JPS6333666B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical Field) The present invention relates to a blood coagulation accelerator for serum separation whose main component is the venom of the Namibium japonica, and a method for producing a blood coagulation accelerator for serum separation whose main component is the venom of the Namibian hawthorn. (Background technology) In general, snakes called habu are taxonomically classified as belonging to the family Viperidae, subfamily Viperidae, and subfamily Cro- viperinae.
talinae), Habu genera (Bothrops, Trimeresurus, etc.)
For example, bothrops
alternatus, B.asper, B.cotiara.B.jararaca, B.
jararacussu, B.lanceolatus, B.neuwiedii, B.
marajoensis, B. moojeni), hub (Trimer−
esurus flavoviridis), T.okinav−
ensis), Sakishima habu (T.elegans), Tokarensis (T.tokarensis), Formosan habu (T.
mucrosquamatus), blue-green herb (T.stejnegeri), etc. Currently, clinical test results of serum components are essential for diagnosis and treatment, and prompt reporting is required, but conventional serum coagulants require a long time for blood to coagulate. However, there have been drawbacks such as the fact that the blood clotting time of patients using anticoagulants becomes significantly longer. In view of this, the present inventor has developed a blood coagulation accelerator containing the crude toxin of P. japonica as the main component for blood coagulation (Japanese Patent Application No. 1983).
−8260). (Problems to be Solved in Background Art) Blood coagulation promoters whose main ingredient is crude toxin of P. elegans have the disadvantage of bathing red blood cells (erythrocyte destructive effect). According to the research conducted by the present inventors, it was found that such hemolytic effect is caused by the involvement of phospholipase A contained in the crude toxin of P. aeruginosa.
However, removal of phospholipase A must be carried out by fractional purification, and the yield of the purified product by this method is reduced to about 1/4 of that of crude poison.
This poses a major problem in terms of economic efficiency. (Disclosure of the Invention) The present invention has been made in view of the above-mentioned points, and has no hemolytic effect on red blood cells, and in terms of blood coagulation properties, it has the same efficacy as a coagulation promoter whose main ingredient is the venom of P. japonica. The object of the present invention is to provide a blood coagulation promoter for serum separation and a method for producing such a promoter. According to one feature of the present invention, there is provided a blood coagulation promoting agent for serum separation, which contains crude or purified toxin of Nambei habu as a main component. According to another feature of the present invention, there is provided a method for producing a blood coagulation promoter for serum separation by collecting a fraction having blood coagulation activity from crude toxin of Nanbei habu and freeze-drying the fraction. . The present invention will be explained in detail below. In addition, "habu poison" or "crude poison" (snake poison) is
It is said to be the poison of Nanbeihab. The present inventor has been researching the improvement and detoxification of therapeutic serum (toxoids) for Habu venom for many years, and during the biochemical analysis of Habu venom, a certain fraction showed strong blood coagulation. Recognizing that it has a stimulatory effect, adding a small amount of this fraction or its purified substance to human blood can cause red blood cells to be hemolyzed without hemolysis.
It has been found that separation of serum necessary for clinical testing can be carried out in a short time (within 30 minutes) and that such purified substances can be obtained in high yield. This substance is produced by dissolving the dried crude toxin of Habu toxin in water, extracting and removing the lipids using an organic solvent, dialyzing the aqueous layer, and adsorbing the dialyzed fluid on a column of Cephadex (trade name). This is then eluted with a buffer solution, and a fraction with strong blood coagulation activity is collected and freeze-dried. This fraction may be further subjected to column chromatography treatment using ion exchange, and the fraction having the highest blood coagulation activity may be collected and then freeze-dried. Furthermore, a predetermined amount of gelatin hydrolyzate, sodium chloride, glycine, etc. may be added to the obtained dried substance, and a freeze-drying process such as vacuum freezing may be performed. In addition, the above dry substance is
Prior to the addition of these additives, human plasma was
It can also be adjusted to solidify within 20±2 seconds. The substance obtained by the above procedure is an enzyme having thrombiwa-like action, and when used,
After dissolving the required amount in physiological saline, prepare a sterile blood clotting enzyme solution. Stabilizers, dispersants, etc. can also be added to this enzyme solution. An example of the composition of 1 ml of the enzyme solution thus obtained is as follows. Sterile blood coagulation enzyme solution 100 μg Gelatin hydrolyzate 1 mg Sodium chloride 8.76 mg Glycine 50 mg The above enzyme solution can be stored as a blood coagulation promoter as it is or by freeze-drying.
Freeze-dried products can be stored semi-permanently if kept below 25°C. Next, Examples and Test Examples of the present invention will be shown. Example 1 g of dried crude toxin of bothrops asper was dissolved in 3 ml of physiological saline, centrifuged at 20,000 rpm for 2 hours at 4°C, and the supernatant was subjected to potato starch zone electrophoresis (tris-barbital buffer). PH
8.8, 4℃, 4 hours, toxic dose 2.0 per 1 m ² of zone
mg) After treatment, the starch in the anode part was collected. Next, this starch was eluted with 100 ml of physiological saline at 4°C.
After dialyzing the liquid against physiological saline for 24 hours,
After centrifugation for 15 minutes, the supernatant was appropriately concentrated using an ultrafilter at 4°C and freeze-dried to obtain 250 mg of a dried product. Next, dissolve the dried product in physiological saline, and
ml to a concentration of 5 mg, remove the precipitate generated by saturation with ammonium sulfate 0.3, collect the precipitate generated by saturating the supernatant with ammonium sulfate 0.8, dissolve in physiological saline, and add to the external solution 0.3% saline at 4°C. After dialysis at 4°C, the mixture was subjected to ultrafiltration at 4°C and concentrated appropriately. After concentration, freeze-drying was performed to obtain a slightly yellowish white powder. The yield was about 70%. Mix 100 μg of this powder with 1 mg of dissolved gelatin water, 8.76 mg of sodium chloride, and 50 mg of glycine, and add double-distilled water to make the entire volume.
The volume was adjusted to 1.0 ml and freeze-dried to obtain a blood coagulation promoter for serum separation. This accelerator was a white powder, and when added to 1 ml of distilled water, it dissolved within 10 minutes and became clear and colorless. Test Example 1 A test was conducted regarding the hemolytic effect on red blood cells regarding the blood coagulation promoter according to the present invention and the blood coagulation promoter obtained from Himehabu. Table 1 shows the chemical composition and test results of each accelerator tested.

