DE2729618C2 - Reagent for the determination of endotoxin - Google Patents

Description

The invention relates to a reagent for the determination of endotoxin, containing Limulus amebocyte lysa in lyophilized form. This reagent is used to determine endotoxins in biological and parenteral fluids or media.

The hemolymph of the horseshoe crab Limulus polyphemus contains only one type of cell, namely amebocytes. The lysis of these amebocytes (or of corresponding Cells of certain, closely related species) releases a substance that is exposed to endotoxin Formation of a gel implements. This reaction between Limulus amebocyte lysate and endotoxin forms the Basis for extremely sensitive in-vitro methods for the detection and / or determination of endotoxin in a variety of pharmaceutically / medically interesting liquids. Von Levin and Bang is in "Clottable Protein in Limulus: Its Localization and Kinetics of Its Coagulation by Endotoxin," Thromb. Diath. Haemorrh 19: 186-197 (1968), the Manufacture of Limulus amebocyte lysate. In US-PS 39 15 805 a method for the detection of Endotoxin in proteinaceous material using this lysate is described.

E. T. Yin et al "Picogram-Sensitive Assay for Endotoxin: Gelation of Limulus Polyphemus Blood Cell-Lysate Induced by Purified Lipopolysaccharides and Lipid A from Gram-negative Bacteria, "Biochim. Biophys. Acta, 261: 284-289 (1972) have lysed the Limulus amebocytes by mechanical rupture of the cells in a tromethamine (Tris) buffer containing calcium and sodium chlorides. They have reported that these deviations from Levin's procedures involve a lysate have shown improved sensitivity and stability.

H. D. Hochstein et al. In "Further Developments of Limulus Amebocyte Lysate Test", Bull. Par. Drug Assn. 27, 139 to 148 (1973), several methods for lysis of Limulus amebocytes described, for example, the use of ultrasonic waves (sonication) to break up the cells that are inside are suspended in a tromethamine buffer. They also describe using a dilution of the lysate distilled water increases the sensitivity, with the maximum sensitivity at a dilution of 1 plus 5 is reached. They have further reported that after a month or more at room temperature, that the normally white lyophilized lysate will discolor and lose activity

By Sullivan and Watson in Factors Affecting the Sensitivity of Limulus Lysate, Applied Microbiology 28: 1023-1026, (1974), the sensitivity of the lysate to endotoxin is so improved been that an inhibitor (with chloroform or other solvents) is extracted and by low

ι concentrations of divalent cations (Ca ⁺² , Mg ⁺² , Mn ⁺² ) are added. These authors have also described the addition of sodium chloride to reduce the turbidity in the blank sample. They also reported that extraction with chloroform

ίο the stability of the lysate in a detrimental way impaired

M. Kobayashi and M. Yamamoto, "Studies on The Gelation Reaction of Limulus Lysate (Pregel) IV: Effect of Alkaline Earth Metals and Chelating Agents on the Gelation Reaction of Limulus Lysate," Yakugaku Zasshi 95, 959-965 (1975) low concentrations of alkaline earth ions (Mg ⁺² , Ca ⁺² , Ba ⁺² or Sr ⁺² ) added to improve the sensitivity of the lysate / endotoxin reaction. DE-OS 25 30 685 describes that the sensitivity of the lysate to endotoxin can be increased if one or more of the following substances are added to the lysate solution as catalysts: manganese, strontium or barium ions, imidazole, a dithiol (Cleland- Reagent), a monothiol (cysteine), or lithium ions. In DE-OS 25 17 785 it is described that the same goal can be achieved if the lysate is passed through a 2 ^ m filter.

]. Eibert »Pyrogen Testing: Horseshoe Crabs vs.

jo Rabbits ", Bull. Par. Drug Assn., 26,253-260 (1972) reports that there is a very good relationship between the Limulus lysate test and the conventional rabbit pyrogen test in the case of a large number of commercially available parenteral preparations, for example 10% strength Fructose and 25% mannil solutions. It was also mentioned by the author that fructose solutions often contain pyrogens, and it is difficult to get the last detectable traces of pyrogens from this product to remove.

