JPH0218080B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying amebocyte lysate, and more specifically, to remove impurities from an extract of amebocytes (amebocyte lysate) obtained from the hemolymph of horseshoe crabs to improve endotoxin specificity. The present invention relates to a method for preparing amebosite lysate with high

Horseshoe crab (Limulus polyphemus,
The horseshoe crab hemolymph coagulation phenomenon that occurs due to the action of Gram-negative bacterial surface substance "endotoxin" (lipo-polysaccharide) on amebocytes present in the hemolymph of Tachypleus tridentatus, T. gigas, etc.) It is interestingly researched. On the other hand, the coagulation phenomenon of amebocyte lysate due to endotoxin is
It is commonly used as the "Limulus test" and is widely used in the medical and pharmaceutical fields as an endotoxin-specific detection method. In addition to endotoxin, mammalian coagulation enzymes (factor The Limulus test has been evaluated as a specific reaction system for endotoxin.

In the process of conducting intensive research to elucidate the coagulation mechanism of amebocyte lysate, the present inventors developed a highly sensitive and highly quantitative method for detecting endotoxin using a synthetic substrate specific to enzymes involved in the horseshoe crab coagulation system. (Refer to Japanese Patent Application Laid-Open No. 54-15797), and the substances on which endotoxin acts directly in the coagulation mechanism of amebosite lysate are:
In addition to the coagulation enzyme precursor that has traditionally been mentioned, there are other endotoxin-sensitive substances that are activated by the action of endotoxin.
Next, the coagulation enzyme precursor is made into an active coagulation enzyme, and by the action of the activated enzyme, coagylogen becomes fibrous coagulin. In other words, the horseshoe crab's coagulation system, like that of mammals, uses a large number of enzyme groups. It was reported that coagulation is a system in which coagulation progresses through a stepwise reaction (FEBS.Letters, 120 ,
217 (1980) S. Iwanaga et al).

In addition to the above-mentioned facts, the present inventors discovered the following new fact and arrived at the present invention. That is, we confirmed for the first time that in addition to endotoxin, glucose polymers having β-1,3-bonds and their derivatives exist as substances that act on amebocyte lysate and cause the lysate to gel. found that there is a β-1,3-glucan-sensitive fraction that is affected by these substances, and that this fraction is different from the endotoxin-sensitive substance fraction.

The present invention removes substances (fraction) that are sensitive to β-1,3-glucan and its derivatives in amebocyte lysate and cause a coagulation reaction of amebocyte lysate even in the absence of endotoxin. The present invention relates to providing amebocyte lysate with high specificity for endotoxin.

The β-1,3-glucan sensitive fraction of the present invention is
The ionic strength is
It is desorbed and eluted from the adsorption carrier by using a 0.15 to 0.3 salt solution, preferably a Tris-HCl buffer containing sodium chloride (NaCl).

Examples of the sulfated polysaccharides include dextran sulfate/agarose, sulfated agarose, etc., which may be used alone or in combination of two or more, and heparin/cepharose CL.
Heparin-bonded cross-linked agarose, such as -6BR○ (Pharmacia Fine Chemicals, Sweden)
May be used in combination with gel.

When β-1,3-glucan is added to this fraction and incubated, and then the coagulation enzyme precursor fraction is added, the coagulation enzyme precursor is activated and a color-forming synthesis prepared from the structure of coagylogen or coagylogen is carried out. Acts on substrates to gel or develop color. The existence of this β-1,3-glucan sensitive fraction was first revealed by the present inventors. The purpose of the present invention is to
Regarding the method for identifying the β-1,3-glucan sensitive fraction, since the purpose is to provide endotoxin-specific amebocyte lysate by removing the β-1,3-glucan sensitive fraction in the lysate. is not limited to the embodiments of the present invention.

For the β-1,3-glucan of the present invention, curdlan yeast cell wall polysaccharide zymosan produced by Pachyman soil bacterium Alcaligenes faecalis obtained from Poria cocos sclerotia, lentinan derived from the fruiting body of Shiitake mushroom, etc. are applied, Pakiman,
For curdlan etc., AE is used to increase water solubility.
Clarke; Phytochemistry, 1 , 175-188 (1967)
It has been confirmed that even when used as carboxymethylated derivatives according to the method described above, both activate β-1,3-glucan-sensitive factors.

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 ml of Sepharose CL-6B (trade name; manufactured by Pharmacia) with 1 part of distilled water, 600 ml of distilled water
ml, 260 ml of 2N NaOH aqueous solution and 60 ml of epichlorohydrin were added, and the mixture was stirred at 40°C for 2 hours. After the reaction, it was transferred onto a glass filter and washed with 700 ml of distilled water until the pH reached around 7. Furthermore, after washing with acetone, it was dried under reduced pressure to obtain 20 g of epoxy-activated Sepharose CL-6B.

(2) Sulfation of epoxy-activated Cepharose CL-6B 20g of epoxy-activated Cepharose CL-2B,
Suspend in 300 ml of pyridine dehydrated with KOH at −20°C.
After cooling to 60 ml of chloroform, add chlorsulfonic acid.
A solution containing 20 ml of the solution was added dropwise over 2 hours with stirring. After dropping, warm to 45℃ and further
Stir for hours. After cooling to -20℃ again, add 15ml of distilled water.
was added, and then 400 ml of 7.5% NaHCO ₃ aqueous solution was added to neutralize. After neutralization, transfer to a glass filter,
500ml of 5% _NaHCO3 aqueous solution, 500ml of distilled water, 50%
Pyridine was completely removed by sequentially washing with 500 ml of ethanol aqueous solution. Next, after washing with 500 ml of distilled water, it was suspended in 300 ml of 0.4M Tris-HCl buffer (PH9.0) and sterilized by autoclaving (121°C, 20 minutes).

