GB2059583A - Inactivating Inhibitors in Endotoxin Clottable Extracts - Google Patents

Abstract

Endotoxin clottable extracts are standardized by inactivating endogenous inhibitor by adjusting the extract pH to above 8.5, incubating and readjusting the pH to near neutrality, determining the coagulase activity of the extract and dividing the extract into endotoxin assay reaction mixture portions each providing coagulase activity at a predetermined level. In most circumstances the coagulogen concentration is also determined and, if the concentration is lower than a predetermined amount, coagulogen is added to the extract.

Description

SPECIFICATION Inactivating Inhibitors in Endotoxin Clottable Extracts This invention is concerned with inactivating inhibitors in endotoxin clottable extracts, e.g.

extracts of the blood cells of organisms such as Limulus polyphemus, the horseshoe crab.

The extracts with which this invention is concerned all have in common an enzyme, coagulase.

This enzyme acts in concert with another extract component to form a gel or clot upon contact with endotoxin. Hence, these extracts are defined as endotoxin clottable extracts. They are ordinarily obtained from the blood amoebocytes of Limulus polyphemus. Conventionally, the amoebocytes are collected from the blood, lysed to free the water soluble intercellular components and centrifuged to remove suspended detritus. A suitable technique for obtaining such extracts from Limulus is disclosed in United Kingdom Patent 1 ,499,846. These extracts also may be obtained from other species, for example Tachypleus tridentatus.

Endotoxin clottable extracts contain two principal components which are responsible for the clot formation. These components are coagulase and coagulogen. Coagulase is believed to be activated by endotoxin to catalyze the proteolytic hydrolysis of coagulogen to a protein fragment which then spontaneously polymerizes to yield a detectable product. Coagulase has been extensively studied and characterized. Illustrative works are those of Tai et al., "Journal of Biological Chemistry" 252(1 4):4773-4776 (1977) and Solum, "Thrombosis Research" 2:55-70 (1973).

Coagulogen as well as a method for its purification is described by Gaffin in "Biorheology" 13:273-280 (1976). Coagulogen essentially serves as a substrate for activated coagulase. It has been particularly useful as a gauge of coagulase activity because it provides its own indicator; the coagulase hydrolyzed fragments form insoluble particles and, eventually, a semisolid clot without the intervention of any additional reagents.

The high sensitivity of endotoxin clottable extracts to endotoxin has been extensively employed in screening assays for pyrogens in human body fluids and biological products such as pharmaceuticals and parenteral solutions. Several conventional methods have been adopted for using the extracts to conduct endotoxin assays. Included among them are those in which the increase in optical density of the extract or the formation of a gel clot is followed upon contact with endotoxin. An exemplary quantitative endotoxin assay entails analysing the specific quantity of protein contained in the clot.

The widespread adoption of such assays on a commercial scale has been hindered by variations in sensitivity encountered among separate lots of Limulus extracts and the difficulty in correcting the variations. Such potency variations have been postulated to be based on seasonal factors, i.e., dependent upon the time of year the Limulus blood is collected. At least one component which is held in part responsible for this variation has been characterized as an "inhibitor" of the clotting reaction.

This nomenclature is based on the apparent action of the inhibitor since wherever it is present losses in endotoxin assay sensitivity are encountered. It has been speculated that the inhibitor may be an enzyme [Nachum et al., "Journal of Invertebrate Pathology" 32:51-58 (1978)] or a lipoprotein that simply binds endotoxin [Sullivan et al., "Applied Microbiology" 28(6):1 023-1026 (1974)]. Whatever its mode of action, the material will hereinafter be designated as an inhibitor.

The Sullivan et al. publication cited above also discloses a technique for reducing the activity of the inhibitor. This method comprises shaking equal volumes of organic solvent and Limulus lysate, followed by centrifugation and recovery of the aqueous phase. This method has been found to be unsatisfactory because it requires handling toxic or highly flammable reagents and calls for a burdensome phase separation. Furthermore, residual organic solvent may remain in the Sullivan et al.

product. Even water-immiscible solvents exhibit a slight solubility in water; in the case of chloroform, this solubility is 0.82 parts per 100 parts of water at 200C. The effect of this residue on the sample to be assayed may be unpredictable or undesirable.

