordingly, the resulting amebocyte lysate
 preparation is capable of reacting with a bacterial endotoxin, e.g., a
 bacterial endotoxin produced by Gram negative bacteria, to produce a
 coagulin clot.
 It is contemplated that any surfactant (otherwise known as a surface active
 agent or detergent) which produces a precipitate when added to crude
 amebocyte lysate, wherein the precipitate once removed from the lysate
 results in a reduction of Factor G activity, may be used in the practice
 of the invention. The surfactant, however, preferably is a zwitterionic
 surfactant, i.e., a surfactant having a headgroup containing both a
 negatively charged chemical moiety and a positively charged chemical
 moiety. Examples of zwitterionic surfactants include betaines and
 sulfobetaines, however, sulfobetaine-type surfactants are preferred.
 Preferred sulfobetaine-type surfactants include, without limitation,
 n-octyl- N, N-dimethyl-3-ammonio-1-propanesulfonate; n-decyl-N,
 N-dimethyl-3-ammonio-1-propanesulfonate; n-dodecyl-N,
 N-dimethyl-3-ammonio-1-propanesulfonate; n-tetradecyl-N,
 N-dimethyl-3-ammonio-1-propanesulfonate; and n-hexadecyl-N,
 N-dimethyl-3-ammonio-1-propanesulfonate. The sulfobetaine-type surfactant
 n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, however, is most
 preferred.
 In one embodiment, the method comprises the additional step of removing
 from or otherwise reducing the concentration of the added surfactant in
 the solution. The surfactant may be removed, for example, by
 chromatographic separation using, for example, a suitable ion exchange
 resin or, alternatively, by any other means known in the art for removing
 a particular surfactant from an aqueous solution. In a preferred method,
 the surfactant is removed by conventional organic solvent extraction. Any
 organic solvent that dissolves the surfactant of interest and is
 compatible with amebocyte lysate may be used in the solvent extraction
 step, however, for the reasons discussed below, chloroform is preferred.
 In another embodiment, the sensitivity to bacterial endotoxin of an
 amebocyte lysate preparation having reduced Factor G activity can be
 enhanced by the addition of exogenous (1.fwdarw.3)-.beta.-D glucan to the
 amebocyte lysate preparation. In particular, (1.fwdarw.3)-.beta.-D glucan
 is added to the lysate preparation in an amount sufficient to enhance the
 sensitivity of the lysate preparation to endotoxin relative to a similar
 amebocyte lysate preparation without exogenously added
 (1.fwdarw.3)-.beta.-D glucan. Without wishing to be bound by theory, it
 appears that exogenously added (1.fwdarw.3)-.beta.-D glucan acts
 synergistically with the endotoxin mediated pathway. It is understood,
 however, that the same amount of (1.fwdarw.3)-.beta.-D glucan when added
 to crude amebocyte lysate, i.e., amebocyte lysate that is reactive with
 both endotoxin and (1.fwdarw.3)-.beta.-D glucan, likely would induce the
 production of a coagulin gel via the Factor G cascade. In effect, during
 the practice of this particular embodiment of the invention, a substrate
 or initiator of the Factor G cascade is added to the amebocyte lysate
 preparation of the invention.
 Although it is contemplated that any amount of (1.fwdarw.3)-.beta.-D glucan
 that enhances the sensitivity of the amebocyte lysate to the endotoxin
 relative to similar amebocyte lysate without the exogenously added
 (1.fwdarw.3)-.beta.-D glucan may be used in the practice of the invention,
 the optimal amount of exogenous (1.fwdarw.3)-.beta.-D glucan for enhancing
 the endotoxin-specific cascade in a particular lysate can be determined by
 routine experimentation. For example, the optimal concentration can be
 determined by adding different amounts of a particular
 (1.fwdarw.3)-.beta.-D glucan to crude amebocyte lysate, i.e., amebocyte
 lysate reactive with both endotoxin and (1.fwdarw.3)-.beta.-D glucan. The
 optimal amount of the (1.fwdarw.3)-.beta.-D glucan to be added to the
 amebocyte lysate preparation of the invention, can be determined using,
 for example, a kinetic turbidimetric assay whereby the optimal amount is
 the amount of (1.fwdarw.3)-.beta.-D glucan that induces the fastest
 coagulin clot formation in crude amebocyte lysate. This assay protocol is
 exemplary, and it is understood that the skilled artisan may use a variety
 of other assays, for example, a gelclot assay, an end-point turbidimetric
 assay, or a chromogenic assay, to determine the optimal amount of
 (1.fwdarw.3)-.beta.-D glucan to be added to the lysate of interest.
 It is contemplated that any (1.fwdarw.3)-.beta.-D glucan that induces the
 Factor G cascade in crude amebocyte lysate can be used to enhance the
 sensitivity of the endotoxin mediated pathway in the amebocyte lysate
 preparation of the invention. Preferred (1.fwdarw.3)-.beta.-D glucans
 include, without limitation, cotton extract; rinses from cellulose acetate
 membranes; curdlan; pachyman; scleratan; leutinan; schizophyllan;
 coriolan; laminaran; and laminarin. Laminarin, however, currently is most
 preferred.
 In another aspect, the invention provides an amebocyte lysate preparation
 having reduced Factor G activity, i.e., an amebocyte lysate having reduced
 reactivity to (1.fwdarw.3)-.beta.-D glucans relative to crude lysate
 produced by the aforementioned methodologies. In one embodiment, such a
 composition may comprise (i) an amebocyte lysate preparation having
 reduced Factor G activity or, most preferably, an amebocyte lysate
 preparation depleted of Factor G activity, and (ii) exogenously added
 (1.fwdarw.3)-.beta.-D glucan, wherein the (1.fwdarw.3)-.beta.-D glucan is
 added in an amount sufficient to enhance the sensitivity of the amebocyte
 lysate preparation to endotoxin relative to a similar amebocyte lysate
 preparation without the exogenously added (1.fwdarw.3)-.beta.-D glucan.
 Determination of the optimal amount of a particular (1.fwdarw.3)-.beta.-D
 glucan for enhancing sensitivity of the lysate to endotoxin has been
 discussed previously.
 In another aspect, the invention provides methods for detecting and/or
 quantitating the amount of a bacterial endotoxin in a sample. The
 improvement in such methods resides in the use of the amebocyte lysate
 preparation of the invention.
 The foregoing and other objects, features and advantages of the present
 invention will be made more apparent from the following detailed
 description of preferred embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION
 As will be more fully described below, this invention is based, in part,
 upon the discovery of an inexpensive and reliable method for producing an
 endotoxin-specific amebocyte lysate preparation. The resulting amebocyte
 lysate preparations are useful in the detection and/or quantitation of a
 bacterial endotoxin in a sample of interest.
 In particular, the method is based upon a protocol for reducing or
 preferably depleting amebocyte lysate of Factor G activity, with the
 resulting lysate being less reactive to (1.fwdarw.3)-.beta.-D glucan than
 untreated amebocyte lysate. In addition, the invention is based, in part,
 upon the discovery that (1.fwdarw.3)-.beta.-D glucan, when exogenously
 added to an amebocyte lysate preparation depleted of Factor G activity
 (for example, by the removal of Factor G, or by the addition of Factor G
 inhibitors, for example, Factor G inhibitors of the type described in U.S.
 Pat. Nos. 5,155,032; 5,179,006; 5,318,893; 5,474,984; and 5,641,643, the
 disclosures of which are incorporated herein by reference) can enhance the
 sensitivity of the resulting amebocyte lysate preparation to endotoxin.