Preparation Example 2 Preparation of dextran sulfate/agarose Method of Anderson et al. [LOAnderson et al.,
Thomb.Res., 7 , 451 (1975)], BrCN
using dextran sulfate (manufactured by Pharmacia;
(molecular weight approximately 500,000) was immobilized on Sepharose CL-6B.

That is, 25 g of dextran sulfate was dissolved in 1 part of ice-cold water, 500 ml of Sepharose CL-6B, and 125 BrCN.
Add 180 ml of acetonitrile solution of
The mixture was stirred for 50 minutes. During the reaction, the pH was maintained at 10.5 by dropping a 10N NaOH aqueous solution. After the reaction, transfer to a glass filter and pass through.
Suspend in 0.5M Tris-HCl buffer (PH8.5) 1,
Stirred at 4°C for 15 hours. Next, pass through a glass filter, wash thoroughly with distilled water, and add 500 ml of distilled water.
ml and autoclaved (121℃, 20 minutes)
and sterilized.

Preparation Example 3 Activity Measurement Method The enzyme activity and activity of each factor in each fraction after chromatography were measured at 37° C. according to the following method. In all enzyme activities, 1 unit is the amount of enzyme that releases 1 μmole of p-nitroaniline per minute.

(i) Activity of endotoxin-sensitive factor (factor B activity): 50μ of each fraction and endotoxin (lipopolysaccharide, LPS) (600ng/ml)
30μ and 0.2M Tris−HCl−13mM MgCL ₂
Mix 100μ of buffer solution (PH8.0) and leave for 15 minutes.
Add a mixture of 20μ of 5mM Boc-Leu-Gly-Arg-pNA and 50μ of fraction A (figure), and after a certain period of time.
The reaction was stopped by adding 0.8 ml of 0.6M acetic acid, and the amount of paranitroaniline released (ε = 10500) was
Measured at 405nm.

(ii) Proclotting enzyme: Mix 50μ of each fraction with 50μ of active factor B and 100μ of Tris buffer (used in (i)),
After standing for 30 minutes, 50μ of 2mM synthetic substrate (used in (i)) was added, and the appearing amidase activity was measured. Note that active factor B is fraction B.
(Figure) 400μ and 0.4M Tris−HCl−26mM
A mixture of 200μ of MgCl ₂ buffer (PH8.0) was prepared by leaving it for 15 minutes.

(iii) Clotting enzyme: 50μ for each fraction
and Tris buffer (used in (i)) 100μ,
50μ of 2mM synthetic substrate (used in (i)) and 50μ of physiological saline were mixed, and after a certain period of time, 0.8ml of 0.6M acetic acid was added to stop the reaction, and the amount of liberated paranitroaniline was measured.

Example 1 Amebosite analysis using sulfated agarose column
Lysate chromatography 10g of horseshoe crab amebosite with 0.05M NaCl
90ml of amebocyte lysate extracted with 0.02M Tris-HCl buffer (PH8.0) containing sulfated sepharose CL-6B equilibrated with the above buffer.
Apply to column (2.2 x 18.0 cm) and add 500 ml of the above buffer.
By washing with (B1), a coagulation enzyme precursor (fraction A) was obtained as a flow-through fraction. Then,
0.02M Tris−HCl buffer containing 0.15M NaCl (PH
8.0) Factor G, a non-endotoxic pro-coagulant activator activated by β-1,3-glucans, eluted at 500 ml (B2)
(Fraction G) was obtained. Additionally, contains 0.45M NaCl
0.02M Tris-HCl buffer (PH8.0) 500ml (B3)
Factor B (fraction B), which is an endotoxin-sensitive factor, was obtained by elution. The chromatogram is shown in the figure. In the figure, curves A, B, and G are
Fractions A, B, and G are shown, respectively.

Fraction A and fraction B obtained as above
By combining these, we were able to obtain an amebocyte lysate specific for the target endotoxin.

Example 2 Chromatography of amebosite lysate using dextran sulfate/agarose column 42g of horseshoe crab amebosite was collected using 0.05M NaCl.
630 ml of amebocyte lysate extracted with 0.02M Tris-HCl buffer (PH8.0) containing By washing with buffer solution 2, a coagulation enzyme precursor (fraction A) was obtained as a flow-through fraction. Next, it was eluted with 0.02M Tris-HCl buffer (PH8.0) containing 0.3M NaCl to obtain a non-endotoxic clotting enzyme proactivator activated by β-1,3-glucans. Factor G (Fraction G)
I got it. Furthermore, 0.02M Tris− containing 0.5M NaCl
Factor B (fraction B), an endotoxin-sensitive factor, was eluted with HCl buffer (PH8.0) 2.
I got it.

Fraction A and fraction B obtained as above
By combining these, it is possible to obtain an amebocyte lysate specific for the endotoxin of interest.

[Brief explanation of drawings]

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

Claims (1)

[Scope of Claims] 1. A horseshoe crab amebocyte lysate or a liquid containing it is subjected to liquid chromatography using a sulfated polysaccharide as an adsorption carrier, and a fraction containing a non-endotoxic coagulation enzyme precursor activator is obtained. 1. A method for preparing an endotoxin-specific amebocyte lysate or a liquid containing the same, which comprises removing endotoxin. 2. The preparation method according to claim 1, wherein the non-endotoxic coagulation enzyme precursor activator is activated by β-1,3-glucans. 3. In liquid chromatography, a salt solution with an ionic strength of 0.15 to 0.3 is used as an eluent to remove a fraction containing a non-endotoxic coagulation enzyme precursor activator. Or the preparation method described in Section 2.