JPS5813516A - Preparation of amebocyte lysate - Google Patents

Abstract

PURPOSE:To obtain the titled substance having a high specificity of endotoxin or a liquid containing the titled substance, by removing a nonendotoxic coagulating enzymic precursor activating factor from amebocyte lysate of a horse crab or a liquid containing it. CONSTITUTION:Ambocyte lysate of a horse crab or a liquid containing it is subjected to the liquid chromatography with a heparin agarose as an adsorption carrier and a salt solution having an ionic strength of 0.15-0.45M as an eluting solution to remove a nonendotoxic coagulating enzymic precursor activating factor capable of causing the coagulating reaction of amebocyte lysate even in the absence of endotoxin by activation with a beta-1, 3-glucan, and give an amebocyte lysate useful in the limulus test in the field of medical science or pharmacology or a liquid containing the amebocyte lysate.

Description

[Detailed Description of the Invention] The present invention relates to a method for purifying amebosite lysate,
For more details, see Ameponite O゛Bullet #At (Ameponai F-2
The present invention relates to a method for preparing amedanite lysate with high endotoxin specificity by removing impurities from lysate.

Kabutoga = (Llmmlms p@1yph@a+m
s, choa*hypl-・ws trLd@mtatw
s, T, gigas, etc.)0 A gram-negative image of amytite present in the lymph fluid.A gram-negative surface substance1. is from the biological defense mechanism.

Interest (being researched. On the other hand, amebonite acupuncture points
ni'-)0. The first impression by Entodo Life Shin is,
``This is a general book on Limulus Test 1, which is widely used in the medical and pharmaceutical fields as an endotoxin-specific detection method.It discusses endotoxin as a substance that causes gelation of amebocyte lysate, and uses it in mammals, etc.
& Enzymes (trigsin, thrombin, etc.)
These II) are known as chain-like enzymes.
Since it has not been found anywhere other than III, the Limulus test has been evaluated as a good allotropic reaction system for endotoxin.

The authors have been conducting extensive research to elucidate the coagulation mechanism of amebunite lysate, and have developed a highly sensitive and highly sensitive method using 41 different acid-containing substrates for enzymes involved in the coagulation system of horseshoe crabs. Detection of highly traversable Entodium Synths--Method (41m1)
1i1$4-1197 11&, and the substances on which endotoxin acts directly in the amebunite lysate coagulant are the coagulant enzyme precursors previously mentioned. Endotoxin susceptibility 1 A substance is present, and it is activated (by the action of endotoxin) and becomes an active coagulant enzyme, and is thus activated and becomes a highly active coagulase. - due to bulk)
Koagi 3-W-Dan becomes a fibrous tsuagie,
In other words, the horseshoe crab omits system is similar to mammals.
1111 that it is a system in which condensation (due to a stepwise reaction involving multiple enzymes) proceeds! (F@bs, L@tt@rs, 1
20 217 (198G) S, Ivaaaga・t
al).

In addition to the above facts, the present inventors further discovered that the primary OIl
I ran straight into the obvious headline. In other words, endotoxin 011 is a substance that acts on amebotate lysate to gel it./-1,! l-
We confirmed for the first time the existence of glucose polymers and their derivatives contained in the ameponite lysate, and found that -1,1-dalcane-sensitive factors that are affected by these substances exist in ameponite lysate, and that this fraction It was found that this fraction differs from the syn-sensitive substance fraction in Entodo.

What is the book Il@? -Rera Ameponite Rai (-to □,.

Sensitive to 07-1.3-glucan and its derivatives, even in the absence of endotoxin 0.
The purpose of this invention is to provide an amebunite lysate that is specific to endotoxin by removing substances (fractions) that cause a quisate Oa solid reaction.

This 1llq40/-1.3-glucan-sensitive fraction contains Limulus po97ems, Tachyplois tocidentatus, etc. When performing liquid chromatography using a heparin-bonded cross-linked agarose gel as an adsorption carrier, such as
．． The 15M-0,5MO salt St is preferably desorbed and eluted from the adsorption carrier using a sodium chloride solution.

Add 7-1,3-glucan to both of these to make indPj-
When the coagulation enzyme precursor fraction is added, the coagulation 1
The mono-intoxicant precursor undergoes activation and is adjusted from the coagielogen O structure in the coagielodan, acts on the hyperchromic synthetic substrate Ili, and gels.

Or develop color. The present inventors were the first to clarify the existence of this /-1,3-glucan-sensitive fraction. (Pharmacia Finekencals) Selmitsai y@Gc-200 (Chisso (
It can also be fractionated using ordinary molecular sieves such as termed sieves. The present invention 01 is turnip toga = 0
By removing the /-1,3-glucan-sensitive fraction in the ameponite lysate, the /-1,3-glucan-sensitive fraction is removed. The confirmation method is not limited to the embodiments of the present invention.

The fDII-1,3-glucan of the present invention is derived from kelp (La-
mlmaria)bAtsumejlis@mIm)etc.01
Tsukennarin (Lamlmarl) obtained from Class 1111
m) and 9W pigs (P@mia...@wsO Sokoku)
Paskygmam obtained from Paskygmam is applied to Paskygmam etc. to increase water sieving property, L C1a
rk@! Fhyt@senghemIstry 1175~
18g (1967) 0 method KN-force k & medium/methylation- even when used as a conductor, both are confirmed to activate /-1,3-darcan-sensitive factors.

The following one shot is an example of the key side! I! According to the example) KA physical Kl! I will clarify.

