JPS5813517A - Inhibitory factor on coagulation present in amebocyte lysate of horse crab - Google Patents

Abstract

NEW MATERIAL:A factor, present in amebocyte lysate of a horse crab, and having the ability to coagulate the lysate or inhibiting the coagulating enzyme in the lysate by acting on the amebocyte lysate. USE:An inhibitory factor on the hemolymphatic coagulating system on the amebocyte caused by the action of the endotoxin. The limulus test utilizing the coagulating phenomenon is used as a specificity detecting method of the endotoxin, and the removal of the inhibitory factor by the collection improves the accuracy of the detecting method. PROCESS:Amebocyte lysate of a horse crab is subjected to the column chromatography with a specific carrier, and a fraction obtained by the elution with a salt solution is then subjected to the chromatography using a molecular sieve for the chromatography to give the aimed inhibitory factor on the coagulation system.

Description

[Detailed Description of the Invention] The present invention v4#i, hemolymph I [#I! ■Factor that makes a mark (#1ill writing factor) and method of collecting it
IIL.More specifically, the ability of the amebonite lysate present in the amebonite lysate to coagulate the amebosite e-lysate and/or act on the amebonite lysate to activate coagulation system enzymes in the lysate. This is a method to reduce the sensitivity and reactivity of Amebonite lyse to Entodochin by collecting and removing the coagulation-inhibiting factor from the microorganism.

Horseshoe crab (Limulus”polyphemus)
.

Tachypl@us trident co'jus,
, TlgiRas group) Oh lymph INK present Amebocyte lysate tube using amebocyte lysate tube that applies the coagulation phenomenon of blood that is caused by the action of the surface substance of Dalam-negative sodium, Entodoxy 7 (13 popolysucharide) l. The so-called ``limulus test'' is widely used in the field of medical science as a substitute for the pyrogen test, which uses open-tubed animals, as a specific method for detecting dichotoids. However, the ``Limulus test'', which is based on the gelation phenomenon of ameponite lysate due to the presence of acetic acid, has many problems before it can be put into practical use on a commercial scale. 'IKIII among so0vlJi points
The goal was to reduce the amount of gelling protein in the lysate to -t' in order to stabilize it and cause the hardening phenomenon, and to remove factors that inhibit gelation. The amount of gelling inhibitory factor in the amount of gelling protein in lysate varies depending on the habitat of the horseshoe crab and seasonal factors. , has the problem of low credibility regarding its quantitative nature as a product.

The author Wj41i et al. first solved the problem of these lights (Ani machine Sakaki) and used a synthetic substrate to develop the endo-Movie vytva.
Disclosure of s-degree quantitative method L (劎111854-15797)
%SaraK has reported on the factors involved in #solidification.

11! O, to 1? (mouth 1110), 8. Iwana
ga @t at).

Based on these studies, it is possible to develop highly sensitive and quantitative enzyme activity by using a specific synthetic substrate to measure the enzymatic activity that depends on the endodoxy ν present in the lysate, regardless of the gelation phenomenon. Since then, we have provided the O-ken aS method. However, this method is not so affected by the presence of Fi writing factors in the normal fixed range of 20% of the amount in 200 mg/day, but when measuring extremely small amounts of 200 mg/dose in the 200 mg/kg amount, the % inhibitory factor is Since the effect of y is reversed by 1, it is clear that there is basically a Kli writing factor in the lysate and one of them is preferable. Regarding the general idea of harmful factors, their existence is not supported, but regarding the situation and its mechanism of action, # jll ell [Nachu
m l e t al * J, 1n'l.

P a t bo, E M L S l (1978)
), the syn-binding lipoprotein theory (8ul)
livan et at.

Appl, Micro″biol, zs, log
s (1G?4)), etc., but it is not considered that the gelation phenomenon has been sufficiently clarified with only w&-.

On the other hand, methods for removing inhibitory factors from amebonite lysate have been studied (U.S. Cost Approval No. 4,107.0).
No. 77, 8ullivaa, etal and JP-A-1987-
42590-1,? - Le 11/9-161 Lau ν et al.),
The former is a method in which the inhibition phenomenon is removed by shaking the lysate using an organic solvent to denature the protein, and the latter is a method in which inhibition is achieved by shaking the lysate with an organic solvent to denature the protein. It is characterized by inactivating the factor and returning it to the coagulase O action range O'pH.

All of these methods have the disadvantage of affecting the concentration of susceptibility factors.

