JPS5928474A - Collection of inhibitory factor of coagulation system existing in amebocyte lysate of limulus polyphemus - Google Patents

Abstract

PURPOSE:To raise the sensitivity and reactivity of amebocyte lysate to endotoxin, by subjecting amebocyte lysate of Limulus polyphemus to liquid chromatography using a polysaccharide sulfate as an adsorption carrier. CONSTITUTION:Amebocyte lysate of Limulus polyphemus or a solution containing it is subjected to liquid chromatography using a polysaccharide sulfate such as dextran sulfate agarose, agarose sulfate, etc. as an adsorption carrier, factors of coagulation system caused by endotoxin existing in amebocyte lysate and inhibitory factors are adsorbed on it, the factors of coagulation system are mainly desorbed with a salt solution having <=0.5 ionic strength, and the inhibitory factors are eluted with a salt solution having >=0.5 ionic strength.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the ability of endotoxin to coagulate amehoyite lysate and/or the ability of endotoxin present in amehoyite extract (more specifically, amehoyite lysate) obtained from the hemolymph of a horseshoe crab. Relating to a method for increasing the sensitivity and reactivity of Amejoite lysate to endotoxin by collecting and removing factors (coagulation inhibitory factors) that act on Amejoite lysate and inhibit its ability to activate coagulation enzymes in the lysate. It is something.

Kabutoga= (Li+nulus polypbemu
s, Tachypleus tridentatus;
Durham-negative bacteria surface substance 1 endotoxin (
The so-called "Limulus test", which uses amejoite lysate, which applies the coagulation phenomenon of hemolymph caused by the action of lipopolysaccharide (lipopolysaccharide)', is used as a specific detection method for endotoxin, and as a substitute for the virodiene test using animals, in the field of medical and pharmaceutical sciences. (This is widely used.

However, the Limulus test, which is based on the endotoxin-induced gelation of amebosite lysate, has many problems before it can be put to practical use on a commercial scale. Among these problems, the most important ones were to keep the gelled protein level in the lysate constant in order to cause a stable coagulation phenomenon, and to remove factors that inhibit gelation. The enemies of gelling proteins and gelation inhibiting factors in lysate vary depending on the horseshoe crab's habitat and seasonal factors, and unless these can be controlled, the reliability of the quantitative quality of amebosite lysate as a commercial product may be compromised. The problem is that the amount is low.

The present inventors have previously elucidated the coagulation mechanism of these lysates and disclosed a highly sensitive method for determining endotoxin using a synthetic substrate (Japanese Patent Application Laid-open No. 15797/1983), and furthermore, we have also investigated the factors involved in the coagulation mechanism. I reported it (S, Iw)
anaga, et at,.

Based on these studies, we have provided a highly sensitive and highly quantitative method for detecting endotoxin by measuring enzyme activity dependent on endotoxin present in lysate using its specific synthetic substrate, without relying on the gelation phenomenon. I came.

However, although this method is not affected much by the presence of inhibitors in the normal measurement range of endotoxin levels, the inhibitors reverse the action of endotoxin when measuring extremely small amounts of endotoxin, so basically, It is clear that the absence of inhibitors in the lysate is preferred. The existence of the concept of an inhibitory factor is supported, and the enzyme theory regarding its true nature and mechanism of action [Nachum, et al., J. Inv.

Patho, 32.51 (1978)) and the endotoxin-binding lipoprotein theory C8ullivan, et al.
aQ,.

Appl, Microbiol, 28.102
3 (1974)), etc., but it is not considered that the gelation phenomenon has been sufficiently clarified just by explaining it.

On the other hand, methods for removing inhibitory factors from amebocyte lysate have been studied (U.S. Pat. No. 4.107,0
No. 77, 5ullivan et al and JP-A-1983
-42590, Earl Clark Brown et al.). The former is a method to remove the inhibition phenomenon by shaking the lysate with an organic solvent to denature the protein, and the latter (
The method is characterized in that after the inhibitory factor is inactivated at a pH in the alkali IJ region that does not affect the coagulation enzyme in the coagulation enzyme, the pH is returned to -I, which is the action range of the coagulation enzyme.

All of these methods have one drawback in that they affect endotoxin-sensitive factors in the lysate.

In the process of researching the mechanism of action of coagulation factors in lysate, the present inventors discovered for the first time the presence of endotoxins (caused by coagulation factors) and the fractionation of their inhibitors, and thus arrived at the present invention.

That is, a specific adsorption carrier made of sulfated polysaccharide is used to adsorb coagulation system factors and inhibitory factors in the lysate, and
．． A salt solution with an ionic strength of 5 or less, preferably N
A Tris-HCl (Tris-HC4) buffer containing aC1 is used to primarily desorb the coagulation system factors, followed by a salt solution with an ionic strength greater than 0.5, preferably a Tris-1cQ buffer containing NaCI. The inhibitory factor can be collected by elution using a solution.

Examples of the sulfated polysaccharide include dextran sulfate/agarose, sulfated agarose, etc., which can be used alone or in combination of two or more, and heparin/Sepharose CL-6B (Pharmacia F
It may also be used in conjunction with a heparin-bonded cross-linked agarose gel, such as ine Cbemicals (Sweden).