 The invention, therefore, provides an endotoxin-specific amebocyte lysate
 preparation for use in reliably detecting and/or quantitating a bacterial
 endotoxin in a sample of interest.
 The amebocyte lysate preparation of the invention is produced by: (a)
 admixing a sample of crude amebocyte lysate, i.e., amebocyte lysate that
 is reactive with both endotoxin and (1.fwdarw.3)-.beta.-D glucan, with a
 surfactant in an amount sufficient to produce a solution containing a
 precipitate; and (b) separating the precipitate from the solution thereby
 to produce an amebocyte lysate preparation which is less reactive with a
 (1.fwdarw.3)-.beta.-D glucan than the crude amebocyte lysate. The
 resulting amebocyte lysate preparation preferably still comprises all
 components necessary for the Factor C cascade and, therefore, is capable
 of producing a coagulin gel following the addition of endotoxin.
 As used herein, the term, "amebocyte lysate" is understood to mean any
 lysate produced by the lysis of blood cells (amebocytes) extracted from
 the hemolymph of a horseshoe crab. Preferred horseshoe crabs include crabs
 belonging to the Limulus genus, for example, Limulus polyphemus, the
 Tachpleus genus, for example, Tachpleus gigas, and Tachypleus tridentatus,
 and the Carcinoscorpius genus, for example, Carcinoscorpius rotundicauda.
 As used herein, the term, "crude amebocyte lysate" is understood to mean
 any amebocyte lysate that is capable of producing a coagulin clot in the
 presence of an endotoxin, for example, an endotoxin produced by Gram
 negative bacteria, and a (143)-.beta.-D glucan, for example, laminarin.
 As used herein, the term, "Factor G" is understood to mean any protein or
 polypeptide that acts as a serine protease zymogen and is capable of
 initiating the production of a coagulin gel-clot in crude amebocyte lysate
 following exposure to (1.fwdarw.3)-.beta.-D glucan. The isolation and
 characterization of horseshoe crab Factor G has been discussed extensively
 in the art (see, for example, Seki et al. (1994) J. Biol. Chem. 269:
 1370-1374, the disclosure of which is incorporated herein by reference)
 and, therefore, is not discussed in detail herein.
 As used herein, the term, "(1.fwdarw.3)-.beta.-D glucan" is understood to
 mean any water soluble polysaccharide, disaccharide or derivative thereof
 that is (i) capable of inducing formation of a coagulin clot in crude
 Limulus amebocyte lysate, and (ii) contains at least two .beta.-D
 glucosides, as defined in formula I below, connected by a
 (1.fwdarw.3)-.beta.-D glycosidic linkage. It is contemplated that such a
 polysaccharide or derivative thereof, in addition to containing a
 (1.fwdarw.3)-.beta.-D glycosidic linkage may also contain glucoside
 moieties connected by a variety of other glycosidic linkages, for example,
 via a (1.fwdarw.4)-.beta.-D glycosidic linkage and/or by a
 (1.fwdarw.6)-.beta.-D glycosidic linkage. It is contemplated that such
 (1.fwdarw.3)-.beta.-D glucans may be isolated from a variety of sources
 including, without limitation, plants, bacteria, yeast, algae, and fungi,
 or alternatively may be synthesized using conventional sugar chemistries.
 ##STR1##
 As used herein, the term "reactive with (1.fwdarw.3)-.beta.-D glucan"
 refers to an amebocyte lysate, which in the presence of a
 (1.fwdarw.3)-.beta.-D glucan is capable of producing a product that can be
 detected in a conventional gel-clot assay, end point-turbidimetric assay,
 kinetic turbidimetric assay or a chromogenic assay. Similarly, as used
 herein, the term "reactive with a bacterial endotoxin" refers to an
 amebocyte lysate, which in the presence of an endotoxin produced by a Gram
 negative bacteria is capable of producing a product that can be detected
 in a conventional gel-clot assay, end point-turbidimetric assay, kinetic
 turbidimetric assay or a chromogenic assay.
 As used herein, the term, "surfactant" is understood to mean any surface
 active agent or detergent that is capable of producing a precipitate when
 admixed with crude amebocyte lysate, wherein the precipitate once removed
 from the lysate results in a reduction of Factor G activity. As used
 herein, the term, "zwitterionic surfactant" is understood to mean any
 surfactant having a headgroup containing both a negatively charged
 chemical moiety and a positively charged chemical moiety. Examples of
 useful zwitterionic surfactants useful in the practice of the instant
 invention include, without limitation, betaines (see, for example, formula
 II below) and sulfobetaines (see, for example, formula III below),
 however, sulfobetaines currently are the most preferred.
 ##STR2##
 wherein n can be 7, 9, 11, 13 or 15.
 As used herein, the term, "precipitate" is understood to mean any insoluble
 material produced following admixture of crude amebocyte lysate with a
 surfactant. It is contemplated that the precipitate may contain Factor G
 and/or other, heretofore undiscovered, components necessary for a
 functional Factor G cascade.
 Preparation of Amebocyte Lysate having Reduced Factor G Activity
 A flow chart showing an exemplary protocol for producing an amebocyte
 lysate preparation having reduced Factor G activity, more preferably, an
 amebocyte lysate preparation depleted of Factor G activity is shown in
 FIG. 1. It is contemplated that any crude amebocyte lysate may be used as
 a starting material in the protocol shown in FIG. 1.
 Crude lysates may be produced using the procedure as originally described
 in Levin et al. (1968) Thromb. Diath. Haemorrh. 19:186, with modification.
 Briefly, blood (hemolymph) is harvested from horseshoe crab in a saline
 solution isotonic with sea water (about 3% NaCl (w/v)) containing an
 anti-coagulant, for example, N-ethylmaleimide, caffeine, or Tween.RTM..
 The amebocytes are washed with the same solution to remove hemolymph
 factors and lysed by osmotic shock via exposure to pyrogen-free water.
 After 24 hours, the amebocyte lysate is separated from cellular debris by
 centrifugation. The preparation of crude lysate also is discussed, for
 example, in Richard B. Prior, Ed., "Clinical Applications of the Limulus
 Amebocyte Lysate Test" CRC Press, pp. 28-36 and pp. 159-166, and in U.S.
 Pat. No. 4,322,217, the disclosures of which are incorporated herein by
 reference.
 In order to produce amebocyte lysate having reduced Factor G activity,
 surfactant is added to crude amebocyte lysate in an amount sufficient to
 produce a precipitate. As mentioned previously, it is contemplated that
 any surfactant or detergent which produces a precipitate upon addition to
 crude amebocyte lysate, wherein the precipitate once removed from the
 lysate results in a reduction of Factor G activity, may be used in the
 practice of the invention. Preferred surfactants include zwitterionic
 surfactants, i.e., surfactants having a headgroup containing both a
 negatively charged chemical moiety and a positively charged chemical
 moiety. Examples of zwitterionic surfactants useful in the practice of the
 invention include betaines and sulfobetaines, however, sulfobetaine-type
 surfactants are the more preferred.
 A family of sulfobetaine type detergents are available commercially from
 Calbiochem.RTM., San Diego, Calif. under the tradename Zwittergent.RTM..
 Sulfobetaine type detergents apparently retain zwitterionic character over
 a wide pH range. Currently preferred sulfobetaine-type surfactants
 include, without limitation, n-octyl- N,
 N-dimethyl-3-ammonio-1-propanesulfonate (also known as Zwittergent.RTM.
 3-08); n-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (also known as
 Zwittergent.RTM. 3-10); n-dodecyl-N,
 N-dimethyl-3-ammonio-1-propanesulfonate (also known as Zwittergent.RTM.