The present inventors have been extensively researching the mechanism of action of coagulation factors in lysate, and have narrowed down the presence of coagulation factors due to entodoxy ν as well as its inhibitory factor lI, and have now arrived at the present invention. In other words, coagulation system factors and inhibitory factors in Lyeide are attached to a salt having an ionic strength of 1.0 M or less using a heteroadsorption carrier using Heparin agarase.
First, the coagulation factor is mainly desorbed using a needle or N*C1 solution, and then 1. .

A salt solution having an ion density of M or more, preferably N*
It is eluted using Cl@ solution to obtain a fraction containing the inhibitory factor as the main component. This fraction is treated with a molecular agent such as agarose, cross-linked agarose, striped strand gel, and cross-linked acrylic azudogel, which are known by trade names such as Sepharo-crosslinked layer fat, Sephatic layer, and Sephacrylic gel (manufactured by Pharmacia). When carrying out a-mudography again using the above-mentioned molecular sieve agent or subjecting the lysate to 711ml17g-mudography using the above-mentioned molecular sieve agent, Schiff sf#J)lJy (molecular weight: 1100-9
00) to NaC/ (molecular weight = 58).
It strongly damages the endo-medium-syn-sensitive factors and the #! It is completely inactivated by #prime treatment. This shows that this inhibitory factor is inhibited by amebonite and
By strongly inhibiting the activation of the lysate group and the awA system factors in the lysate, and by removing this inhibitory factor from the lyeid using the gentle method of the present invention, p4-sensitive and stable amebocytes can be produced. You can get Lysate.

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

III Preparation Example 1) Substrate and carrier Boc-Leu-Gly-Arg-p
Nmu HCJ was purchased from Seikagaku Corporation. Heberin-agaa 7'J-su is Cephas 1-su (8@pha-
Rose) e CL-@B' (Pharmacia) was prepared using the method of Marck et al. and autotared for 20 minutes.
Blood was collected according to the protocol, and the blood cells obtained were I0005M.
) Lis-salt @ (Tris-HCj) 1lli liquid (
3) Constant activity method Enzyme activity and stage factor of the fraction after chromatography The activity of Kljmol was constant at 37°C under Qin conditions according to the method below.
・Paranito Aeri 7 (p-+aitroan eyes)
The amount of enzyme liberating e) was defined as 1 unit.

Nakashiy (ribobolinatucaride, LP8) (・ooag
/mz > 5osz parallel OJMTris-HCj-
1BmMgcj, mild 1111 (pHs, o) Z
After crystallizing oo pg and leaving it for 15 minutes, 5mM Boc-
The reaction was stopped by adding 0.8 rM of 6M acetic acid for a certain period of time, and the release amount (#=50, Boo) 40511
It remained constant at m.

00# member-prime precursor (Proclotting e@-
trem ym・): 4 fractions so sz and activated carbon fatata-B
! $0J11. ) Used in Squirrel Buffer II (Q)
) 100j11116, and after standing for 30 minutes, add zmM Kaiwei substrate (used in (i)) so sl, and aS
Factor B is fraction B, (Fig. h) 400 μI to 0,
4 M Tr l s -HCl-! 41mM MgCJ
, Buffer II [(p)ls,o) 200 IAl,
LP8 (400ng/mj) Snow o.

#jOalltls4JIk and IIILl also ot
m%/% Fraction 60111 and Trim buffer (used in (Engineering)) 100jl 13mM4rjill! Substrate ((J
> used) 80j141! Salt water 5ozt
After a certain period of time, O, sM#alo, and 8mj were added to stop the reaction, and the amount of baraetol aniline released was determined. (iv) Anti-L12 fa
ctor): sample and both fractions, fraction B%0.4M
'rres -HCl -311mMMgCj, 4)! iOμ11 SaraK! ! m
%e) used in the M substrate (0) 20at and L12 (
006Mg1@0.400nK/ml) was mixed and left for 21 minutes. The reaction was stopped by adding amZ, and the amount of released paraethyl O7μsv was kept constant.
Heparin Y-Agarose CL-5B with 1 unit of inhibiting activity
-suA (iX, 17cm) IC% 0.05M
Added 9840 ml of Twist rhinocytosis tB and performed #l elution using 0.15 MTh and iM sodium chloride S* (-5) Then, added IM sodium chloride 9m elution section (18G-360 tube). Atsu J6-7 cm-s @ CL-6B column (3,!1x 1zs4
When the gel filtration was detected according to the detection method (described above) for each of the inhibitor sections using 111), it was found that tubes 1 to 204 of the salt extraction section did not contain any enzyme system. At t1, a bitter classification is issued to %fh-ν, and this and Moribetsu El! The B~l5OfK gastrointestinal and armpit segments were eluted (Figure 2).

