JPH03399B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for collecting a factor that inhibits the hemolymph pseudosolid system caused by endotoxin (pseudosolid inhibitory factor) from an amebocyte extract (amebocyte lysate) obtained from the hemolymph of a horseshoe crab. More specifically, a factor present in the amebocyte lysate that inhibits the ability of endotoxin to pseudo-solidify the amebocyte lysate and/or the ability to act on the amebocyte lysate and activate a pseudo-solid enzyme in the lysate ( The present invention relates to a method for increasing the sensitivity and reactivity of amebocyte lysate to endotoxin by collecting and removing pseudo-solid inhibitory factors. Horseshoe crab (Limulus Polyphemus,
We use amebocyte lysate, which utilizes the pseudo-solidification phenomenon of hemolymph caused by the action of endotoxin (lipopolysaccharide), a surface substance of Gram-negative bacteria, on amebocytes present in the hemolymph of Tachypleus tridentatus, T. gigas, etc.) The so-called "limulus test" is widely used in the field of medicine and pharmacy as a specific detection method for endotoxin and as a substitute for the virodiene test using animals. 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 amount of gelling protein in the lysate constant in order to cause a stable pseudo-solidification phenomenon, and to remove factors that inhibit gelation. The amount of gelling protein and gelation inhibiting factors in the lysate varies depending on the habitat of the horseshoe crab and seasonal factors, and unless these can be controlled, the quantitative nature of the amebocyte lysate as a commercial product will be affected.
It has the problem of low reliability. The present inventors previously elucidated the pseudo-solidification mechanism of these lysates and disclosed a highly sensitive method for quantifying endotoxin using a synthetic substrate (Japanese Patent Application Laid-Open No. 15797-1982).
Furthermore, we have reported on factors involved in pseudo-solidification [S. Iwanaga, et al., FEBS. Letters,
120, 217 (1980)]. Based on these studies, we have provided a method for detecting endotoxin that is highly sensitive and quantitative, by measuring enzyme activity that is 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 significantly affected by the presence of inhibitory factors in the measurement range of ordinary endotoxin amounts, when measuring extremely small amounts of endotoxin, the inhibitory factors override the effects of endotoxin.
It is clear that it is basically preferable that no inhibitor is present in the lysate. The existence of the inhibitory factor yin thought is supported, and the enzyme theory [Nachum, et al.
al., J.Inv. Patho., 32 , 51 (1978)] and the endotoxin-binding lipoprotein theory [Sullivan, et al.,
Appl. Microbiol., 28 , 1023 (1974)], etc., but it is not considered that the explanation is sufficiently clear just by explaining the gelation phenomenon. On the other hand, methods for removing inhibitory factors from amebocyte lysate have been studied (U.S. Patent No.
No. 4107077, Sullivan.et al and JP-A-56-42590
No., Earl Clark Brown 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.
The latter is characterized by inactivating the inhibitor at a pH in an alkaline range that does not affect the pseudo-ligase in the lysate, and then returning the pH to the action range of the pseudo-ligase. All of these methods have the drawback of affecting endotoxin-sensitive factors in the lysate. In the course of research into the mechanism of action of pseudo-solid factors in lysate, the present inventors discovered for the first time the existence of pseudo-solid factors due to endotoxin and the fractionation of their inhibitors, and thus arrived at the present invention. That is, a specific adsorption carrier consisting of a sulfated polysaccharide is used to adsorb pseudosolid factors and inhibitory factors in the lysate, and a salt solution having an ionic strength of 0.5 or less, preferably Tris-HCl containing NaCl ( Tris-HCl) buffer is used to primarily desorb the pseudosolid factors, followed by a salt solution with an ionic strength greater than 0.5, preferably containing NaCl.