 3-12); n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (also known
 as Zwittergent.RTM. 3-14); and n-hexadecyl-N,
 N-dimethyl-3-ammonio-1-propanesulfonate (also known as Zwittergent.RTM.
 3-16). The sulfobetaine-type surfactant is n-tetradecyl-N,
 N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent.RTM. 3-14), however,
 is most preferred.
 The optimal concentration of surfactant necessary to produce a precipitate
 when combined with crude amebocyte lysate can be determined by routine
 experimentation. For example, the skilled artisan may simply add
 increasing concentrations of a particular surfactant to crude amebocyte
 lysate until a precipitate is produced. The amebocyte lysate preparation
 following removal of the precipitate can then be tested for reduced Factor
 G activity using any of the conventional assays, for example, gel-clot,
 end-point turbidimetric, kinetic turbidimetric, or chromogenic assays,
 well known and thoroughly document in the art. With regard to the
 sulfobetaine type-detergents, preferred detergent concentrations range
 from about 0.01% to about 0.6% (w/v) and most preferably from about 0.05%
 to about 0.25% (w/v). Specifically, with regard to the sulfobetaine-type
 surfactant n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate
 (Zwittergent.RTM. 3-14), this detergent is preferably added to the crude
 lysate to a final concentration of from about 0.05% (w/v) to about 0.20%
 (w/v), most preferably about 0.12% (w/v) to produce a precipitate.
 Optimal precipitation conditions may be determined by varying one or more
 of detergent concentration, temperature, and time of incubation. For
 example, preferred incubation conditions include incubation at
 temperatures below about 20.degree. C., most preferably about 2-15.degree.
 C. for about 2-24 hours. These conditions appear to preserve the activity
 of the lysate preparation during the precipitation step. For example, in
 the case of Zwittergent.RTM. 3-14, the resulting mixture preferably is
 incubated at 2-8.degree. C. for 8-24 hours. Following incubation, the
 resulting precipitate can be removed by any conventional technique known
 in the art, for example, centrifugation followed by the removal of
 supernatant from a pellet of precipitate.
 In a preferred embodiment, the concentration of the surfactant remaining in
 the supernatant, if necessary, is reduced to a level that permits the
 Factor C cascade to be operative. The extent of surfactant removal to
 produce a lysate containing an operative Factor C cascade may be
 determined by routine experimentation. For example, the amount of
 surfactant remaining in the supernatant can be determined, for example, by
 standard thin layer chromatography (TLC). Under certain circumstances it
 may be preferable to remove at least about 35% of the surfactant from the
 supernatant, more preferably at least about 70%, and most preferably at
 least about 90%. For example, consistent removal of at least about 90% of
 the surfactant from the lysate, enables one to produce formulations of
 amebocyte lysate preparations with relatively defined components. As a
 result, it may be easier to produce batches of amebocyte lysate
 preparations having the same or similar activities. It is understood,
 however, that it is possible to control the sensitivity of the resulting
 lysate preparation to endotoxin by altering the concentration of residual
 detergent in the lysate preparation. For example, the more detergent
 removed, the more sensitive the lysate to endotoxin. Accordingly, in order
 to prepare a lysate having a desired sensitivity to endotoxin, the amount
 of detergent removed from the lysate preparation may be altered by varying
 the detergent extraction conditions discussed below.
 It is contemplated that the skilled artisan may use any conventional
 procedure for removing a particular surfactant or more particularly,
 Zwittergent.RTM. from the mixture. For example, it is contemplated that
 the surfactant may removed by, for example, conventional ion exchange
 chromatography or, more preferably by organic solvent extraction.
 Furthermore, it is contemplated that the surfactant may be removed prior
 to, subsequent to, or during the removal of the precipitate from the
 mixture.
 In a preferred embodiment, Zwittergent.RTM. surfactant is removed from the
 mixture simultaneously with the precipitate by organic solvent extraction.
 For example, following the production of the precipitate, the resulting
 mixture may be extracted with an organic solvent that dissolves surfactant
 and is compatible with amebocyte lysate. In an exemplary protocol, organic
 solvent is added to the mixture of amebocyte lysate and surfactant, and
 the combination thoroughly mixed. After extraction, the combination may
 separate to produce a three phase system comprising an organic phase, an
 interface of precipitate, and an aqueous phase. When an organic solvent,
 for example, chloroform is employed, the resulting aqueous phase is
 located above the organic phase, with the precipitate residing at the
 interface of the organic and aqueous phases.
 It is contemplated that any organic solvent which dissolves the surfactant
 and which is compatible with amebocyte lysate (i.e., does not impair the
 Factor C cascade) may be used to remove the surfactant. Also, it has been
 noted that certain organic solvents may be used to remove or inactivate
 lysate inhibitors, which when removed or inactivated enhance the
 sensitivity of the resulting lysate to endotoxin. See, for example, U.S.
 Pat. Nos. 4,107,077 and 4,279,774, the disclosures of which are
 incorporated herein by reference. Accordingly, it may be preferable, but
 not essential that the organic solvent used to remove the surfactant also
 remove or inactive the lysate inhibitors. Preferred solvents include,
 without limitation, chloroform, iodoform, bromoform, lower alkyl halides
 such as methyl bromide, methyl chloride, methyl iodide, ethyl chloride,
 ethyl iodide, propyl chloride, propyl bromide and propyl iodide, ethylene
 dichloride, methylene dichloride, benzene, monohalobezenes such as
 chlorobenzene, bromobenzene and iodobenzene, lower alkyl ethers such as
 dimethyl ether and diethyl ether, carbon tetrachloride, trichloroethane,
 trichloroethylene, toluene and hexane. Choice of the optimal organic
 solvent for a particular surfactant may be determined by routine
 experimentation
 In a preferred embodiment, when using the sulfobetaine detergent
 Zwittergent.RTM. 3-14, chloroform is the preferred organic solvent for use
 in the organic extraction. Chloroform is not only compatible with the
 lysate and capable of dissolving Zwittergent.RTM. 3-14, but also is
 capable of removing and/or inactivating known LAL inhibitors in amebocyte
 lysate. It is understood that the amount of detergent removed can be
 altered, thereby altering the endotoxin sensitivity of the lysate, by
 varying the chloroform to lysate ratio during organic solvent extraction.
 Following extraction, the resulting organic and aqueous phases are allowed
 to separate. Separation may be speeded up by centrifugation. For example,
 phase separation may be speeded up by centrifugation at about 500 G to
 about 2,000 G, most preferably about 1,200 G. Following centrifugation,
 the aqueous phase is harvested. For example, when chloroform is used, the
 upper aqueous phase may be harvested by decanting the aqueous phase
 thereby leaving behind the interfacial material and the lower chloroform
 organic phase. The decanted upper aqueous phase, preferably is subjected
 to a second round of centrifugation, for example, at about 5,000 G to
 about 9,000 G, most preferably about 7,000 G. The resulting aqueous phase
 then is separated and harvested from residual interfacial material and/or
 organic phase, and either stored or formulated as desired. It is
 appreciated, however, that the optimal number and extent of the
 centrifugation steps for a particular system may be determined by routine
 experimentation.
 The resulting upper aqueous phase can then stored at reduced temperature or
 formulated immediately. For example, when frozen lysate may retain
 activity indefinitely, whereas, when stored at 2-8.degree. C., the lysate
 may retain activity for several weeks. The reduction in Factor G activity
 of the resulting amebocyte lysate preparations can be determined using any
 of the techniques described hereinbelow.