The presence of this inhibitor in extracts which had been considered "standardized" has prevented the extracts from in fact performing as reliable standards. Further, the known "standardized" extracts have not been prepared with suitable consideration of the coagulogen and true coagulase levels in the products. Typical "standardized" Limulus lysates are disclosed in U.S. Patents 3,944,391 and 4,038,147. These products have been prepared by simply assaying a lysate batch against a serial dilution of endotoxin to determine the gross potency of the lysate. The principal difficulty with this approach is that it is essentially passive; the extract is in no way treated or processed to yield a single predetermined signal, e.g., development of a given nephelometric endpoint upon reaction with a given endotoxin concentration.This renders quality control more difficult since new standard curves must be prepared at the user level for each new lot of lysate. It is commercially desirable to be able to supply lysate from a variety of lots which will continuously produce the same results in an endotoxin determination using constant endotoxin in contrations even though the original, untreated lots would have produced a scattering of signals, some higher and some lower than the desired, predetermined level.

Thus it is one object of this invention to produce endotoxin clottable extracts which will yield a reproducible predetermined analytical signal for a given endotoxin concentration within an analytically significant range and to replace the previously employed process for inactivation of the Limulus inhibitor with one which does not require toxic or hazardous reagents, which is less expensive in both labour and materials and which leaves no potentially interfering residue in the product.

Applicants have surprisingly found that the influence of the inhibitor may be removed from Limulus extracts by adjusting the pH of the extract above about 8.5. After the desired degree of inhibitor inactivation has occurred, the pH of the treated extract is reduced to a point at which the coagulase is active. The resulting extract is noteworthy because it contains no organic solvent residue, yet is substantially free of inhibitor.

This method can be employed to manufacture a standardized extract. This method for making standardized endotoxin clottable extract comprises: (a) substantially inactivating the inhibitor content of a native, endotoxin clottable extract; (b) determining the coagulase activity of the extract; and (c) separating the extract in portions calculated to contain a predetermined coagulase activity.

Usually it is also desirable to determine that the coagulogen concentration in the extract is at least in excess of a predetermined level.

The resulting standardized product is a quantity of endotoxin clottable extract which is substantially free of inhibitor and contains a predetermined coagulase activity. Again it is desirable that the volume of extract also contain at least an excess of a predetermined amount of coagulogen.

The discovery that the inhibitor can be destroyed by a pH adjustment was most surprising. Levin et al. ["J. Lab. Clin. Med." 75(6):903-91 (1970)] disclose several attempts to remove an inhibitor of the Limulus lysate clotting reaction found in blood plasma. While Levin et al. teach inactivating the inhibitor with chloroform, precipitation with trichloroacetic acid was unsuccessful at removing or inactivating the inhibitor. Parenthetically, the inhibitor in plasma has since been linked to the inhibitor which is found in Limulus extracts (U.S. Patent 4,107,077). Further, not only does Levin et al. suggest that acid is ineffective against the inhibitor, for others have concluded that acidification of lysate irreversibly inactivates coagulase [Solum, "Thrombosis Research" 2:55-70 (1973)].This is, of course, the opposite result from that which is desired herein. Surprisingly, however, when the pH of the endotoxin clottable extract is raised, rather than lowered, coagulase activity is preserved while the inhibitor is irreversibly inactivated.

The endotoxin clottable extract which is the starting material for the improved processes and compositions herein may be any composition containing coagulase. This composition is termed an extract because it is generally obtained by separating the soluble components of Limulus amoebocytes from insoluble elements such as cell walls. Nonetheless, it is not essential that the insoluble elements be removed, or even that the cells be lysed if a coagulase assay is employed which is not dependent upon the formation of a clot or suspension of polymerized coagulogen particles. Further, the extract need not contain coagulogen if an analytical substrate other than coagulogen is employed to follow the coagulase activity. It is, however, essential that the starting material contain coagulase.

These endotoxin clottable extracts generally contain an inhibitor. The activity of the inhibitor is removed in accordance with this invention by adjusting the pH into the alkaline range, incubating until the desired degree of inactivation is achieved and then reducing the pH into the neutral region where coagulase once again is active. A significant advantage of this process is that the pH adjustments ordinarily do not precipitate elements of the extracts, including inhibitor. Thus, unlike the known chloroform procedure it is not necessary to separate any portion of the reaction mixture after inactivation of the inhibitor. A brief centrifugation or crude filtration step may occasionally be desirable if the inhibitor is inactivated at a pH above 11 and the treated extract is to be subsequently used in a nephelometric or optical density method for endotoxin assay.In most circumstances, however, no special treatment of the extract is needed after inactivation of the inhibitor by pH adjustment. It is not necessary that the inhibitor be 100% inactivated. It is generally satisfactory that the lysate be substantially free of inhibitor, generally at least 75% inactivated. The degree of inactivation needed will depend upon the sensitivity for endotoxin which is desired and the variability in inhibitor content within the lots being standardized. Thus, the degree of inactivation will be a matter of choice.