The inhibitory factor can be collected by elution using Tris-HCl buffer. Examples of the sulfated polysaccharides include dextran sulfate/agarose, sulfated agarose, etc., which may be used alone or in combination of two or more types, and heparin/cepharose CL-
It may also be used in conjunction with heparin-bound cross-linked agarose gels such as 6B (Pharmacia Fine Chemicals, Sweden). The inhibitory factor collected as described above strongly inhibits the activation step by endotoxin of amebocyte lysate and endotoxin-sensitive factors present in the lysate, and can be treated with alkaline, enzymes such as bronase, subtilisin, etc. completely deactivated. This shows that this inhibitory factor strongly inhibits the activation of amebocyte lysate and pseudosolid factors in the lysate by endotoxin, and that this inhibitory factor can be removed from the lysate by the gentle method of the present invention. A highly 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 ml of Sepharose CL-6B (trade name; manufactured by Pharmacia) with 1 part of distilled water, 600 ml of distilled water
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, transfer to a glass filter and add 700ml of distilled water.
Washed until the pH was 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 Epokin-activated Cephalose CL-6B 20 g of Epoxy-activated Cephalose CL-6B was suspended in 300 ml of pyridine dehydrated with KOH, and after cooling to -20°C, 20 ml of chlorosulfonic acid was dissolved in 60 ml of chloroform. The solution was added dropwise over 2 hours with stirring. After the dropwise addition was completed, the mixture was heated to 45°C and further stirred for 2 hours. After cooling to −20° C. again, 15 ml of distilled water was added, and then 400 ml of 7.5% NaHCO ₃ aqueous solution was added for neutralization. After neutralization, it was transferred onto a glass filter and washed successively with 500 ml of 5% NaHCO ₃ aqueous solution, 500 ml of distilled water, and 500 ml of 50% ethanol aqueous solution to completely remove pyridine. Next, after washing with 500ml of distilled water, 0.4M Tris-HCl buffer (PH
9.0) Suspend in 300ml and autoclave (121
℃, 20 minutes) and sterilized. Preparation Example 2 Preparation of dextran sulfate/agarose Method of Anderson et al. [LOAnderson et al.,
Thromb.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.
Suspended 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 was defined as the amount of enzyme that released 1 μmol of p-nitroaniline per minute. (i) Activity of endotoxin-sensitive factor (factor B activity): 50 μl of each fraction and 30 μl of endotoxin (lipopolysaccharide, LPS) (600 ng/ml)
and 100μl of 0.2M Tris-HCl-13mM _MgCl2 buffer (PH8.0) and left for 15 minutes.
Add a mixture of 20μl of 5mMBoc-Leu-Gly-Arg-pNA and 50μl of fraction A (figure), and after a certain period of time add 0.8ml of 0.6M acetic acid to stop the reaction, and calculate the amount of paranitroaniline released (ε = 10500). Measured at 405nm. (ii) Proclotting en-zyme:
Mix 50 μl of each fraction, 50 μl of active factor B, and 100 μl of Tris buffer (used in (i)).
After standing for 30 minutes, 50 μl 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) 400μl and 0.4MTris-HCl- _26mMMgCl2 buffer (PH8.0) 200μl, LPS (400ng/ml) 200μl
A mixed solution prepared by leaving the mixture for 15 minutes was used. (iii) Clotting enzyme: 50μl of each fraction
and 100 μl of Tris buffer (used in (i)),
50 μl of 2 mM synthetic substrate (used in (i)) and 50 μl of physiological saline were mixed, and after a certain period of time, 0.8 ml of 0.6 M acetic acid was added to stop the reaction, and the amount of liberated paranitroaniline was measured. (iv) Anti-LPS factor: sample and fraction A, fraction B, 0.4MTris-HCl-
50 μl each of 26mM _MgCl2 buffer (PH8.0), and
20μl of 5mM synthetic substrate (used in (i)) and LPS
Mix 30μl of (400ng/ml) and leave for 25 minutes.