 Enhancement of the Sensitivity of Amebocyte Lysate Preparations to
 Endotoxin.
 As discussed hereinabove, it has been discovered that the sensitivity to
 endotoxin of an amebocyte lysate preparation having reduced Factor G
 activity may be enhanced by the addition of an exogenous
 (1.fwdarw.3)-.beta.-D glucan. An increase in sensitivity is understood to
 mean that the lysate with additive reacts faster to produce a product at
 lower endotoxin concentrations than lysate without additive. In
 particular, (1.fwdarw.3)-.beta.-D glucan can be added to the lysate
 preparation in an amount sufficient to enhance the endotoxin sensitivity
 of the lysate preparation relative to a similar amebocyte lysate
 preparation without the exogenously added (1.fwdarw.3)-.beta.-D glucan. It
 is understood, however, that the same amount of (1.fwdarw.3)-.beta.-D
 glucan when added to crude amebocyte lysate likely would induce the
 production of a coagulin gel via the Factor G cascade. In effect, during
 the practice of this particular embodiment of the invention, surprisingly
 a substrate or initiator of the now reduced or depleted Factor G cascade
 is added to the amebocyte lysate preparation of the invention. It is
 contemplated that this type of enhancement may occur with any lysate
 depleted of Factor G activity (e.g., wherein Factor G is removed from
 lysate or wherein the lysate contains Factor G inhibitors).
 The identification of suitable (1.fwdarw.3)-.beta.-D glucans as well as the
 identification of optimal concentrations of (1.fwdarw.3)-.beta.-D glucans
 for enhancing endotoxin sensitivity may be determined by routine
 experimentation using the methodologies described hereinbelow. With regard
 to the type of useful (1.fwdarw.3)-.beta.-D glucans, it is contemplated
 that any (1.fwdarw.3)-.beta.-D glucan that induces a Factor G mediated
 cascade in crude amebocyte lysate can be used in the practice of this
 aspect of the invention. Currently preferred (1.fwdarw.3)-.beta.-D glucans
 include, without limitation, natural polysaccharides obtained from cell
 walls of, for example, various bacteria (for example, Alcaligenes genus,
 and Agrobacterium genus), yeasts (for example, shiitake) with specific
 examples of natural polysaccharides including, for example, curdlan,
 pachyman, scleratan, leutinan, schizophylan, and coriolan. Other natural
 polysaccharides include storage polysaccharides of algae, for example,
 brown algae, Euglena, diatoma, with specific examples of storage
 polysaccharides including, for example, laminaran, laminarin, and
 paramilon. Preferred (1.fwdarw.3)-.beta.-D glucans also include, for
 example, polysaccharide derivatives in which at least one group selected
 from a carboxymethyl group, a carboxyethyl group, a methyl group, a
 hydroxyethyl group, a hydroxypropyl group, and a sulfopropyl group, is
 introduced into a natural polysaccharide or storage polysaccharide using
 conventional methodologies well known in the art. See, for example, Munio
 Kotake "Daiyukikagaku" Vol. 19 7.sup.th ed. Asakura Shoten, May 10 (1967)
 pp. 70-101; A. E. Clarke et al. (1967) Phyto-chemistry 1: 175-188; and T.
 Sasaki et al. (1967) Europ. J. Cancer, 15: 211-215. Other naturally
 occurring polysaccharides may be derived from cotton wool, for example, in
 cotton wool extracts, and certain cellulose-based filters used in the
 processing of medicinals. See, for example, Roslansky et al. (1991) J.
 Clin. Micro. 29: 2477; Henne et al. (1984) Artif. Organs 8: 299; and
 Ikemura et al. (1989) J. Clin. Micro. 27: 1965. Furthermore, it is
 contemplated that the aforementioned polysaccharides and derivatives
 thereof may be used either alone or in combination with others to enhance
 endotoxin sensitivity.
 Although it is contemplated that any amount of (1.fwdarw.3)-.beta.-D glucan
 that enhances the sensitivity of the amebocyte lysate to the endotoxin
 relative to similar amebocyte lysate without the exogenously added
 (1.fwdarw.3)-.beta.-D glucan can be used in the practice of this aspect of
 the invention, the optimal amount of exogenous (1.fwdarw.3)-.beta.-D
 glucan for enhancing the endotoxin-specific cascade in a particular lysate
 can be determined by routine experimentation. For example, the optimal
 concentration can be determined by adding different amounts of a
 particular (1.fwdarw.3)-.beta.-D glucan to crude amebocyte lysate, wherein
 the optimal amount is the amount of (1 .fwdarw.3)-.beta.-D glucan that
 induces the fastest coagulin clot or color formation in crude amebocyte
 lysate. See, for example, Examples 4, 5 and 6, disclosed hereinbelow.
 These assay protocols are considered to be exemplary, and it is understood
 that the skilled artisan may use a variety of assays, for example,
 gel-clot, kinetic turbidimetric, end-point turbidimetric or chromogenic
 assays, to determine the optimal amount of (1.fwdarw.3)-.beta.-D glucan to
 be added to the lysate of interest.
 Once the optimal concentration of a particular (1.fwdarw.3)-.beta.-D glucan
 for enhancing endotoxin sensitivity has been determined, then the same of
 amount of the (1.fwdarw.3)-.beta.-D glucan can then be added to an
 amebocyte lysate preparation having reduced or eliminated Factor G
 activity thereby to enhance the endotoxin sensitivity of the resulting
 mixture. It is contemplated that the endotoxin sensitivity of any
 amebocyte lysate having reduced Factor G activity may be enhanced by the
 addition of (1.fwdarw.3)-.beta.-D glucan. For example, it is contemplated
 that the endotoxin sensitivity of any amebocyte lysate having reduced
 Factor G activity may be enhanced by the addition of exogenous
 (1.fwdarw.3)-.beta.-D glucan.
 Formulation of Amebocyte Lysate
 The resulting lysate may be formulated using conventional methodologies
 well known and thoroughly discussed in the art. See, for example, R. B.
 Prior, Ed., (1990), "Clinical Applications of the Limulus Amebocyte Lysate
 Test", CRC Press, and U.S. Pat. No. 4,322,217, the disclosures of which
 are incorporated herein by reference. Methods for enhancing sensitivity of
 amebocyte lysate may include, without limitation, the aging of crude
 amebocyte lysate, adjustment of pH, adjustment of divalent cation
 concentration, adjustment of coagulogen concentration, chloroform
 extraction, and the addition of serum albumin, biocompatible buffers
 and/or biological detergents.
 For example, typical formulation additives may include, without limitation,
 about 100-300 mM NaCl, about 10-100 mM divalent cations (e.g., Mg.sup.2+
 or Ca.sup.2+), biocompatible buffers, e.g., Tris, to give a final pH of
 about 6.0 to 8.0, and, if the lysate is to be freeze dried, then sugars,
 e.g., mannitol or dextran. It is contemplated that, the choice of
 appropriate formulation additives may be determined by routine
 experimentation.
 Lysate, once formulated, typically is lyophilized for long term storage.
 The lyophilized amebocyte lysate formulations may be reconstituted prior
 to use by the addition of, for example, pyrogen-free water, or any other
 pyrogen-free biocompatible buffer.
 Methods for Measuring Bacterial Endotoxins
 It is contemplated that the amebocyte lysate preparations of the invention
 may be used to detect and/or quantitate the amount of endotoxin in any
 sample of interest. For example, it is contemplated that the lysate may be
 used to detect and/or measure the concentration of endotoxin in any
 pharmaceutical preparation, for example, an organically produced drug or a
 recombinantly produced protein, and/or any medical device of interest. In
 addition, the lysate may be used to detect and/or measure the
 concentration of endotoxin in biological samples of, for example, blood,
 serum, plasma, urine, semen, ascitic fluid, peritoneal fluid, sputum,
 breast exude, and spinal fluid.