【table】

[Table] In the test, approximately 30 μl of the solution obtained by dissolving 1 vial of each sample in 1.0 ml of distilled water was added to 5 ml of human blood containing 2 units/ml of heparin, mixed by inversion, and incubated at room temperature for approximately 30 μl. Let stand for a minute, then centrifuge (3000 rpm/
5 minutes). Among the coagulation accelerators obtained from Hymehabu, samples 4, 8, and 9 that were purified did not cause hemolysis, but the yield of purified toxin was reduced to about 1/4 of that of crude toxin in each case. Sample 7 was subjected to the same purification treatment, but hemolysis occurred and the yield was reduced to about 1/4. Test Example 2 The blood coagulation promoter obtained according to the above example was added to normal human plasma, and the plasma coagulation time was measured. Using a mixture of commercially available standard normal citrated plasma and citrated plasma from more than 10 people,
After the plasma and accelerator were pre-incubated at 37°C, 0.025 ml of accelerator was added to each 0.1 ml of plasma and the clotting time was measured by the tilted test tube method, and both coagulated within 20 seconds. Test Example 3 The blood coagulation promoter obtained according to the above example was dissolved in 1.0 ml of double distilled water, and 25μ was added to the blood 5.0 μl immediately after blood collection.
ml, it could be solidified within 30 minutes at room temperature. (Effects) As described above, the blood coagulation promoter according to the present invention has excellent effects in that it does not cause any hemolytic effect on red blood cells and the coagulation time is equivalent to that of the coagulation promoter obtained from Hymehabu. It is something that you have. Further, according to the present invention, the coagulation accelerator can be obtained in high yield, which is a very excellent effect.

Claims (1)

[Scope of Claims] 1. A blood coagulation promoter for serum separation containing crude or purified toxin of Nanbei Habu as a main component. 2. A method for producing a blood coagulation promoter for serum separation, which comprises collecting a fraction having blood coagulation activity from crude toxin of Nanbei habu and freeze-drying the fraction.