The reaction was stopped by adding 0.8 ml of 0.6M acetic acid, and the amount of free paranitroaniline was measured. Under these conditions, activation of factor B (using 2 ng of LPS)
The activity inhibiting 50% was defined as 1 unit. Example 1 Chromatography of amebosite lysate using sulfated agarose column 10g of horseshoe crab amebosite was treated with 0.05M NaCl
Sulfated Sepharose CL-6B was obtained by equilibrating 90 ml of amebocyte lysate extracted with 0.02M Tris-HCl buffer (PH8.0) containing
Apply to column (2.2 x 18.0 cm) and add 500 ml of the above buffer.
By washing with (B1), a pseudo-immobilized 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 pseudozymogen activator activated by β-1,3-glucans, eluted at 500 ml (B2)
(Fraction G) was obtained. Furthermore, it contains 0.45MNaCl
Factor B (fraction B), which is an endotoxin-sensitive factor, was obtained by elution with 500 ml (B3) of 0.02M Tris-HCl buffer (PH8.0). Finally, 500ml of 0.02M Tris-HCl buffer (PH8.0) containing 2M NaCl.
The target pseudo-solid inhibitory factor was obtained by elution with (B4). The chromatogram is shown in the figure. In the figure, curves A, B, G, and X represent fraction A, fraction B, fraction G, and pseudosolid inhibitory factor, respectively. Fraction A and fraction B obtained as above
By combining these, we were able to obtain endotoxin-specific amebocyte lysate. 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 0.02 MT Tris-HCl buffer (PH8.0) containing By washing with Solution 2, a pseudo-solidified enzyme precursor (Fraction A) was obtained as a flow-through fraction. then
0.02M Tris−HCl buffer (PH
8.0) Factor G (Fraction G), a non-endotoxic pseudoenzyme precursor activator that is eluted with 2 and activated by β-1,3-glucans.
I got it. Furthermore, 0.02MTris− containing 0.5M NaCl
Factor B (fraction B), an endotoxin-sensitive factor, was eluted with HCl buffer (PH8.0) 2.
I got it. Finally, 0.02MTris− containing 2MNaC1
The target pseudo-solid inhibitory factor was obtained by elution with HC1 buffer (PH8.0) 2. Fraction A and fraction B obtained as above
By combining these, we were able to obtain endotoxin-specific amebocyte lysate. Example 3 Chromatography of amebosite lysate using a sulfated agarose column 15g of horseshoe crab amebosite was collected at 0.05MNaC1
130 ml of amebocyte lysate extracted with 0.02MTris-HCl buffer (PH8.0) containing sulfated sepharose CL-6B equilibrated with the above buffer.
Column (2.2 x 21.0cm) containing 0.5MNaC1
Elution was performed with 500 ml of 0.02M Tris-HCl buffer (PH8.0) to obtain a mixture of pseudo-immobilized enzyme precursor, factor G, and factor B. Then containing 2MNaC1
The target pseudo-solid inhibitory factor was obtained by elution with 500 ml of 0.02M Tris-HCl buffer (PH8.0). Example 4 Chromatography of amebosite lysate using dextran sulfate/agarose column 40g of horseshoe crab amebosite was 0.05MNaC1
610 ml of amebocyte lysate extracted with 0.02 MT Tris-HCl buffer (PH8.0) containing
0.02M Tris−HCl buffer (PH
8.0) 2 to obtain a mixture of pseudo-solid enzyme precursor, factor G and factor B. Then, 0.02M Tris-HCl buffer (PH
The target pseudo-solid inhibitory factor was obtained by elution with 8.0)2.

[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. A method for controlling endotoxin present in amebocyte lysate, which is characterized by subjecting horseshoe crab amebocyte lysate or a liquid containing it to liquid chromatography using sulfated polysaccharide as an adsorption carrier. A method for collecting a factor that inhibits the ability to pseudo-solidify the amebocyte lysate or the ability to act on the amebocyte lysate and activate a pseudo-solid enzyme in the lysate. 2. The collection method according to claim 1, characterized in that in liquid chromatography, fractions eluted by a salt solution having an ionic strength exceeding 0.5 are collected.