 It is contemplated that the amebocyte lysate of the invention may be used
 to detect and/or quantitate a bacterial endotoxin in a sample using any
 lysate-based assay now known or later developed that detects one or more
 products of the Factor C cascade. It is contemplated, however, that the
 amebocyte lysate preparation of the invention may be used to advantage in
 any conventional gel-clot, end-point turbidimetric, kinetic turbidimetric,
 or chromogenic assay, known and/or used in the art. The particulars of
 each of these four types of assays are described below.
 (i) Gel-Clot Assay
 This technique is described in Prior, R. B., Ed., supra, pp. 28-34, the
 disclosure of which is incorporated by reference herein, and, therefore,
 is not described in detail herein. Briefly, the gel-clot assay comprises
 the steps of (i) mixing amebocyte lysate preparation with the sample to be
 analyzed, (ii) incubating the resulting mixture at a temperature of
 0.degree. to 40.degree. C., preferably 25.degree. to 40.degree. C., for a
 predetermined time, for example, one hour, and (iii) visually inspecting
 whether or not a gel-clot has been produced.
 (ii) End Point Turbidimetric Assay
 This technique is described in Prior, R. B., Ed., supra, pp. 28-34 and,
 therefore, is not described in detail herein. Briefly, the end point
 turbidimetric assay comprises the steps of (i) mixing amebocyte lysate
 preparation with a sample to be investigated, (ii) incubating the
 resulting mixture at a temperature of 0.degree. to 40.degree. C.,
 preferably 25.degree. to 40.degree. C., for a predetermined time, and
 (iii) measuring the increase in turbidity as a result of in coagulation,
 if any, using a conventional coagulometer, nepherometer,
 spectrophotometer, or the like.
 (iii) Kinetic Turbidimetric Assay
 This technique is described in Prior, R. B., Ed., supra, pp. 28-34 and,
 therefore, is not described in detail herein. Briefly, the kinetic
 turbidimetric assay comprises the steps of (i) mixing amebocyte lysate
 preparation with a sample to be investigated, (ii) incubating the
 resulting mixture at a temperature of 0.degree. to 40.degree. C.,
 preferably 25.degree. to 40.degree. C., over a predetermined time range,
 and (iii) measuring a time required for either a turbidity change caused
 by coagulation to reach a preselected value or a ratio in change of the
 turbidity, using a conventional coagulometer, nepherometer,
 spectrophotometer, or the like.
 (iv) Chromogenic Assay
 This technique is described in Prior, R. B., Ed., supra, pp. 28-34, and
 U.S. Pat. Nos.: 4,301,245; 4,717,658; and 5,310,657, the disclosures of
 which are incorporated herein by reference and, therefore, is not
 described in detail herein. Briefly, the chromogenic assay comprises the
 steps of (i) mixing amebocyte lysate preparation with a sample to be
 investigated, (ii) incubating the resulting mixture at a temperature of
 0.degree. to 40.degree. C., preferably 25.degree. to 40.degree. C., for a
 predetermined time, then, if necessary, adding a reaction inhibitor, and
 (iii) measuring a substance released by protease activity from the
 synthetic substrate calorimetrically, or the like.
 EXAMPLES
 Practice of the invention will be more fully understood from the following
 examples, which are presented herein for illustrative purposes only, and
 should not be construed as limiting the invention in any way.
 Example 1
 Preparation and Characterization of Amebocyte Lysate Depleted of Factor G
 Activity
 This example describes a preferred method for producing an amebocyte lysate
 having reduced Factor G activity. Throughout the following procedure, all
 reagents and apparatus, where appropriate, are produced or treated to be
 pyrogen free. Such methodologies are well known in the art and, therefore,
 are not discussed in detail herein. For example, apparatus may be made
 pyrogen-free by baking in an oven at .gtoreq.200.degree. C. for four
 hours, and reagents may be made pyrogen-free by treatment by ultra
 filtration (.ltoreq.20 kD cut off), oxidation with peroxide, or treatment
 with NaOH.
 Crude amebocyte lysate was prepared by harvesting hemolymph from horseshoe
 crab. The resulting hemolymph was centrifuged to produce an amebocyte
 pellet. The amebocytes then were reharvested, rerinsed and recentrifuged.
 After second rinsing and harvesting steps, the resulting amebocytes were
 lysed by osmotic shock, and the resulting crude amebocyte lysate stored at
 2-8.degree. C. until further use.
 Thereafter, Zwittergent.RTM. 3-14 was added stepwise to the crude lysate to
 a final concentration of 0.12% (w/v). The resulting solution was stored at
 2-8.degree. C. for 8-24 hours. Thereafter, the solution was mixed with
 chloroform (4-5 parts lysate to 1 part chloroform) by gentle stirring for
 10-30 minutes at 2-8.degree. C. The resulting mixture was then centrifuged
 at 1,200 G for 15 minutes, whereupon the resulting upper aqueous and lower
 organic phases were separated by interfacial precipitate material. The
 aqueous phase then was harvested by decanting, and recentrifuged at 7,000
 G for at least 30 minutes to achieve clarity. After centrifugation, the
 resulting aqueous phase was decanted from the residual organic phase and
 interfacial material. The extent of detergent extraction was estimated by
 thin layer chromatography (TLC). Briefly, samples of chloroform extract
 and lysate were applied to a TLC plate (Whatman PE SIL GLUV), developed
 with methanol:water (10:1(v/v)), and the detergent visualized with UV
 light. By this procedure, no residual detergent was detectable in the
 lysate.
 Fractionation of the crude amebocyte lysate, the precipitate produced by
 the surfactant, and the supernatant containing the amebocyte lysate by
 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
 suggests that Factor G is actually contained in the precipitate and,
 therefore, is removed from the lysate. Densitometric profiles of each lane
 of the resulting Coomassie Blue stained SDS-PAGE gel are shown in FIG. 3.
 In FIG. 3, lane 1 represents the densitometric profile of molecular weight
 markers, wherein peak 2 represents a protein having a molecular weight of
 66 kD, peak 3 represents a protein having a molecular weight of 45 kD,
 peak 4 represents a protein having a molecular weight of 36 kD, peak 5
 represents a protein having a molecular weight of 29 kD, peak 6 represents
 a protein having a molecular weight of 24 kD, peak 7 represents a protein
 having a molecular weight of 20 kD, peak 8 represents a protein having a
 molecular weight of 14.2 kD, and peak 9 represents a protein having a
 molecular weight of 6.5 kD.
 In FIG. 3, lane 2 represents a fractionated sample of crude amebocyte
 lysate wherein peaks 12 and 15 apparently represent the two subunits of
 Factor G (one having a molecular weight of about 76 kD and the other
 having a molecular weight of about 36 kD). Lane 3 represents a
 fractionated sample of amebocyte lysate supernatant following detergent
 precipitation. According to lane 3, it appears that the supernatant is
 depleted of the 76 kD and 36 kD Factor G subunits. Lane 4 represents a
 fractionated sample of the lysate precipitate. According to lane 4, it
 appears that the precipitate contains the 76 kD and 36 kD subunits of
 Factor G (peaks 29 and 33/34, respectively). According to these profiles,
 it appears that Factor G was actually precipitated from the lysate by the
 surfactant.
