JPH0324628B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a new and useful horseshoe crab amebocyte lysate and a method for producing the same. This invention relates to a high-quality horseshoe crab amebosite lysate and a method for producing the same. Horseshoe crab (Limulus polyphemus,
Tachypleustridentatus, T.gigas, etc.) amebocyte lysate (blood cell extract, hereinafter referred to as lysate) is a component of the outer cell membrane of Gram-negative bacteria and is highly pyrogenic due to extremely small amounts of "endotoxin" (lipopolysate). lysate) and coagulates the lysate. The "Limulus test" for detecting endotoxin by utilizing this phenomenon is widely used in the medical and pharmaceutical fields. In addition to endotoxin, other mammalian coagulation enzymes (factor Since no endotoxins have been detected, the Limulus test has been evaluated as a method for specifically detecting endotoxins. (Means for Solving the Problem) In the process of conducting intensive research to elucidate the coagulation mechanism of lysate, the present inventors discovered that there is a substance in lysate that directly acts on endotoxin, and that the action of endotoxin causes It undergoes activation, and through the action of the thus activated enzyme, coagyurogen becomes fibrous coagyurin,
In other words, it was reported that in the horseshoe crab's blood coagulation system, coagulation proceeds through a stepwise reaction of a large number of enzyme groups, similar to that in mammals (FEBS Letters. 120 217-220
(1980) S. Iwanaga et al). In addition to the above-mentioned facts, the present inventors further discovered that, in addition to endotoxin, (1→3)-
Gelation is also caused by β-D-glucan, and a (1→3)-β-D-glucan sensitive substance (hereinafter referred to as G factor) is present in the lysate; 3) It was discovered for the first time that it is an enzyme precursor activated by -β-D-glucan, which activates pro-clotting enzyme to form clotting enzyme, which acts on coagulogen and gels as coagulin. . The present invention is based on the above discovery, and is a lysate that does not contain G factor, which causes a coagulation reaction of the lysate even in the absence of endotoxin. can be detected with high sensitivity. That is, the present invention is an endotoxin-specific lysate that does not contain factor G. In the present invention, to produce an endotoxin-specific lysate that does not contain factor G, the lysate is subjected to liquid adsorption using a heparin-loaded adsorption carrier such as heparin-cepharose CL-6B (Pharmacia Huain Chemicals, Sweden). Fraction A containing pro-clotting enzyme subjected to chromatography, (1→3)-
divided into a G fraction containing β-D-glucan sensitive substances and a B fraction containing endotoxin sensitive substances,
By combining the A and B fractions, a lysate containing no G factor can be produced. For example, the A fraction is eluted with buffer, then the G fraction is eluted with 0.15M NaCl solution, and then the B fraction is eluted with a 0.15M NaCl solution.
It is eluted with 0.45M NaCl solution. Furthermore, NaCl of amebosite lysate
The additive solution was added to Cefacryl S-200 (Pharmacia Huain Chemicals Co., Ltd.), Cellulofain.
Subject to molecular sieve chromatography such as GC-200 (Chitsuso Co., Ltd.), the A fraction eluted first and the B fraction eluted together with the NaCl fraction are combined to determine endotoxin specificity, which does not contain factor G. lysate can be produced. To measure endotoxin using the amebocyte lysate that does not contain factor G produced as described above, it is necessary to use a standard Limulus test to cause the sample containing endotoxin to gel, or to use a chromogenic synthetic substrate. (For example, Boc-
Leu-Gly-Arg-pNA) can be used to develop color and be measured. (Effects of the Invention) Since the lysate of the present invention does not contain factor G, it can be used to specifically measure endotoxin in a specimen with high sensitivity. (Examples) Hereinafter, the present invention will be explained in more detail with reference to Preparation Examples and Examples. Preparation Example 1 Preparation of Japanese Horseshoe Crab Amebosite Lysate Approximately 100 ml of hemolymph was collected from a Japanese horseshoe crab (Tachypleus tridentatus (body weight: approximately 2 kg)) according to the method of Special Publication No. 51-40131, while strictly avoiding contamination. . Amebocytes (blood cells) were separated by centrifugation and washed with a 3% sodium chloride solution to obtain an amebocyte pellet. Add 0.05M Tris-HCl buffer (PH7.2) to this amebocyte pellet (1/10 volume of the original blood lymph fluid), stir well with a sterilized homogenizer, centrifuge at 8000 rpm for 30 minutes, and collect the supernatant to lysate. TTL (extract liquid) was obtained. Preparation example 2 American horseshoe crab (Limuluspolyphemus)
Preparation of amebosite lysate Amebosite lysate LPL was obtained in the same manner as in Preparation Example 1. Preparation Example 3 Carboxymethylated (1→3)-β-D
- Preparation of glucan (CMP) from Bukuriyo, Y. Shibata, J. Ferment.
Pachyman was prepared according to the method of Technol. 50 , 388 (1972), purified using dimethyl sulfoxide, AEClarke BAStone,
CMP was obtained by carboxymethylation using the method described in Phytochemistry 1 , 175-188 (1967). Example 1 Production by liquid adsorption chromatography using a heparin-Sepharose CL-6B column 50 ml of the lysate TTL obtained in Preparation Example 1 was
It was diluted 3 times using 0.05M Tris-HCl buffer (PH7.2) and applied to a heparin-Sepharose CL-6B column (5.0 x 17 cm) that had been previously buffered with the same buffer. The elution operation was performed in the following steps. That is, fraction A was obtained by elution using 0.05M Tris-HCl buffer (PH7.2). Next, it was eluted using 0.15M NaCl to obtain a G fraction. Next, it was eluted using 0.45M NaCl to obtain a B fraction. These fractions were analyzed using the method previously reported by the present inventors [FEBS Letters 120 , 217-220 (1980), S.
Experiments 1 to 7 were conducted according to Iwanaga et al.
The properties of the eluted fraction were investigated. The results are shown in Tables 1 and 2. Table 1 shows the results of experiments conducted with fractions A, G, and B alone or in combination in the presence of endotoxin derived from E.Coli 0111-B4, and Table 2 shows the results of experiments conducted with fractions A, G, and B, alone or in combination, in the presence of endotoxin derived from E.Coli 0111-B4. ,G and B
This figure shows the results of experiments conducted in the presence of carboxymethylated (1→3)-β-D-glucan (CMP), either alone or in combination. The experiment was conducted as follows. 50μ of each fraction, endotoxin or CMP (final concentration: 24ng/ml), 0.4mM chromogenic synthetic substrate (Boc-Leu-Gly-Arg-pNA), 80mM Tris-HCl buffer (PH: 8.0) and Reactions containing 5.2mM magnesium chloride ( _MgCl2 ) and having a total volume of 250μ were incubated at 37°C for 30 minutes. Next, 0.8 ml of 0.6M acetic acid was added to stop the reaction, and the absorbance at 405 nm was measured. In a combination experiment, 50μ of each fraction of A, B and G were mixed and amidase activity was measured under the same conditions as described above. The ability to form clots in each experiment was measured using highly purified coagulon as follows. The reaction mixture was a mixture of fractions A, B, and G alone or in combination of 50 μ of each fraction, CMP (400 n
g/ml) 30μ, Tachypleus coagulogen (2mg/ml) 100μ and 26mM
0.4M Tris-HCl buffer (PH8.0) containing _MgCl2
The sample was determined to have a positive reaction if clots appeared within 1 hour at 37°C, with a total volume of 230μ.