The inhibitory factor collected as described above strongly inhibits the activation stage of amepozyte lysate and the endotoxin-sensitive factors present in the lysate by endoto-V-sin, and is effective against alkali treatment, pronase, sptilisin, etc. Completely inactivated by enzyme treatment. This suggests that this inhibitory factor strongly inhibits endotoxin-induced activation of amebocyte lysate and coagulation system factors in the lysate, and that removing this inhibitory factor from the lysate by the gentle method of the present invention can further enhance A sensitive and stable amebosite lysate can be obtained.

Hereinafter, the present invention will be explained in more detail with reference to Preparation Examples and Examples.

Preparation Example 1 Preparation of sulfated agarose (1) Preparation of epoxy-activated Sepharose CL-6B After washing 400 m7 of Sepharose CL-6B (product name + 7 manufactured by Almacia) with distilled water IQ, wash 600 m7 of distilled water.
1, 260 ml of 2N NaOH aqueous solution and 6 ml of epichlorohydrin were added, and the mixture was stirred at 4°C for 2 hours. After the reaction, transfer to a glass filter and add 7 oor distilled water.
nl''Cp117.Furthermore, after washing with acetone, it was dried under reduced pressure to obtain Nigexy-activated Sepharose-y, CL-(2) Epoxy-activated Sepharose CL-6B sulfated epoxy-activated Sepharose-C
20 g of L-6B was suspended in 30 QmJ of pyridine dehydrated with KOH and cooled to -20C, and then a solution of 2Qm/chlorosulfonic acid dissolved in chloroform 6ON was added dropwise over 2 hours with stirring. After the dripping is finished,
The mixture was heated to 45 tr and further stirred for 2 hours. 120 again
After cooling to C, distilled water 15'/rLl! After adding 7.
Add 400 ml of 5% Na HC03 aqueous solution and neutralize.

Ta. After neutralization, transfer to a glass filter and add 5% Na
HCo 3 aqueous solution 5ooml, distilled water 500ml, 5
0% ethanol aqueous solution 500m7! The pyridine was completely removed by washing sequentially with . Next, 500 TrL of distilled water
After washing with 300 ml of 0.4 M Tris-HCQ buffer (pH 9.0), the mixture was sterilized by autoclaving (12 IC, 120 minutes).

Preparation Example 2 Preparation of dextran sulfate/agarose Anderson et al.'s method CL, 0. Anderson eta
l, Thromb, 几es, 7,451 (1
Dextran sulfate (manufactured by Pharmacia; molecular calmness approximately 500,000) was immobilized on Sepharose CL-6B using BrCN according to 975).

That is, dextran sulfate 25y was dissolved in ice-cold water IQ,
Sepha0-su CL-6B 500ml and BrCN1
Add 180 mJ of 25F acetonitrile solution and
Stirred at 0C for 50 minutes. In addition, during the reaction, l Q NNa
By dropping an OH aqueous solution, it was maintained at r) 1 (10,5. After the reaction, it was transferred onto a glass filter and filtered, and then 0.5 M Tri 5-HCI buffer (pI (
8,5) Suspend IQ and fflP at 4c for 15 hours.
F- did. Next, it was filtered through a glass filter, thoroughly washed with distilled water, suspended in 500 rrtl of distilled water, and sterilized by autoclaving (121c, 20 minutes).

Preparation Example 3 Activity Measuring Method After chromatography, the enzyme activity and activity of each factor were determined as follows:
Measurement was carried out under the conditions of 7C according to the following method. In all enzyme activities, 1 unit was defined as the amount of enzyme that released 1 μmol of varanitroaniline (p-n1troanlline) per minute.

ter B activity): 50 μC of each fraction and endotoxin (lihoborinatucharite, LPS) (600 ng/+n
1) 3otty and 0.2 M Tr 1s-1-I
Cj, -13mM MgCQ2 buffer (pHs
, o) Mix 100 μi, leave for 15 minutes, then add 5
mMBoc-Leu-Gly-Arg-pNA 20μ
A mixture of 50 μl of Fl and A (Figure) was added, and after a certain period of time, 3 ml of 0.6 M acetic acid Q was added to stop the reaction. It was measured.

(11) Proclotting enzyme precursor
n-zyme): 50 μC for each fraction and active factor
B50μ area, Tris buffer (used in (1)) 1
00 μα was mixed and left for 30 minutes, 50 μα of 2 mM synthetic substrate (used in (1)) was added, and the appearing amidase activity was measured. In addition, active factor B is fraction B (figure) 400μα and 0.4MTri s-)-
ICQ-26mM MgCQ2 buffer (pH 8,0)
200 μQ, LPS (400ng/mJ)20
A mixed solution of 0 ttQ was prepared by allowing it to stand for 15 minutes).

50ttQ, 'I'ris buffer (used in (1)) 100/JQ, 2mM synthetic substrate (used in (1)) 5011Q, and physiological saline 50μα were mixed, and after a certain period of time, 0. The reaction was stopped by adding 0.8-6M acetic acid, and the amount of paranitroaniline released was measured.