 Example 2
 Formulation of Amebocyte Lysate Depleted of Factor G Activity
 Following preparation of the amebocyte lysate preparation of the invention,
 the resulting lysate was formulated as follows:

PERCENTAGE OF
 COMPONENTS COMPONENTS
 Extracted Limulus Amebocyte Lysate 37%
 Water 8%
 0.4 M Mg.sup.2+ /0.6 M Tris-HEPES Buffer 10%
 6% Dextran 6%
 3% NaCl 39%
 Laminarin 0.000004%-0.000013%
 The resulting formulation was stored at 2-8.degree. C. until use.
 Example 3
 Reactivity of Crude Amebocyte Lysate and Lysate Depleted of Factor G
 Activity with Bacterial Endotoxin
 This example demonstrates that amebocyte lysate of the invention still
 reacts with bacterial endotoxin, as determined by the gel-clot assay.
 Three batches of amebocyte lysate having reduced Factor G activity
 (referred to as L4941LB, L1081LB, L1711LB) as produced by the method of
 Example 1 and formulated as described in Example 2 were tested for
 endotoxin reactivity using a gel-clot assay. Batches L4941LB and L1081LB
 were also formulated with 0.4% bulking protein and 0.01% Zwittergent.RTM.
 3-14.
 The endotoxin reactivity of each batch of lysate was determined
 simultaneously with a control batch of lysate, referred to as reference
 lysate lot 13, obtained from the USFDA. The endotoxin standard used in
 each determination was obtained from USFDA, and is referred to herein as
 EC-6. Briefly, a standard solution of EC-6 endotoxin was prepared.
 Thereafter, endotoxin was added to the reference lysates to give a final
 endotoxin concentration of 0.5, 0.25, 0.125, 0.06, or 0.03 EU/mL.
 Similarly, endotoxin was added to the test lysates to give a final toxin
 concentration of 0.06, 0.03, 0.015, or 0.007 EU/mL. After mixing, the
 resulting mixtures were incubated at 37.degree. C. for one hour, after
 which the presence or absence of a clot was noted. All the samples were
 treated the same.
 In order to assure the integrity of the assay, the assays were performed in
 a specific order. For example, a first batch of four reference lysate
 samples (denoted by test number 1 in Tables 1, 3, and 5) was analyzed
 first, then a batch of ten lysate test samples formulated as described in
 Example 2 (denoted by vial numbers 1-10 in Tables 2, 4, and 6) was
 analyzed, and finally a second batch of four reference lysate samples
 (denoted by test number 2 in Tables 1, 3, and 5) was analyzed.
 Each test sample was analyzed side-by-side with a reference lysate.
 Accordingly, data relating to the reactivity of reference lysate 13 (Table
 1) was derived contemporaneously with data relating to the reactivity of
 the test lysate batch number L1711Lb (Table 2). According to Table 1, the
 reactivity of reference lysate 13 had an endpoint value (E.Pt) of 0.06
 EU/niL for the assay (i.e., within a recognized acceptable level).
 According to Table 2, the reactivity of test lysate L1711LB (10 samples)
 had an endpoint value estimated at 0.06 EU/mL. Accordingly, the test
 lysate L1711LB was within an acceptable level of sensitivity.
 TABLE 1
 Reference Lysate Lot 13, Endotoxin Lot EC-6
 TEST 0.5 0.25 0.125 0.06 0.03 E.Pt.
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 MEAN (Log.sub.2): -4
 S.D. (Log.sub.2 Data): --
 Geometric Mean: 0.06
 STATED ENDPOINT: 0.06 EU/ml
 TABLE 2
 Test Lysate Lot L 1711LB, Endotoxin Lot EC-6
 VIAL 0.125 0.06 0.03 0.015 E. Pt.
 1 + + - - 0.06
 2 + + - - 0.06
 3 + + - - 0.06
 4 + + - - 0.06
 5 + + - - 0.06
 6 + + - - 0.06
 7 + + - - 0.06
 8 + + - - 0.06
 9 + + - - 0.06
 10 + + - - 0.06
 MEAN (Log.sub.2): -4
 S. D. (Log.sub.2 Data): --
 Geometric Mean: 0.06
 STATED ENDPOINT: 0.06 EU/ml
 Similarly, data relating to the reactivity of reference lysate 13 (Table 3)
 was derived contemporaneously with data relating to the reactivity of the
 test lysate batch number L1081LB (Table 4). According to Table 3, the
 reactivity of reference lysate 13 had an endpoint value (E.Pt.) of 0.06
 EU/mL for the assay (i.e., within a recognized acceptable level).
 According to Table 4, the reactivity of test lysate L1081LB (10 samples)
 had an endpoint value estimated at 0.015 EU/mL. Accordingly, the test
 lysate L1081LB was more sensitive to endotoxin than the reference lysate.
 TABLE 3
 Reference Lysate Lot 13, Endotoxin Lot EC-6
 TEST 0.5 0.25 0.125 0.06 0.03 E.Pt.
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 MEAN (Log.sub.2): -4
 S.D. (Log.sub.2 Data): --
 Geometric Mean: 0.06
 STATED ENDPOINT: 0.06 EU/ml
 TABLE 3
 Reference Lysate Lot 13, Endotoxin Lot EC-6
 TEST 0.5 0.25 0.125 0.06 0.03 E.Pt.
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 MEAN (Log.sub.2): -4
 S.D. (Log.sub.2 Data): --
 Geometric Mean: 0.06
 STATED ENDPOINT: 0.06 EU/ml
 In addition, data relating to the reactivity of reference lysate 13 (Table
 5) was derived contemporaneously with data relating to the reactivity of
 the test lysate batch number L4941LB (Table 6). According to Table 5, the
 reactivity of reference lysate 13 had an endpoint value (E.Pt.) of 0.06
 EU/mL for the assay (i.e., within a recognized acceptable level).
 According to Table 6, the reactivity of test lysate L4941LB (10 samples)
 had an endpoint value estimated at 0.03 EU/mL. Accordingly, the test
 lysate L4941LB was more sensitive to endotoxin than the reference lysate.
 TABLE 5
 Reference Lysate Lot 13, Endotoxin Lot EC-6
 TEST 0.5 0.25 0.125 0.06 0.03 E.Pt.
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 MEAN (Log.sub.2): -4
 S.D. (Log.sub.2 Data): --
 Geometric Mean: 0.06
 STATED ENDPOINT: 0.06 EU/ml
 TABLE 5
 Reference Lysate Lot 13, Endotoxin Lot EC-6
 TEST 0.5 0.25 0.125 0.06 0.03 E.Pt.
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 1 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 2 + + + + - 0.06
 MEAN (Log.sub.2): -4
 S.D. (Log.sub.2 Data): --
 Geometric Mean: 0.06
 STATED ENDPOINT: 0.06 EU/ml
 All three batches of lysate, L4941LB, L1081LB and L1711LB met the 24 hour
 negative control requirements as required by the USFDA.
 Example 4
 Reactivity of Crude Amebocyte Lysate and Lysate Depleted of Factor G
 Activity with Laminarin
 This example demonstrates that Limulus amebocyte lysates produced by the
 method of the invention, unlike crude lysates, are insensitive to the
 presence of laminarin in the sample. In addition, this example defines the
 optimal amount of laminarin (as determined from the crude lysate sample)
 that may be exogenously added to the lysate preparation of the invention,
 thereby to enhance the sensitivity of the lysate preparation to endotoxin.