The pH of a typical lysate of Limulus amoebocytes is approximately 6.0-7.0. The alkaline pH adjustment of this invention may be made in any conventional fashion, for example by titering an aqueous alkaline solution into the extract with steady stirring. However, crystalline or solid basic substances may be employed in place of solutions. The former is desirable where it is intended to later lyophilize the extract since this technique will not add to the water volume to be removed.

The alkaline material itself may be organic or inorganic, although dilute aqueous solutions of alkali metal hydroxides such as sodium hydroxide are preferred. An examplary organic alkaline substance which can be employed is an alkyl amines. The specific identity of the substance is not critical; it need only raise the pH without causing undesirable side reactions which could inactivate coagulase or coagulogen to a degree which might burden the use of the lysate in an endotoxin assay. It is preferred that a buffer not be included in either the extract or the alkaline substance as this will increase the amount of reagent required to make either or both pH adjustments.

While the alkaline material should exert no deleterious effect on the coagulase, endotoxin or coagulogen, if coagulogen is present, it is possible to select a cation which may in fact be of advantage in the assay. For example, manganese or magnesium hydroxide can be employed as at least a portion of the alkaline material, since the manganese or magnesium ions also serve as a cofactor for the coagulase. Unfortunately, the use of divalent metal cofactor hydroxides is limited because of their low solubility. In any case a diva lent cation cofactor for coagulase should preferably be added to the extract before or concommitant with the alkaline pH adjustment, whether or not the cation-containing composition contributes any alkalinity. The cofactor, however, is not essential.

The extract is incubated at an elevated pH until the desired degree of inhibitor inactivation has occurred. It is preferred that all of the inhibitor be inactivated before incubation is halted. The degree of inactivation varies with incubation time, temperature and pH; for example, the longer the incubation and the higher the temperature and pH the more rapid is the inactivation. Optimizing these parameters is matter of routine experimentation for the skilled artisan; the conditions will depend upon the nature of each lot of lysate.

The pH may be adjusted to any point greater than about pH 8.5, generally about from 8.5 to 11 and preferably about pH 9.0-10.0. Both coagulase and coagulogen are stable at these hydrogen ion concentrations. However, since other proteins in the extracts may precipitate at a pH of about 11, it may be useful to select a lower pH and incubate for a greater period.

The incubation time sufficient to inactivate the inhibitor will, of course, vary depending upon the amount of inhibitor present and the desired degree of inactivation. Generally, from 20 minutes to 4 hours is sufficient; 90 minutes is preferred at pH 9 and 40C.

The incubation temperature may range from about OOC. to the temperature at which coagulase is thermally inactivated; the latter point has been previously determined to be at about 600C. The temperature should be maintained at about from OOC to 400C, and is preferably held at about 40C by immersion of the reaction vessel in an ice bath.

After incubation, the pH should be returned a leyel at which coagulase is active. Active coagulase is defined as coagulase capable of reversibly binding endotoxin or coagulase capable of so binding endotoxin and, in addition, expressing this binding by catalytic action on coagulogen or other suitable substrate. Ordinarily the catalytic alternative is meant. The pH readjustment is most conveniently done by adding an acid using any of the techniques employed to initially raise the pH. Generally the pH is readjusted by addition of dilute aqueous acid to the alkaline extract while stirring. A suitable concentration of acid is about 0.1 N. The optimum final pH is one which will yield the optimum sensitivity of the lysate in an endotoxin assay.This is ordinarily coextensive with the optimum pH for coagulase activity, about from pH 6.0 to 7.5 and preferably 7; the lysate is then preferably lyophilized or stored frozen.

The substance used to lower the pH should, like the alkaline substance, not be deleterious to coagulase, or coagulogen if the latter is present. Generally, inorganic acids such as hydrochloric, sulfuric or nitric acid are satisfactory although organic acids may also be employed. The substance may be buffered and in fact, the buffer need not be at an acid pH but may be prepared for use at the final pH desired, e.g. pH 7, and used in an amount sufficient to reduce the pH to that level. Alternatively, a buffer such as tris may be added after the pH has been adjusted to about 7. A divalent cation cofactor for coagulase may also be added to the extract at this time.

