JPH0898688A - Stabilization of bromelain solution - Google Patents

Abstract

PURPOSE: To stabilize the subject solution used for detection of irregular antibody in inspection of human blood transfusion by adding a water-soluble mercury compound in an amount by which the enzymatic activity is not inhibited and a polyhydric alcohol to a bromelain solution. CONSTITUTION: A water-soluble mercury (e.g. thimerosal, etc.) in an amount not inhibiting the enzymatic activity of bromelain is added as a stabilizer to a purified bromelain solution and further, as necessary, a polyhydric alcohol (e.g. diethylene glycol) is added as a stabilizing auxilizry thereto. The mixture is dissolved in a phosphoric acid buffered physiological saline (pH5.5) and an insoluble matter is removed by centrifugal separation and pH thereof is controlled to 5.0. The supernatant is filtered by a filter and aseptically poured into a vessel to provide the objective stabilized bromelain solution. The bromelain solution is frequently used for detecting an irregular antibody by utilizing a phenomenon wherein IgG type antibody and erythrocyte are agglutinated by the action of bromelain which decomposes the surface glycopeptides of erythrocyte containing sialic acid and N-acetylneuraminic acid, etc., in inspection of human blood transfusion.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing the protease activity of a bromelain solution which is frequently used for detecting irregular antibodies in human blood transfusion tests.

[0002]

Bromelain solution has a function that, when added to a red blood cell suspension, bromelain acts on a red blood cell membrane to decompose and release glycopeptides containing sialic acid and N-acetylneuraminic acid. As a result, when the amount of sialic acid on the red blood cell membrane decreases, the zeta potential of negatively charged red blood cells decreases, the distance between blood cells shortens, and IgG type antibodies crosslink with red blood cells to cause aggregation, which is utilized. It has been used as a method for detecting irregular antibodies. Current,
As the bromelain solution used in the blood transfusion test, a solution prepared by diluting the extracted bromelain powder from a pineapple fruit or stem into an appropriate concentration in a laboratory, a commercially available bromelain solution purified to an appropriate concentration, or an appropriate A bromelain solution may be used, which is prepared by lyophilizing to a desired concentration, lyophilized, and dissolved before use.
The usefulness of bromelain used for detection of irregular antibody in blood transfusion test is closely related to the activity value of protease.If the activity value of this enzyme is too low, the detection of irregular antibody becomes impossible and If too much, a nonspecific agglutination reaction unrelated to the irregular antibody occurs. Therefore, the bromelain solution has a low appearance rate of non-specific reactions and is used clinically at a concentration at which an irregular antibody can be detected. This enzyme is relatively stable in the form of a dry powder, but becomes easily inactive by heating at 36 ° C in the form of a solution.
In storage at -25 ℃, hydrogen ion temperature (p
H), additives, etc. affect the stability of the enzyme. The solution is stable in a frozen state (-20 ° C or lower). It is most stable when freeze-dried and stored at low temperature. When stored in the form of a solution at 2 ° C to 25 ° C, the most stable pH is 4.0, and the higher the pH, the worse the stability.
When bromelain with a pH of 4.5 or less is used in fresh serum, the detection ability of irregular antibodies is lowered, and therefore pH is usually 5.0 to 5.0.
Often used in 5.5.

[0003]

From the viewpoint of rationalization of the blood transfusion test, it is desirable to use bromelain already in a solution state at the time of the test, but in the solution state of the concentration used for the blood transfusion test, satisfactory stability is still obtained. I have no Bromelain solution for detecting irregular antibodies. The present invention is p
By examining H, additives, etc., a bromelain solution that is stable at 2 ° C to 25 ° C and suitable for detecting irregular antibodies in a blood transfusion test is prepared, and the blood transfusion test is rationalized.

[0004]

Means for Solving the Problems The inventors of the present invention have stable detection of irregular antibodies in a blood transfusion test and low incidence of non-specific reaction for 72 days or more when stored at 23 ° C. and 13 months when stored at 5 ° C. Succeeded in preparing a bromelain solution,
The inventors have found that this solution is a highly useful reagent in blood transfusion tests because of its ease of use, reliable detection of irregular antibodies, and low incidence of non-specific reactions, and completed the present invention.

That is, the present invention provides a method for stabilizing a bromelain solution, which comprises adding to a bromelain solution an amount of a water-soluble mercury compound that does not substantially inhibit the enzyme activity as a stabilizer. It is a thing. In addition, in the present invention, in addition to the stabilizer,
A polyhydric alcohol may be added as a stabilizing aid.