M. Kobayashi et al "Studies on the Gelation Reaction of Limulus Lysate (Pre-Gel) III: Influence of Various Substances on the Gelation Reaction of Limulus Lysate ", Yakugaku Zasshi 95: 720-727 (1975) from the effects of a number of substances, e.g. B.

Glucose and maltose, on the endotoxin / lysate gelation reaction reported. You have with maltose solutions with a concentration of 2.7 to 43.2% and with Glucose solutions with a concentration of 2.5 to 25% no sensitivity fluctuations were found.

However, the sensitivity was lower with the 50% glucose solution. You still have endotoxin detected at various stages in the manufacture of certain products containing maltose.

J.D. Sullivan et al. "Purification and Properties of the Clotting Enzyme from Limulus Lysate," Biochemical and Biophysical Research Communications, 66: 848-855 (1975), have a heat-labile one from the lysate Enzyme isolated which mediates the endotoxin-clottable protein gelation reaction. Other authors have other variations of methods using Limulus Amebocyte Lysate as well as theirs Applications described. Compare e.g. B. J. F. Cooper et al. »The Limulus Test for Endotoxin (Pyrogenic) in Radiopharmaceuticals and Biologies, "Bull. Parenteral Drug Assn. 26: 153-162 (1972) and Harris et al. in the U.S. Patent 3,944,391.

The limulus hemo-

solution obtained lymph is not very stable, & h. their Ability to gel in the presence of endotoxin decreases rapidly at ambient temperatures. This stands Consistent with the above-described discovery that the lysate contains a heat-labile enzyme that has a On the other hand, the stability of the lysate is an essential factor in the coagulation mechanism reduced temperatures greatly increased In US-PS 39 15 805 it is reported that the lysate for months at 4 ° C can be stored without a significant loss of activity

Limulus Amebocyte Lysate is in lyophilized form available in stores for some time. The lyophilized lysate is more stable than the liquid lysate and therefore it is better adapted to the conditions normally encountered in distribution and use. S & are z. B. 1.2 ml of lysate lyophilized in a serum glass. The closed glass with the dry one The residue is usually stored at low temperatures (below 5 ° C) to prevent decomposition of the Minimize lysates.

The invention is based on the object of a reagent for the determination of endotoxin, which Limulus contains amebocyte lysate in lyophilized form, to provide that one considerably has improved thermal stability

This object is achieved according to the invention by a reagent of the type described above, which is characterized in that it is a lyophilized mixture of Limulus amebocyte lysate, lactose as inert, water-soluble, organic stabilizer and a tris buffer consisting of tromethamine and tromethamine hydrochloride.

According to a preferred embodiment of the invention, the reagent also contains sorbitol.

In US-PS 3133 001 is already the Use of crystallizable sugars as stabilizers in the freeze-drying of biological Material, but the enzymes described in this patent are of a different type chemical composition as the Limulus lysate, which forms part of the reagent according to the invention, In addition, the Limulus lysate used according to the invention differs from the ones mentioned in the above The enzymes described in the publication also differ in terms of their functional properties. As the person skilled in the art knows that in principle there are no general predictions in the enzyme field About the behavior of enzymes can be taken that the stability and activity ratios differentiate from enzyme to enzyme, the person skilled in the art of this publication was unable to provide any information on the solution of the present invention the underlying task. The use of glucose as a stabilizer in the Freeze-drying of bacterial cultures is also described in K. Neumann, plan of freeze-drying, 2nd ed. 1955, pages 132 and 133. Notes on the use of lactose in a reagent of the However, the type considered according to the invention are not found in this publication.

The reagent can also be a source of divalent cations and a salt of an alkali metal contain.

The reagent according to the invention can be obtained in such a way that a Limulus amebocyte lysate solution, which contains a small amount of an inert water-soluble organic stabilizer and that this solution is lyophilized.