Once the inhibitor activity of an endotoxin clottable extract has been substantially destroyed, the extract is suitable for use in preparing a standardized extract. It is preferred that the inhibitor be inactivated by the pH adjustment discussed above. However, any other method such as the known chloroform extraction technique can be used. Unless the inhibitor activity has been destroyed, the competing reaction kinetics of the inhibitor-endotoxin reaction and the coagulase-endotoxin reaction will make it impossible to standardize the extract over a range of endotoxin concentrations.For example, if the inhibitor-containing extract is assayed for coagulase activity at a coagulase-saturating amount of endotoxin, for example 3 mg/ml, and then filled into containers in an amount such that predetermined enzyme activity will be added to each container, whatever the activity of the original extract lots, the product is found to contain only about 80% of the assayed activity when endotoxin concentrations over the range of 25 to 100 pg/ml are determined. This variance is postulated to be due to diversion of endotoxin by the inhibitor, thus resulting in lower apparent endotoxin levels than are actually present in the original sample. When the endotoxin concentration is so high that all coagulase present is activated, i.e. the coagulase is saturated, diversion of excess endotoxin by inhibitor will not affect the coagulase activity.However if the coagulase is to be used as an indicator of endotoxin concentration, the enzyme a fortioriwill not be saturated over the range of endotoxin concentrations expected. In such case the interference by the inhibitor necessarily will be manifested in the endotoxin assay. Similarly, if the extract is standardized at a first, non-saturating endotoxin level and then assayed at a second level of endotoxin, an extract with inhibitor will yield a different result than one without. This anomaious result is believed to result from the balance of the coagulase and the inhibitor kinetics: At the first endotoxin level activation of coagulase might be favoured while at the second level the action of the inhibitor might be favoured.

If the intended assay method is based on coagulogen hydrolysis by coagulase, the standard extract also should contain a predetermined coagulogen concentration which is in such an excess that the assay for endotoxin is not limited by coagulogen over the expected endotoxin range. If coagulase is standardized by simply determining the enzyme activity at one endotoxin concentration and an insufficient quantity of coagulogen is present to indicate true coagulase activity at other more elevated endotoxin levels, the results at such elevated levels will indicate less endotoxin than is actually present.

This failing could have a severe adverse impact in the diagnosis of bacteremia or in the quality control screening of parenteral products.

It has been found that coagulogen concentrations are generally proportional to coagulase activity in native Limulus extracts. Thus in many cases it is not desirable to do more than to confirm that sufficient coagulogen is present; in such cases it would not be necessary to add supplementary coagulogen to the extract. However, it is conceivable that inadvertent exposure of such extracts to extraneous endotoxin during collection and processing might deplete the coagulogen and thereby create a need for coagulogen supplementation. Thus the coagulogen concentration should be determined to be at least a predetermined level or in great excess of a predetermined concentration.

The predetermined amount of coagulogen will depend upon the endotoxin concentrations expected in the samples to be assayed. If the samples are expected to be only slightly contaminated, on the order of 1-100 pg/ml, then a coagulogen content in of about 150,ug/ml lysate is ordinarily satisfactory.

Usually, a coagulogen concentration in excess of 100 Mg/ml lysate will enable the product to be used with most endotoxin-contaminated samples of biological materials. The minimum amount of coagulogen is defined at its lower level by the detection limits of the coagulase assay for endotoxin, and this can be expected to improve as the assay is developed further in the future. On the other hand, the maximum amount to be used is limited only by the maximum solubility of the coagulogen in aqueous solutions; even exceptionally high endotoxin concentrations, for example 1.0 mg/ml, could be theoretically determined by coagulogen hydrolysis-product formation, particularly where the product is removed from the reaction mixture as the hydrolysis proceeds.Since a coagulogen content of over 100-400 ,ug"ml will not adversely affect the assay and the lower coagulogen concentration is defined by the state of the art in detecting coagulogen hydrolysis products, the selection of a particular predetermined coagulogen content is presently a matter of choice.

Coagulogen may be determined by modifying the Munford assay for endotoxin disclosed in "Analytical Biochemistry"91:509-515 (1978). Instead of assaying unknown endotoxin, as provided in this method, a fixed endotoxin concentration is used with 1251-labelled coagulogen, unknown coagulogen and coagulase. Following an incubation period, the clot-bound radioactivity is determined as in a typical competitive-type radio-immunoassay. A large quantity of coagulogen will displace labelled coagulogen from coagulase-mediated precipitation, and vice versa with comparatively lower levels of coagulogen. Thus, the greater the clot-bound radioactivity the lower is the coagulogen concentration. The method is quantifiable by use of purified coagulogen standards.