Sample and both fraction A, fraction B, 0.4 M Tr Is-HC
Q-26mM MgCIL2 buffer (pI(8,0) each 50119., plus 5mM synthetic substrate (used in (1)) 20pQ and LPS (400n% nR) 30μ2
After mixing and leaving for 25 minutes, 0.81 nl of 0.6M acetic acid was added to stop the reaction, and the amount of liberated paranitroaniline was measured.

Under these conditions, the activity that inhibits the activation of Factor B (using 2 ng of LPS) by 50% was defined as #1.

horseshoe crab amebocyte 109 in 0.05M NaCl
0.02 M Tri s JICQ buffer containing (
Amebozite lysate 90d obtained by extraction with pH 8.0) was applied to 5 μm (2.2×18.0 Crrl) of sulfated Sepharose CL-6B equilibrated with the above buffer, and then extracted with 500 ml (Bl) of the above buffer. By washing, a coagulation enzyme precursor (fraction A) was obtained as a flow-through fraction. Then 0.02 M' containing 0.15 M NaCl
l'ri 5-HCQ buffer (pi(8,0) 50
Factor G (fraction G), which is a non-endotoxic coagulation enzyme precursor activator activated by β-1,3-glucans, was eluted at 0 ml (B2). Furthermore,
0.02M Tris-H containing 0.45 MNa'CI
It was eluted with 500 ml (B3) of CQ buffer (pHs, o ) to detect factor B (which is an endotoxin-sensitive factor).
Fraction B) was obtained. Finally, 0.02 containing 2M NaCI
MTr I s -I- (CI buffer (+) H8, O)
The target coagulation inhibitory factor was obtained by elution with 500 ml (B4). The chromatogram is shown in the figure.

In the figure, curves A, B, G, and X represent fraction A, respectively.
Fraction B, fraction G represent coagulation inhibitory factors.

By combining the two obtained fractions and fraction B in the manner described above, it was possible to obtain an endotoxin-specific amepozite lysate.

Amebosite 42y of spiny horseshoe crab in 0.05M NaCl
0.02 MT'r Is-HCQ buffer containing (
630ml of amebocyte lysate extracted at pHs, o) was transferred to a dextran sulfate/Sepharose CL-6B column (5X23.5Cr
n) and washing with the above-mentioned buffer 2Q, a coagulation enzyme precursor (fraction A) was obtained as a flow-through fraction. Next item 0.02M Tris-HC containing 0.3 MNaCI
Elute with Q buffer (pl(8,0)2Q, β-1,3
-Factor G (Fraction G), a non-endotoxic clotting enzyme precursor activator activated by glucans
I got it. Furthermore, 0.02 M containing 0.5 M NaCl
Factor B, an endotoxin-sensitive factor, was eluted with Tris-HCQ buffer (-18,0) 2Q.
(Fraction B) was obtained. Finally, 0.0 containing 2MNaC]
2M Tris-HC1 buffer (pH 8,0) 2Q
The desired coagulation inhibitory factor was obtained.

By combining both fractions A and B obtained in the manner described above, it was possible to obtain an endotoxin-specific amebocyte lysate.

15 horseshoe crab amoebocytes at 0.05M NaCI
0.02 M Tri 5-IJCQ containing 0.02 M Tri 5-IJCQ, obtained by extraction with buffer (pt18.0)) 130 m7! was equilibrated with the above buffer solution.
m) and 0.02 MTr containing 0.5 M NaCI.
is-HCQ buffer (pHs, o) 50 (
lt/! A mixture of coagulation enzyme precursor, Factor G and Factor B was obtained by elution. Then 2M NaCl
0.02M Tris-HCO, buffer (pH 8,
0) The target coagulation inhibitory factor was obtained by elution at 500 resistance.

Horseshoe crab amebosite 40y in 0.05M NaCl
0.02M Tr l 5-HCQ buffer (pif
8. Ameboduite lysate 6 obtained by extraction with Q)
10 ml of a DexFran sulfate/Sepharose CL-6B column (5 x 23 cm) equilibrated with the above buffer solution, Nikake, Q, 0.02 M Tri containing 5 M NaCI.
Elution was performed with s-1-ICQ buffer (pH 8,0) 2Q to obtain a mixture of clotting enzyme precursor, Factor G, and Factor B. Then 0.02M containing 2M NaCl
The desired coagulation inhibitory factor was obtained by elution with 2Q of Tr I 5-HCQ buffer (pH 8,0).

[Brief explanation of the drawing]

The figure is a chromatogram of amebocyte lysate using a sulfated agarose column in Example 1.

Claims (1)

[Scope of Claims] 1. Amehoyite lysate of horseshoe crab or a liquid containing it is subjected to liquid chromatography using a sulfated polysaccharide as an adsorption carrier.
A method for collecting a factor present in a lysate that inhibits the ability of endotoxin to coagulate the amejoite lysate or the ability to act on the amejoite lysate and activate coagulation system enzymes in the lysate. 2. The collection method according to claim 1, characterized in that in liquid chromatography, both fractions eluted by a salt solution having an ionic strength exceeding 0.5 are collected.