【table】

[Table] As can be seen from Experiments 1 to 7 above, fractions A and B alone do not exhibit clotting enzyme activity (amidase activity) or clot formation even when endotoxin is added, but the combination of B and A does not show endotoxin. Coagulase activity occurs against the enzyme. However, (1→3)
For -β-D-glucan, A and B neither alone nor together exhibit coagulase activity or clot-forming ability. On the other hand, the G fraction is (1→3)-β-D-
Expresses clotting enzyme activity and clot-forming ability for glucans. Based on these results, by liquid adsorption chromatography of heparin-sepharose/CL-6B, the G fraction eluted with 0.15M NaCl was not sensitive to endotoxin and was found to be (1→3)-β-
It can be seen that this is a fraction containing G factor that is sensitive to D-glucan. Example 2 Production by molecular sieve chromatography using Sephacryl S-200 50 ml of the LPL NaCl solution of Preparation Example 2 was placed in a 4.3 x 114 cm
Gel filtration was performed using a Sephacryl S-200 column. The chromatogram is shown in the figure. Each fraction was examined for clotting enzyme activity and clot-forming ability according to the experimental method of Example 1. Result fraction number 86
~130 is A, fraction number 131-162 is G and fraction number
163-217 were collected as the B fraction. The NaCl fraction was calculated from the electrical conductivity of each fraction. Fraction B is adsorbed to Cephacryl S-200, but is desorbed by NaCl and is eluted into almost the same fraction as NaCl.

[Brief explanation of drawings]

The figure is a chromatogram of lysate LPL obtained by gel filtration using a Sephacryl S-200 column. 〇−〇……A fraction, ●−●……B fraction, △−△
...G fraction, -...NaCl fraction.

Claims (1)

[Scope of Claims] 1. An endotoxin-specific horseshoe crab amebocyte lysate that does not contain a (1→3)-β-D-glucan sensitive substance. 2 Horseshoe crab amebosite lysate,
A fraction A containing pro-clotting enzyme, a G fraction containing a (1→3)-β-D-glucan-sensitive substance, and an endotoxin-sensitive substance were separated by liquid adsorption chromatography using an adsorption carrier carrying heparin. A method for producing an endotoxin-specific horseshoe crab amebocyte lysate that does not contain a G fraction, the method comprising dividing the lysate into a B fraction containing the lysate, and combining the A and B fractions.