If the coagulogen concentration is lower than the predetermined level it will be necessary to increase the coagulogen level in the extract. For example, a coagulogen-rich extract or purified coagulogen may be mixed with the extract to achieve the desired increase in coagulogen concentration. Alternatively, the coagulogen may be concentrated by ultrafiltration.

In preparing a standardized, endotoxin clottable extract it is necessary to determine the coagulase activity. This may be accomplished by any of the known coagulase assay methods; these methods have frequently bee used to determine endotoxin concentration as well. Here, however, a known, preferably saturation concentration of endotoxin is used. A saturating endotoxin concentration is generally in excess of about 1 mg/ml endotoxin, preferably 3 mgjml. The assay results may be converted into arbitrary enzyme units since for standardization purposes it is the relative rather than absolute enzyme activity which is relevant. Once the coagulase activity of the extract has been determined the extract is proportioned into an endotoxin assay reaction mixture in an amount calculated to produce the selected enzyme activity.For example, the treated extract may be added directly to the endotoxin assay reaction mixture in a volume calculated to yield the predetermined activity. Since it is more expeditious for the analyst to always add a fixed volume of extract, containers for storing the extract can be filled with an amount of extract calculated to contribute the predetermined activity upon reconstitution in a constant amount of aqueous solution; a proportional fill relieves the user of the burden of proportional reconstitution. The extract may then be lyophilized.

Reference is now made to the following examples.

Example I Two lots of Limulus polyphemus amoebocytes were separately collected and lysed by the method of United Kingdom Patent 1,499,846. One lot was designated a reference Iysate.The remaining lot was employed as the starting material in the preparation of standardized lysate in accordance with the method of this invention. It was designated lot 8-113.

Both lots were first assayed for coagulase activity. 50 mM Tris buffer at pH 7.5 and containing 10 mM MgCl2was used wherever buffer is mentioned below. E. collendotoxin (055:B5, Difco Laboratories) was dissolved in buffer. Coagulogen was prepared in a concentration of 8.0 mg/ml in buffer by the method of Solum, supra, except that the crude lysate was precipitated with ammonium sulfate at 80% of saturation before redissolution and placement on the Sephadex column, the column then eluted with phosphate buffer and the eluate further purified on a carboxymethyl cellulose column.

0.1 ml of a 3.0 mg/ml endotoxin solution, 0.1 ml of coagulogen solution, 0.1 ml of lysate and 2.7 ml of buffer were mixed and incubated for 8 minutes. After incubation the change in optical density at 360 nM and 37 OC was recorded. One enzyme unit was arbitrarily defined as a 0.001 absorbance unit change in optical density at 360 nM. The reference lot and lot 8-11 3 exhibited an enzyme activity of 665 and 440 units/ml, respectively.

Since a plot of optical density against lysate fill volume, i.e., dilution, is not a straight line, the quantity of reference and 8-113 lysate which will yield the same coagulase activity will not be directly proportional to the arbitrarily assigned optical density units described above. Because of this dilution effect the amount of 8-113 lysate needed to produce the same activity as the reference lysate is readily calculated from the above-described plot as only 2.65 ml. Thus 2 ml and 2.65 ml of reference and 8-113 lysate, respectively, were added to the containers. The vial contents were then lyophilized.

Next the inhibitor activity of the lots was inactivated by the novel method of this invention. Two vials of the reference lysate and two vials of lysate to be standardized were reconstituted in 2.5 ml of sterile H2O. One vial of each lot was adjusted to pH 9.0 with 0.1 N NaOH and incubated at 40C for 90 minutes. Then the pH in each vial was reduced to 7.5 by stirring in 0.1 N HCl at 40C. 2.5 ml of buffer was added to each vial and the lysate assayed against the above E. coli endotoxin at endotoxin concentrations of 0,25, 50 and 100 pg/ml.

The assay method was essentially that shown in United Kingdom Patent 1,499,846. Table 1 below compares the optical density at 650 nM of the treated and control lysates.