*Meeting! 1-4013140mK甐Cfi本jll
# Butoj engineering (Ta@kyplworking ws trtd@mts
tws) (weighs approximately! ~), strictly avoid contamination,
Collect the leopard's lymph fluid and centrifuge it.
Separate the candy and night and wash with 3% sodium chloride**.
You can get 90 Ameponite pellets from this site.
Beret) K O, OSM Trla-ICj Ilw
-ff@r, pm '1.2 to 1/i of the original car lymph fluid
Add o spring, stir well with a sterilized light powder, 8
．． After stirring for 30 minutes in Goer's group, the supernatant was used as Amebonite 2 Ize) TTL.

Preparation Example 27Me9 Turnip Dead Crab (Limmlwsp・t
ypb・Peng-) Ame I Night Twize-) 0$1931 Amebosite Twize) was prepared by the same procedure as Preparation Example 1.
It was set as LPL.

Preparation putari jitsukara* Y* 8hlbata, J,
F@rm@nt.

Dingeek@@1. ．． 60388 (1972) 0 method, -1pm@b-ymam was cleaved and purified using DMSO.
@m1stry 1.157-18g (1967) was used to perform *lupoxymethylation to obtain CMP.

Preparation Example 1 TTL Light TTL S0111#t
O,O5MTrI s -HCj elk II IE
Dilute 3 times using PH'I-2 and first add Oka-Yu 1i1.
Heparin Sepharose CL-6B:JF9 (buffered) at 1:1 was applied. The elution operation was performed in the following steps. Namely.

■ Column conditioning using 11I buffer solution,
Obtained AK1 minute.

■0. Elute using ISMNaCj to obtain 6 fractions.

■Obtain 8 fractions using 046MNaC.

These 0 fractions were analyzed using the method previously reported by Akika Motoya et al. (F@b
s L@tt@rs ■1217-220 (1980
), S.

Iwamaga@tal) was used to conduct 1 to 70 configuration tests on the properties of the fractions extracted from the air, and the results were reported in the 1st I! and good as shown in Table 2. This is an experiment conducted in the presence of endotoxin by recombining fractions A, G, and S, *110111-14, and the results thereof. and b were combined again to form dimethylated 9(l→3)-7-D-dulcan (CM
The experiment was carried out in the presence of P S ) and its results are shown.The experiment was carried out as follows.

5 ois each fraction, endotoxin or CMP (final concentration 11!2407ml), 04mM chromogenic oral fluid.

110mM ()Trls-HCJ ()Lis-HCl) mild miml (pig s Hatake, 0) and 5.2 KM chloride!
Contains gnesium (MgC bird) 1 feeding damage amount - IIA! 50
The sjO reaction chamber was incubated at 3 °C for 30 minutes. Next, 6 M acetic acid was added to stop the reaction, and the luminous intensity at 405 mmK was measured. Composition experiment KTh & winter.

For ByL, 5osi of each CO fraction was mixed with vhK.

Under the same conditions as in 9 above, the activity was kept constant, and the tarot-forming ability of the reaction fraction was purified at a high temperature, and the large core agielogne was used at 9A, then at 0 °C to maintain a constant 0 reaction. , each fraction A, Bl and 0% or a good combination of them! ) 100#jjh and 26-MgCJ,
0.4M) Lys-hydrochloric acid 1111 (PII8.O
), and the total size was 3!30μノ. Rit is 3
If the large pigeon coop is incubated at 7C for less than 1 hour, the sample is considered to have a positive reaction.

1III IA O--2G
O- 4mu 10G O- 5A+Il 326 +1!
G+Il 414 +7
A〇+1 662 + 2nd I
I (XIG”') I A O-2G 2
84 13 B Q
-4A+G 455
+5 A+I
O-g G+il 373
+7 A+G18 491
10B alone does not exhibit clotting enzyme activity (Adendai activity), but when subjected to the action of endotoxin, 9B together with 9B produces coagulation 1iIWII bulk activity. However.

/- Both Ao and b are affected by the action of 1,3-glucan.

They exhibit clotting enzyme activity either alone or in combination.

The G fraction expresses coagulase activity under the action of /-1,3-glucan. For these reasons, heparin-Sepharose/CL6B4Da-attached chromatography
It can be seen that both fractions desorbed by -0,15M to 0.45M NaCJ are I-1,3-glucan-sensitive fractions that are not sensitive to endotoxin. -S*ph
Gel filtration can be performed using aeryls-zoo Katsum.

The chromatogram is shown in the figure, and the properties of each component were clarified according to the construction experiment method of Example 1.

Shown as corresponding A, B and G fractions *@NaC fractions were calculated from the electrical conductivity at the position corresponding to the column volume. B fraction is S@ph Hatake*ryl S-200
It is thought that it shows adsorption to NaCj (K) and releases a large amount of Si.

[Brief explanation of the drawing]

The figure is a chromatogram of Amebosite lysae) LP obtained by gel filtration using a %S@pha*ryl S-200 column. 0-OA fraction --- B fraction Δ --- Δ G fraction NaCJ fraction

Claims (1)

[Claims] (1) ★A unique ameponite acupoint (-toxin) characterized by the removal of non-endotoxic coagulation bulk precursor activating factors from staghorn crab ameponite lysate or liquids containing it. or a liquid containing the same.
-1,3-Dulcan 111 adds IJ to O'
The OII method described in the patent claim that uses the fact that there is c as a 411 image. AL-JI buto S engineering, amebonite, lysate, or a liquid containing hepatic acid and aga4 as an attachment carrier and L ion tonicity (LIBM~EI4$MO salt S* as a single case) 2. The preparation method according to claim 1, which comprises subjecting the sample to E1 to remove a portion containing a non-enzymatic coagulation-decorating precursor activator.