The invention thus provides a reagent for the determination of endotoxin, which Limulus amebocyte lysate made available in lyophilized form, which as a result of its lactose content Stabilizers provide improved thermal stability and has high sensitivity

As a general rule it can be said that the level of stabilizer used does not

in is critical Only a small amount is required. Usually an amount in the range of 0.01 to 0.1% stabilizer is met in the lysate solution prior to Lyophilization the desired purpose. Lyophilization is achieved by using a non-hygroskoj pischen stabilizer facilitated

The sensitivity of the reagents to endotoxin is increased when using low concentrations added by divalent and monovalent cations Calcium ions are the preferred divalent

; o gen ions, although other alkaline earth ions, e.g. B. Magnesium ions, or other divalent ions, can be used. Sodium ions do are the preferred monovalent ions, but other monovalent ions can also be used, particularly alkali metal ions such as lithium ions can be used. The chlorides (CaCb, NaCl etc.) are suitable sources for these ions to be added, although other salts can also be used. These are electrolytes preferably added in such amounts that, if

3 «the lyophilized lysate is reconstituted, the concentration of the divalent cation (e.g. of Ca ^{+ 2} ) is in the molar range from 0.0001 to 0.1 and that the concentration of the monovalent cation (e.g. of Na *) is in the molar range Range from 0.001 to 0.1.

Naturally, all additives must be free of endotoxin. Methods to ensure freedom from endotoxins ensure are known. So z. B. inorganic additives (CaCb, NaCI) by at least 120 minutes Heating the dry salts to 250 ° C can be made endotoxin-free. Organic additives must are usually dissolved because of their melting points, and the solution must be im for 60 minutes or more Autoclave can be heated to a temperature of about 121 ° C in order to remove any endotoxins destroy.

The invention is illustrated in the example.

example

Please note: All procedural measures described are carried out under such conditions that ß it is ensured that the end product is sterile and free of endotoxins.

The hemolymph of healthy specimens of Limulus polyphemus is placed in an anticoagulant Saline solution collected as described by Levin and Bang (1969, see above). The amebocytes are collected and washed in a centrifuge with the anticoagulant saline solution.

The separated amebocytes are immersed in water

bo is suspended and osmotic cell disruption is achieved by multiple exposure to a combination of mechanical and ultrasonic agitation completed. The cell debris is removed from the lysate separated by means of a centrifuge and the lysate fraction

b-5 ions are collected and stored at 0 to 4 ° C.

The sensitivity of the lysate to endotoxin is determined according to the method described below certainly. The lysate is required to be adjusted to the desired

he sensitivity by dilution or mixing adjusted with a different approach of the lysate with different sensitivity. The solution is adjusted to the pH range from 6.5 to 7.5 using tromethamine [tris (hydroxymethyl) aminomethane] and Buffered tromethamine hydrochloride and 0.01 molar calcium chloride, 0.077 molar sodium chloride and of lactose 0.0015 molar (0.05% weight / volume) made. All additives have been freed from end-dioxins beforehand.

The buffered lysate solution prepared as described above is divided into the serum glass each contain 1.2 or 5.2 ml of the solution. The divided solution is lyophilized. After lyophilization the vials are closed and cooled (Ibis 5 ° C).

The lyophilized lysate is in the form of a white powder or white fragile pellets.

Demonstration of the improved stability:

A lot of lyophilized lysate was prepared from horseshoe crabs as in the example above, which had been caught near Wallops Island, Virginia. The approach was called "L" designated. Two other approaches that were made in a similar manner, with the exception of that the lactose had been omitted were designated as "U" and "A", respectively.

Storage tests were performed on the three approaches at two different elevated temperatures. Periodically, the lyophilized residues were collected in tubes at random from each batch were selected, redissolved and the sensitivity of the reconstituted lysate was determined by means of the Gelation reaction determined with standard endotoxin solutions as described below.

The following procedures were carried out under conditions such that asepsis was guaranteed and that the introduction of endotoxin from outside was excluded.