Table I Lysate With Coagulaseand En do toxin Coagulase and Concentration pHAdjustment ControlLysate pg/ml Reference 8-113 Reference 8-113 100 0.826 0.812 0.683 0.562 50 0.577 0.565 0.486 0.359 25 0.362 0.361 0.285 0.207 0 0.161 0.124 0.135 0.112 As can be seen by these results, the alkaline treatment in conjunction with the proportional distribution of coagulase both increased the sensitivity of the lysates to endotoxin and eliminated the variability encountered with native lysate.With the pH adjustment, the standard lot continued to exhibit coagulase activity approximately equal to that of the reference, in the order of from 97.9 to 99.7% of the reference activity, while without the adjustment the 8-113 lot contained an apparent activity at 25, 50 and 100 pg endotoxin/ml, respectively, of only 72.6%, 73.9% and 82.3% of the reference lot activity.

Example II The procedure of Example I was repeated with the reference lot and another lot of lysate, designated 8-112. As in Example I with lot 8-113, the coagulase activity of lot 8-11 2 had been proportioned to the same level as the reference lot. However, in this example the inhibitor was inactivated by a chloroform extraction. Chloroform was thoroughly mixed with the reference lot and lol 8-112 in a 1:2 proportion of chloroform to lysate, and incubated at 40C for 60 minutes. The lysatechloroform mixtures were centrifuged to remove precipitate and undissolved chloroform and afterwards treated and assayed in the same fashion as in Example I. The results are tabulated below.

Table II Lysate With Chloroform Control L ystate Endotoxin Extraction (No ExtractionJ Concentration pg/ml Reference 8-112 Reference 8-112 100 1.224 1.400 1.044 0.773 50 1.008 1.111 0.695 0.528 25 0.774 0.717 0.469 0.391 0 0.214 0.165 0.195 0.170 The chloroform extraction technique resulted in a lysate showing greater variation among assays at various endotoxin concentrations than encountered with the pH adjustment method of Example I, i.e., lot 8-112 was 14.4% and 10.2% more active than the reference lot and 7.3% less active at, respectively, 100, 50 and 25 pg/ml of endotoxin. However, this example illustrates that chloroform extraction can also be employed to prepare the standard endotoxin clottable extracts of this invention.

Claims (14)

Claims

1. A method for inactivating the inhibitor content of an endotoxin clottable extract which comprises adjusting the pH of the extract above 8.5 to inactivate the inhibitor, followed by lowering the pH to a level at which coagulase is active.

2. A method for inactivating Limulus amoebocyte lysate inhibitor which comprises increasing the lysate pH to 8.5 to 11, incubating the lysate until the desired degree of inactivation has occurred and then lowering the lysate pH to about 7.

3. A method for inactivating the inhibitor content of Limulus amoebocyte extract which comprises inactivating the inhibitor without causing a precipitate to form in the extract.

4. An aqueous solution of coagulase which is substantially free of inhibitor and completely free of organic solvent.

5. An aqueous solution of coagulase according to claim 4 including coagulogen.

6. A standardized composition for the determination of endotoxin, comprising an endotoxin clottable extract having a predetermined coagulase activity and being substantially free of inhibitor.

7. A standardized composition according to claim 6 which is dry.

8. A standardized composition according to claim 6 or 7 which also includes at least an excess of a predetermined amount of coagulogen.

9. A method for preparing a standardized endotoxin clottable extract which comprises: (a) substantially inactivating the inhibitor component of the extract; (b) determining the coagulase activity of the extract; (c) evaluating coagulogen content of the extract; and (d) separating the extract into portions calculated to contain a predetermined coagulase activity.

10. The method of claim 9, wherein the extract is separated into portions by dispensing volumes of extract into containers so that each container holds a predetermined coagulase activity.

11. The method of claim 10, wherein the extract is lyophilized after being dispensed into the containers.

12. The method of claim 9, 1 0 or 11, wherein the coagulogen content of the extract is evaluated by quantitatively assaying for the coagulogen.

13. A method for preparing a standardized Limulus amoebocyte lysate composition which comprises first inactivating the lysate inhibitor and then adjusting the coagulase activity to a predetermined level.

14. The method of claim 13, wherein the coagulogen level is adjusted to in excess of a predetermined concentration.

1 5. A method for preparing a standardized endotoxin clottable extract which comprises: (a) substantially inactivating the inhibitor component of the extract; (b) determining the coagulase activity of the extract; (c) determining the coagulogen content of the extract and, if the coagulogen content is not in excess of a predetermined level, adjusting said content so that it is in excess of said level.

1 6. The method of claim 1 5, wherein the coagulogen content is adjusted by adding coagulogen.