The bromelain to be the subject of the stabilization method of the present invention is not particularly limited and may be any of those commonly used, and examples thereof include commercially available purified bromelain. Bromelain is usually dissolved in a weakly acidic, eg pH 5.5 buffer, eg 1/150 molar phosphate buffered saline, at a concentration of 0.2-0.7 w / v%.

Examples of the water-soluble mercury compound used as a stabilizer include mercuric chloride and thimerosal.
These are used in an amount that does not substantially inhibit the enzyme activity of bromelain. For example, mercuric chloride is about 0.00
005-0.0002w / v%, thimerosal is about 0.00
Used in an amount of 05-0.002 w / v%.

As the polyhydric alcohol used as a stabilizing aid, sugar and sugar alcohol (eg, sorbitol,
Mannitol, lactose, saccharose, glucose) and other polyhydric alcohols (eg, diethylene glycol, propylene glycol). Although not particularly limited, the polyhydric alcohol is usually used in an amount of up to 1 w / v%.

To prepare a bromelain solution by the stabilization method of the present invention, for example, purified bromelain (commercial purified bromelain) is added to a pH 5.5 / 150 mol phosphate buffered saline solution at 0.2 to 0.7 w. Dissolve at a concentration of / v%. Then, a stabilizer is added in an amount suitable for stabilizing the enzyme (eg, thimerosal 0.001w / v%, hydrogen chloride 0.0001w without interfering with the detection of blood group irregular antibodies).
% / V%), and if desired, polyhydric alcohol is further added in an amount suitable for stabilizing the enzyme (eg, 0.1 w / v%), and insoluble matter is removed by centrifugation. By adjusting the pH to 5.0 ± 0.2 and sterilizing by filtration, a bromelain solution that can easily detect irregular antibodies in a blood transfusion test and screen antibodies can be obtained. The obtained Bromelain solution can be used for blood transfusion test in the same manner as conventional methods.

[0010]

EXAMPLES Next, the present invention will be described in more detail with reference to examples. Example 1 Trial Product 1: Purified Bromelain (Lot 3) 4.5 g, thimerosal 0.01 g, diethylene glycol (DEG) 1
g = 1/150 mol Phosphate buffered saline (pH 5.
5) Dissolve in 1000 ml, remove the insoluble matter by centrifugation, adjust the pH to 5.0, filter the supernatant with φ = 0.22 μm filter paper, and aseptically divide it into a 10 ml drop bottle for 10 ml. I made a note. Trial product 2: Prepared in the same manner as trial product 1 using 4.5 g of purified bromelain (Lot 3) without adding thimerosal and DEG. Trial product 3: Purified Bromelain (Lot 3) 4.5 g and thimerosal 0.01 g were used, and trial product 1 without addition of DEG
Prepared as in. Trial product 4: Prepared in the same manner as trial product 1 by using 4.5 g of purified bromelain (lot 3), adding thimerosal and adding 1 g of DEG.

Normal serum (serum containing no irregular antibody)
1 drop of 2v / v% physiological saline suspension of known antigen (O, CcDEe) erythrocytes was added to 2 drops, 1 drop of the prepared Bromelain trial product was added, and the mixture was heated at 37 ° C for 15 minutes and 1000 rpm1
As shown in Table 1, the non-specific agglutination reactions of prototypes 1, 2, 3 and 4 stored at 5 ° C for 0, 3, 6, 9, 13 months when centrifuging for minutes and visually determining an agglutination image are shown in Table 1. It was low.

[Table 1]

On the other hand, anti-D antibody and anti-E of irregular antibody
A known antigen (O, CcD
Ee) Add a drop of 2 v / v% saline suspension of red blood cells,
Add 1 drop of each prepared Bromelain trial product, 37 ℃ 15
Using the method of warming for 1 minute and centrifuging at 1000 rpm for 1 minute to determine the aggregated image with the naked eye, 0, 3, 6, 9, 13 at 5 ° C.
Bromelain trial products 1, 2, 3 and 4 stored for months
The agglutination value of was as shown in Table 2. Trial product 1 is 13
The aggregation values of the anti-D antibody and the anti-E antibody were kept stable by the end of the month. Trial products 2, 3 and 4 showed a decrease in agglutination activity after 3 to 9 months, and the former product was excellent in preservability in the order of trial products 3>4> 2. From these results, when analyzing the factors that affect the shelf life of the bromelain solution, thimerosal was useful as a stabilizer, and DEG had the second best preservative effect, and the addition of both thimerosal and DEG was the best.