 A standard solution of laminarin was produced by dissolving 1.0 g of
 laminarin in 100 ml of 5 mM NaOH to produce a 10 mg/mL solution. The
 resulting solution was autoclaved at 121.degree. C. for one hour. After
 autoclaving, the pH was adjusted to 7.0 with 0.1M Tris buffer. This
 solution was then diluted in pyrogen-free water to give a final laminarin
 stock solution of 0.2 mg/mnL. A dilution series of laminarin was produced,
 and the reactivity of crude lysate and the lysate preparation of the
 invention to laminarin was determined both by gel-clot and
 kinetic-turbidimetric assays.
 I. Gel-clot Assay
 Gel-clot assays were performed by combining either crude lysate or lysate
 preparations of the invention with different amounts of laminarin. Crude
 lysate (batch K2222L) was formulated essentially as described in Example
 2, except that the "Extracted Limulus Amebocyte Lysate" was replaced with
 crude lysate and the concentration of laminarin was varied between
 samples. The lysate preparations of the invention (batch L4941LB, batch
 L1081LB, and batch L1711LB) were formulated essentially as described in
 Example 3, except the concentration of laminarin was varied between
 samples. The samples were incubated at 37.degree. C., and the presence or
 absence of clots determined after one hour. Each experiment was performed
 in duplicate, and the results summarized in Table 7.
 TABLE 7
 LAMINARIN
 Conc. K2222L L4941LB L1081LB L1711LB
 Dilution mg/mL 0.03 0.03 0.015 0.06
 1 50 4.00E-03 - - - - - - - -
 2 100 2.00E-03 - - - - - - - -
 3 200 1.00E-03 - - - - - - - -
 4 5 6 7 8 400 800 1600 3200 6400 5.00E-04 2.50E-04 1.25E-04
 6.25E-05 3.13E-05 ##STR3## + + + + + + + + + + - - - - - - -
 - - - - - - - - - - - - - - - - - - - - - - -
 9 12800 1.56E-05 - - - - - - - -
 10 25600 7.81E-06 - - - - - - - -
 11 51200 3.91E-06 - - - - - - - -
 12 102400 1.95E-06 - - - - - - - -
 13 204800 9.77E-07 - - - - - - - -
 14 409600 4.88E-07 - - - - - - - -
 15 819200 2.44E-07 - - - - - - - -
 16 1638400 1.22E-07 - - - - - - - -
 17 3276800 6.10E-08 - - - - - - - -
 18 6553600 3.05E-08 - - - - - - - -
 Shaded areas represent sample active range.
 The results in Table 7 indicate that none of the batches produced by the
 method of the invention (i.e., L4941LB, L1081LB and L1711LB) were reactive
 with exogenously added laminarin. In contrast, the batch of crude lysate
 (K2222L) was reactive with laminarin over the dilution range of 400
 through 6400 in a gel-clot assay.
 II. Kinetic-turbidimetric Assay
 Reactions were initiated by combining either formulated crude lysate (batch
 K2222L) or formulated lysate preparations of the invention (batch L4941LB,
 batch 1081LB, and batch L1711LB) with different amounts of laminarin. The
 assay was formed using a Biotek elx-808 incubating nicroplate reader in
 accordance with the manufacturers instructions. Each assay was run, at
 least, in duplicate. During the assay, the samples were incubated at
 37.+-.0.2.degree. C., and data was collected over a period of one hour.
 The assay was performed simultaneously using additional lysate samples
 incubated in the presence of known amounts of endotoxin standards.
 Endotoxin standards were used to generate a standard curve covering a
 4-log range, for example, from 50 to 0.005 EU/mL. After data collection,
 the data was analyzed using Biotek Kc3-cre kinetic software (available
 from Charles River Endosafe, Charleston, S.C.). The sample concentrations
 were interpreted as "endotoxin values" by the instrument and were
 represented by the units of EU/mL because they were estimated from the
 endotoxin standard curve. The resulting concentration values reflect the
 reactivity of the lysate to laminarin, and are referred to as "endotoxin
 equivalent values".
 By plotting the laminarin endotoxin equivalent values versus dilution
 factor, it is possible to generate a bell-shaped curve with crude lysate.
 The peak of the curve provides the optimal amount of laminarin which can
 be added to amebocyte lysate depleted of Factor G activity, thereby to
 enhance the sensitivity of the lysate.
 The results of the experiment are shown in FIG. 4. According to FIG. 4,
 none of the batches produced by the method of the invention (i.e.,
 L4941LB, L1081LB and L1711LB) were reactive with exogenously added
 laminarin. In contrast, the batch of crude lysate (K2222L) was reactive
 with laminarin over the dilution range of 100 through 51200, with the
 dilution value of 3200 producing the maximal endotoxin equivalent value.
 Accordingly, this value represents the optimal amount of laminarin to be
 added to amebocyte lysate depleted of Factor G activity to provide maximal
 sensitivity to endotoxin in a kinetic turbidimetric assay.
 Example 5
 Reactivity of Crude Amebocyte Lysate and Lysate Depleted of Factor G
 Activity with LAL Reactive Material
 This example demonstrates that Limulus amebocyte lysates produced by the
 method of the invention, unlike crude lysates, are insensitive to the
 presence in the sample of LAL reactive material (LAL-RM) produced by
 rinsing a cellulose-acetate filter with water. In addition, this example
 defines the optimal amount of this LAL-RM (as determined from the crude
 lysate sample) that may be exogenously added to the lysate preparation of
 the invention, thereby to enhance the sensitivity of the lysate
 preparation to endotoxin.
 LAL-RM was prepared by passing one liter of pyrogen-free water through a
 conventional sterile, non-pyrogenic cellulose acetate hollow fiber
 membrane adapted for use in renal dialysis. The resulting rinse then was
 used to create a dilution series of cellulose acetate rinse. The
 reactivity of crude lysate and the lysate preparation of the invention to
 the cellulose acetate rinse was determined both by gel-clot and
 kinetic-turbidimetric assays.
 I. Gel-clot Assay
 The gel-clot assays were performed as described in Example 4I above, except
 in the formulations laminarin was replaced by cellulose acetate rinse. The
 assays were performed by combining either formulated crude lysate (batch
 K2222L) or formulated lysate preparations of the invention (batch L4941LB,
 batch L1081LB, and batch L1711LB) with different amounts of cellulose
 acetate rinse. The samples were incubated at 37.degree. C., and the
 presence or absence of clots assessed after one hour. Each experiment was
 performed in duplicate and the results summarized in Table 8.
 TABLE 8
 Cellulose Acetate Rinse
 Conc. K2222L L4941LB L1081LB L1711LB
 Dilution mg/mL 0.03 0.03 0.015 0.06
 1 2 3 4 5 6 7 8 50 100 200 400 800 1600 3200 6400
 4.00E-03 2.00E-03 1.00E-03 5.00E-04 2.50E-04 1.25E-04 6.25E-05
 3.13E-5 ##STR4## + + + + + + + + + + + + + + + + - - - - - -
 - - - - - - - - - - - - - - - - - - - - - - - - -
 - - - - - - - - - - - - - - - - -
 9 12800 1.56E-05 - - - - - - - -
 10 25600 7.81E-06 - - - - - - - -
 11 51200 3.91E-06 - - - - - - - -
 12 102400 1.95E-06 - - - - - - - -
 13 204800 9.77E-07 - - - - - - - -
 14 409600 4.88E-07 - - - - - - - -
 15 819200 2.44E-07 - - - - - - - -
 16 1638400 1.22E-07 - - - - - - - -
 17 3276800 6.10E-08 - - - - - - - -
 18 6553600 3.05E-08 - - - - - - - -
 Shaded areas represent sample active range.