Table I.

Storage temperature 30 to 32 ° C

The lyophilized residue of 1.2 ml of lysate was by adding 1.2 ml of water and then carefully panning over a period of a few seconds to dissolve the residue π complete, reconstituted aliquots (0.1 ml) of the reconstituted lysate were divided into a series of small test tubes, which were then incubated in a water bath at 37 ° C until the Temperature equilibrium was reached. In every rea-

H) gensglas was 0.1 ml of a series of series diluted endotoxin standard solutions made from E. coli endotoxin or FDA standard-K. pneumonia endotoxin, given. The test tubes were swirled gently to dilute the solutions.

It was then incubated undisturbed at 37 ° C. for 60 min. At the end of this period, each test tube was examined for the occurrence of gelation by gently and slowly tilting the tube a few degrees. If gelation was observed, then the test tube has been rotated through an angle of 180 ⁰ C, or by such until the gel began to distort. A gel was said to be "solid" if rotation through 180 ° C did not result in distortion. A partial gel that distorted when the tubes were rotated up to 180 degrees was said to be viscous.

The test results for endotoxin concentrations below certain specified values are in the Tables I and II compiled. If the tests are at

jn higher endotoxin values than indicated in the tables solid gels were formed in all cases.

Sensitivity of three batches of lyophilized Limulus amebocyte lysate after storage at elevated temperature for varying periods of time:

Please note: The designation "G" means that a firm gel has been formed. "V" means a viscous one Gel and "N" means no gel was formed. "-" means that no test was performed.

l.ysiit- Appearance Day Gelation results 0.05 0.01 FDA endoloxin (ng / ml) 0.39 0,! 9 , Hi sat / of the dry No. E. coli endotoxin (ng / ml) G N G N No. Residue CU G N 0.78 G G II White 0 G G N G V - White 1 G G N G V - White 2 G G N G V V White 3 G G N G V - White 4th G N N G N - White 5 G N N V N - White 6th G G N V G V White 7th G G N N V N A. White 0 G G N G N - White 1 G G N G N - White 2 G G N G V N White 3 G N N G N N White 4th G N N G N N White 5 G N N V N N White 6th G N White 7th N N

rortsel / img Appearance Repayment Cie results 0.01 R) A-I • ndotoxin (ng / ml) 0.14 Lysate of the dry No. i · :. coli l'ndotoxin (ng / mlI N N ans.it/ Residue 0.1 0.05 G 0.78 0.34 G No. White 0 G G N G G N L. White 1 G G N G G N White 2 G G V G G N White 3 G G N G G N White 24 G G G G White 28 G G G G

Table II

Storage temperature 35 to 37 ° C

Lysate Appearance Day Gelation results 0.05 ι (ng / ml) FDA endotoxin 0.78 (ng / ml) 0.19 ansat / of the dry No. E. coli endoloxir G 0.01 G N No. Residue 0.1 G N 1.56 G 0.39 V U White 0 G G V G G G - White 1 G V N G G G - White 2 G N N G N N N White 3 G V N G V N N White 4th G N N G N N N X * 5 G N N V N N N X * 6th N G N N G N V X * 7th N G N N G N - A. White 0 G V N G G G - White 2 G V N G G N N White 3 G V N G N N N X * 4th G N N G N N N gray-white 5 G N N V N N N gray-white 6th N G N N G N N gray-white 7th N G N N G N G L. White 0 G G G G G G N White 1 G G N G G G N White 2 G G N G G G N White 3 G G N G G G N White 25th G N G G White 28 G G G X * - white to gray-white.

Claims (2)

Patent claims: 1. reagent for the determination of endotoxin, containing Limulus amebocyte lysate in lyophilized Form, characterized in that it is a lyophilized mixture of Limulus amebocyte lysate, Lactose as an inert, water-soluble, organic stabilizer and a tris buffer, consisting of tromethamine and tromethamine hydrochloride, represents 2. Reagent according to claim 1, characterized in that it further contains sorbitol.