[Table 2]

Example 2 Trial product 5: 4.5 g of purified bromelain (lot 3), 0.001 g of mercuric chloride and 1 g of DEG were dissolved in 1000 ml of 1/150 molar phosphate buffered saline (pH 5.5),
The insoluble matter was removed by centrifugation, the pH was adjusted to 5.0, and the supernatant was filtered with φ = 0.22 μm filter paper and aseptically dispensed into 10 ml 10 ml drop bottles. Trial product 6: Purified bromelain (Lot 3) (4.5 g), thimerosal (0.01 g) and D-mannitol (1 g) were used and prepared in the same manner as in trial product 5. Trial product 7: Purified bromelain (lot 3) (4.5 g), thimerosal (0.01 g) and lactose (1 g) were used and prepared in the same manner as in trial product 5. Trial product 8: Using purified bromelain (lot 3) 4.5 g, thimerosal 0.01 g, and sorbitol 1 g, trial product 5
Prepared as in. Trial product 9: Using 4.5 g of purified bromelain (lot 3), 0.01 g of thimerosal, and 1 g of propylene glycol,
It was prepared in the same manner as the trial product 5.

These test products were tested for non-specific reaction and anti-D antibody and anti-E antibody agglutination values by the same test method as in Example 1, and the results shown in Tables 3 and 4 were obtained.

[Table 3]

[Table 4] All of trial products 5, 6, 7, 8 and 9 are stored at 5 ℃
No non-specific reaction was observed for 3 months. In addition, the anti-D antibody and anti-E antibody agglutination values are 5, 6, 7, 8 and 9
None of them showed a decrease during storage at 5 ° C for 13 months. From these results, when the storage effect on the bromelin solution was analyzed, 0.0001 w / v% of mercuric chloride showed an enzyme stabilizing effect equivalent to 0.0001 w / v% of thimerosal, and D-mannitol, lactose, sorbitol. , W / v% of each of propylene glycol is 0.1 w / v of DEG
It showed a stabilizing effect equivalent to%.

Example 3 Trial product 10: 4.5 g of purified bromelain (lot 1) and 1/150 of 1 g of a known stabilizer sodium azide
Dissolve in 1000 ml of morphophosphate buffered saline (pH 5.5), remove insoluble matter by centrifugation, adjust the pH to 5.0, filter the supernatant with φ = 0.22 μm filter paper, and add to 10 ml. Aseptically dispensed into a 10 ml dropper bottle. Trial products 11, 12, 13 and 14: Using purified bromelain (Lot 2), using the same formulation and preparation method as trial product 10, trial product 11 and similarly purified bromelain (Lot 3)
Was used to prepare a trial product 12, a purified bromelain (lot 4) was used to prepare a trial product 13, and a purified bromelain (lot 5) was used to prepare a trial product 14. Test product 15: 4.5 g of purified bromelain (lot 1) and 0.01 g of thimerosal were dissolved in 1000 ml of 1/150 mol phosphate buffered saline (pH 5.5), the insoluble matter was removed by centrifugation, and the pH was adjusted to 5 Adjusted to 0.0 and the supernatant φ = 0.2
It was filtered with a 2 μm filter paper and aseptically dispensed into a 10 ml drop bottle for 10 ml. Trial products 16, 17, 18 and 19: Using purified bromelain (lot 2), using the same formulation and preparation method as trial product 15, trial product 16 and purified bromelain (lot 3) were used. A trial product 18 was prepared using lot 4) and a trial product 19 was prepared using purified bromelain (lot 5) and dispensed.

Each of these prototypes was stored at 23 ° C., and the anti-D aggregation value of the preserved product was measured. Table 5
, The trial products 10 and 14 containing sodium azide as an additive remained at least active for 77 days at 23 ° C., but the trial products 11, 12 and 13
Had lost the aggregating activity after 34 days. On the other hand, all the trial products 15 to 19 to which thimerosal was added maintained the aggregation activity at 23 ° C. for 72 days.

[Table 5] From these results, the storage stability of the prototype containing sodium azide is affected by the lot of purified bromelain, but the storage stability of the prototype containing thimerosal is good at least at 23 ° C for 72 days. No effect was observed. From the above results, it was determined that thimerosal as an additive has a better preservation of anti-D antibody activity than sodium azide.

[0017]

EFFECTS OF THE INVENTION According to the present invention, the stability of the bromelain solution can be kept extremely good, and the blood transfusion test can be rationalized.

Claims (2)

[Claims] 1. A method for stabilizing a bromelain solution, which comprises adding to the bromelain solution an amount of a water-soluble mercury compound that does not substantially inhibit the enzyme activity thereof. 2. The method for stabilizing a bromelain solution according to claim 1, which further comprises adding a polyhydric alcohol.