 The results of Table 8 indicate that none of the batches produced by the
 method of invention (i.e., LA4941LB, L1081LB and L1711LB) were reactive
 with exogenously added cellulose acetate rinse as determined by gel-clot
 assay. In contrast, the batch of crude lysate (K2222L) was reactive with
 cellulose acetate over the dilution range of 50 ugh 6400 in a gel-clot
 assay.
 II. Kinetic-turbidimetric Assay
 This assay was performed as described in Example 4II above, except in the
 formulations laminarin was replaced by cellulose acetate rinse. Reactions
 were initiated by combining either crude lysate (batch K2222L) or lysate
 preparations of the invention (batch L4941LB, batch L1081LB, and batch
 L1711LB) with different amounts of cellulose acetate rinse. The results of
 the assay are presented in FIG. 5. According to FIG. 5, none of the
 batches produced by the method of the invention (i.e., LA4941LB, L1081LB
 and L1711LB) were reactive with exogenously added cellulose acetate. In
 contrast, the batch of crude lysate (K2222L) was reactive with cellulose
 acetate over the dilution range of 50 through 102400 with the dilution
 value of 400 producing the maximal endotoxin equivalent value.
 Accordingly, this value represents the optimal amount of cellulose acetate
 to be added to amebocyte lysate depleted of Factor G activity to provide
 maximal sensitivity to endotoxin in a kinetic turbidimetric assay.
 Example 6
 Reactivity of Crude Amebocyte Lysate and Lysate Depleted of Factor G
 Activity with Cotton Extract
 This example demonstrates that Limulus amebocyte lysates produced by the
 method of the invention, unlike crude lysates, are insensitive to the
 presence in the sample of cotton extract. In addition, this example
 defines the optimal amount of cotton extract (as determined from the crude
 lysate sample) that may be exogenously added to the lysate preparation of
 the invention, thereby to enhance the sensitivity of the lysate
 preparation to endotoxin.
 Cotton extract was prepared as follows. Approximately, 1 gram of cotton
 (about 4 one inch cotton balls) was added to 40 ml of 1 N NaOH in a 50 ml
 polystyrene conical tube. Thereafter, the mixture was incubated at room
 temperature (about 20.degree. C.) for 10 days. Prior to use, the solution
 was decanted from the cotton into a fresh, pyrogen-free tube (baked
 200.degree. C., 6 h) and neutralized with HCl to produce a stock solution
 of cotton extract. Further dilutions were made in pyrogen-free water
 (available from Charles River Endosafe, Charleston, S.C.). The reactivity
 of crude lysate and the lysate preparation of the invention to cotton
 extract were determined both by gel-clot and kinetic-turbidimetric assays.
 I. Gel-clot Assay
 The gel-clot assays were performed as described in Example 4I above, except
 in the formulations laminarin was replaced by cotton extract. The assays
 were performed by combining either formulated crude lysate (batch K2222L)
 or formulated lysate preparations of the invention (batch L4941LB, batch
 L1081LB, and batch L1711LB) with different amounts of cotton extract. The
 samples were incubated at 37.degree. C., and the presence or absence of
 clots determined after one hour. Each experiment was performed in
 duplicate and the results summarized in Table 9.
 TABLE 9
 COTTON EXTRACT
 Conc. K2222L L4941LB L1081LB L1711LB
 Dilution mg/mL 0.03 0.03 0.015 0.06
 1 50 4.00E-03 / / / / / / / /
 2 3 4 5 100 200 400 800 2.00E-03 1.00E-03 5.00E-04 2.50E-04
 ##STR5## + + + + + + + + - - - - - - - - - - - - - - -
 - - - - - - - - -
 6 1600 1.25E-04 - - - - - - - -
 7 3200 6.25E-05 - - - - - - - -
 8 6400 3.13E-05 - - - - - - - -
 9 12800 1.56E-05 - - - - - - - -
 10 25600 7.81E-06 - - - - - - - -
 11 51200 3.91E-06 - - - - - - - -
 12 102400 1.95E-06 - - - - - - - -
 13 204800 9.77E-07 - - - - - - - -
 14 409600 4.88E-07 - - - - - - - -
 15 819200 2.44E-07 - - - - - - - -
 16 1638400 1.22E-07 - - - - - - - -
 17 3276800 6.10E-08 - - - - - - - -
 18 6553600 3.05E-08 - - - - - - - -
 Shaded areas represent sample active range.
 The results in Table 8 indicate that none of the batches produced by the
 method of the invention (i.e., L4941LB, L1081LB and L1711LB) were reactive
 with exogenously added cotton extract. In contrast, the batch of crude
 lysate (K2222L) was reactive with cotton extract over the dilution range
 of 100 through 800 in a gel-clot assay.
 II. Kinetic-turbidimetric Assay
 This assay was performed as described in Example 411 above, except in the
 formulations laminarin was replaced by cotton extract. Reactions were
 initiated by combining either crude lysate (batch K2222L) or lysate
 preparations of the invention (batch L4941LB, batch L1081LB, and batch
 L1711 LB) with different amounts of cotton extract. The results of the
 assay are presented in FIG. 6. According to FIG. 6, none of the batches
 produced by the method of the invention (i.e., L4941LB, L1081LB and
 L1711LB) were reactive with exogenously added cotton extract. In contrast,
 the batch of crude lysate (K2222L) was reactive with cotton extract over
 the dilution range of from at least 100 through 3200. According to the
 results, it appears that cotton extract also may be used to enhance the
 sensitivity of Factor G depleted lysate to endotoxin.
 Example 7
 Endotoxin Sensitivity of Amebocyte Lysate Depleted of Factor G Activity
 With and Without (1.fwdarw.3)-.beta.-D Glucan
 This example demonstrates that the sensitivity to endotoxin of an amebocyte
 lysate depleted of Factor G activity can be enhanced by the addition of
 exogenous (1.fwdarw.3)-.beta.-D glucan.
 Briefly a batch of amebocyte lysate prepared in accordance with Example 1
 was formulated essentially as described in Example 2. A control batch was
 formulated without the exogenously added laminarin. The reactivities of
 each lysate to different amounts of endotoxin were measured by
 kinetic-turbidimetric assay, essentially as described in Example 4II.
 Briefly, different amounts of endotoxin (USFDA lot EC-6) were added to
 each sample of lysate and the reaction end point determined by Bio-tek
 KC3-cre kinetic software. The results are shown in FIG. 7. The circles
 represent samples of lysate not supplemented with laminarin (control
 samples), and the boxes represent samples of lysate supplemented with
 laminarin (test samples).
 According to FIG. 7, at high concentrations of endotoxin (i.e., about 50
 EU/ml) there appears to be little difference in reaction time between
 lysate supplemented with laminarin and lysate not supplemented with
 laminarin. However, at low concentrations of endotoxin (i.e., about
 .ltoreq.0.05 EU/mL), the lysate supplemented with laminarin reacted
 significantly faster with endotoxin than lysate not supplemented with
 larinarin. Accordingly, these results demonstrate that it is possible to
 enhance the sensitivity of an amebocyte lysate depleted of Factor G
 activity by the addition of exogenous (1.fwdarw.3)-.beta.-D glucan.
 Equivalents
 The invention may be embodied in other specific forms without departing
 from the spirit or essential characteristics thereof. The foregoing
 embodiments are therefore to be considered in all respects illustrative
 rather than limiting on the invention described herein. Scope of the
 invention is thus indicated by the appended claims rather than by the
 foregoing description, and all changes that come within the meaning and
 range of equivalency of the claims are intended to be embraced therein.