Example 2 Effect of lysate on removing inhibitors When measuring the amidase activity by mixing the respective fractions obtained in Example 5, the bitter fraction was placed in the middle as shown in the table* ia, some inhibition of the activity may be observed in the case of hardware, but a very high amidase activity is maintained in the mixed material that does not contain the inn and bitter sections.'i.

■Table end) Effects of coagulation inhibitors on the activation of the R. mucus aggregation system induced by xin.
All 518.1-minute + B LP Hatake
ass so ox.

Caputo 1t-o-rai (-to SOmj 8*pka-d@
x G-1@Kurau A (4,3X114Cm) K addition mu o
, osM) Filter the gel using Lith buffer $1 (pH 8,0) sodium decachloride (@, IsM) 1111.
ee, if we perform the detection using the previous difference detection method, we will eat %A pure yw bitter category in the same category as the salt 0Ill output category (we will put %A pure Iyw bitter category in the first meeting). Detected (I
lm ) @

[Brief explanation of the drawing]

■1: Hebashi Y-7 galose/CL-6B column
%this Column taro of horseshoe crab hemocyte fluid! Showing Todaram. All snow was set using Sephas CL-6B column and gel filtration! The column chromatography column of AM Nasilicum chloride #I output section (180 to 60 tubes) in ilK is shown. aasa, Sephadex x (8sphadex)G
-Gel filtration using a 5O column to show the column of captotIJI&Ly layer -Total 1 Coagulation lI# elementary precursor ←→ Factor B -111 Cyclodextrin-4 inhibitor- NaCj Procedural Amendment 1981 $ Monthly Japanese Patent Office Official 1! Mr. Kazuo 1, Incident Display Showa S6 Patent Application No. 11jH17jj, -2,1A
I! 0 Name 3. Relationship with the person making the amendment Patent applicant name Seikagaku Kuwa Co., Ltd. (name) 8. Contents of the amendment! , #4 The Detailed Description of the Invention column in the specification will be amended as follows. (1) “-” from page 7, line 1 from the bottom to page 8, line 2, l
・Intermediate endotoxin-susceptible factor ・・Activation enzyme system” Activation stage by endotoxin
and correct it. a) Correct "-1chymotrypsin" on page 7, line 3 of the specification to "sptilisin". --) Delete "(Figure 3)" on the 6th line from Specifications Library 13. (4) The following Example 4 is added after the description of Example 3 on page 13 of Qll Bookmark. 0.0 1M NaC of horseshoe crab ameposite 10t
1 in 0.02 M Trls-HCI buffer (pH
Amebosite lysate 90 obtained by extraction with 8,0)
- was applied to a heparin Sepharose CL-6B column (2,2X18.01) equilibrated with the above buffer solution, and 0
．． Elution with 0.02 M Tris-HCI buffer (118,0) containing 5 M NaCl gave a mixture of clotting prozyme, Factor G and 7 Actor B, followed by 0.02 M Tris containing 2 M NaCl. -M
The desired coagulation inhibitory factor was obtained by elution with CI buffer 111 (pi (11,0) 500-.) 1. In the brief explanation column O of Figure 11i of the specification, page 14 of the specification, 4- Delete line 12. ■Delete Figure 3 in 0 drawings.

Claims (1)

[Scope of Claims] (2) The ability to make the amebonite lysate turn around or the ability to turn the amebonite lysate present in the amebonite lysate. 11J% - A factor that inhibits the ability to activate bulk. The fractions desorbed from the insects are combined with a NaC1 latent solution with a strength of 0, and the fractions are molecularly sieved to 1, av) When a mudgraphy is performed using a Tarafie carrier, the self-cytarode cost is obtained. Position E where strine or sodium chloride exits S
2. The factor O according to claim 1, which is a strongly basic factor that increases in a minute when it is released. (2) Submit the horseshoe crab lysate to a column using a molecular sieve for tarmadography, or
1.
．． In an amebotate lysate, which is characterized in that, after collecting one fraction eluted by a salt solution IE having an ionostrength of 0M or more, both fractions are sealed in a column termadography using a molecule-talomad support body. A method for the extraction of all system inhibitory factors by ylc. (2) The support described in claim 3, wherein the carrier for molecular sieve a-mardography is different from agarose, striped agarose, structured gel, and cross-linked acrylamide gel. 4. The collection method according to claim 3, wherein the soybean solution is a sodium chloride solution.