JPS62291568A - Preservation of microcapsule - Google Patents

Abstract

PURPOSE:To stably preserve a microcapsule for a long period of time, by holding the microcapsule having an enclosed substance in a preserving solution having osmotic pressure 3-50% higher than that of the enclosed member. CONSTITUTION:A microcapsule may be a small body constituted of at least one or more of a membrane having a semipermeable property. An enclosed substance may be any one and is not limited specially but pref. a hydrophilic substance. A preserving solution may be any solution having osmotic pressure 3-50% higher than that of the enclosed substance in the microcapsule and is not limited specially. As mentioned above, by preserving the microcapsule in the solution having osmotic pressure 3-50% higher than that of the enclosed substance, the substance in the microcapsule is held therein by slight osmotic pressure difference and storage stability can be enhanced by 5 times or more as compared with the using an isotonic solution.

Description

[Detailed description of the invention] 3. Detailed description of the invention [Purpose of the invention] (Industrial application field) The present invention relates to a method for preserving microcapsules.

More specifically, the present invention relates to a preservation method in which the osmotic pressure of a preservation solution is changed.

(Prior Art) The present inventors previously disclosed in Japanese Patent Application No. 58-224509 a liposome reagent, which is a microcapsule with a hydrophilic antigen or antibody immobilized on its surface and a hydrophilic labeling substance encapsulated inside. have disclosed an immunoassay reagent and an immunoassay method using this method. In this method, the liposome reagent described above is first reacted with the antigen or antibody in the sample and complement added separately and destroyed, and the encapsulated label substance flows out. Since the amount of the labeled substance that has leaked out is in a corresponding function with the amount of the analyte in the sample within a certain concentration range, it is possible to quantitatively reconstitute the analyte by quantifying the labeled substance that has leaked out. According to this immunoassay method, a test substance in a sample can be easily quantified in a short time.

(Problems to be Solved by the Invention) By using the above-mentioned liposome reagent, a test substance in a sample can be easily quantified in a short time. However, this liposome reagent has the problem of lack of storage safety as a reagent and deterioration of sensitivity since the encapsulated substance gradually flows out. Therefore, when using it for measurement after a certain storage period, the background as a liposome reagent will be high, so it is necessary to centrifuge, wash, and resuspend to reduce the background before use. Ta. If such operations are repeated, a problem arises in that although the function of the sensitized antigen or antibody is sufficiently maintained, the concentration of the encapsulated substance decreases, resulting in a decrease in sensitivity.

The present invention was made in view of the above-mentioned problems, and an object thereof is to provide a preservation method that can be stably preserved for a long period of time.

[Structure of the invention]

(Means for solving the problem of redundancy) The present invention provides microcapsules characterized in that the microcapsules having an encapsulating member are held in a preservation solution having an osmotic pressure 3 to 50% higher than the osmotic pressure of the encapsulating member. This is a method of preservation.

The microcapsule in the present invention may be any endoplasmic reticulum composed of at least one layer of pancreas having semipermeable membrane properties, for example, composed of phospholipids and/or glycolipids and cholesterol. Examples include liposomes and red blood cells.

Furthermore, the enclosing member may be of any material, and is preferably a hydrophilic substance, although it is not particularly limited. Examples include light-absorbing substances, fluorescent substances, oxygen, substrates, and the like.

The preservation solution of the present invention is not particularly limited, and may be anything that has an osmotic pressure that is 3 to 50% higher than the osmotic pressure of the encapsulating member in the microcapsule. For example, do the following <:am. Phosphate buffer, Penal buffer,
Prepare a storage solution by adding appropriate amounts of Na'', Ni-Ag'', Ca'', Ca-, etc. to a buffer such as gelatin veronal buffer, Tris-HCl buffer, Hepes buffer, or glycine buffer.1 The osmotic pressure of the preservation solution of the present invention is 3 to 50% higher than the osmotic pressure of the enclosing member, preferably 3 to 30% higher, and is generally 278 to 416IIIO9!l1oQ.The value of the osmotic pressure is determined by the freezing point depression method. decide.

The freezing point depression method is based on the following principle.

The osmolarity of a solution is proportional to its molarity, and the molarity is proportional to its freezing point depression, so the osmolarity and freezing point depression are in a proportional function. The osmotic pressure is in units of 03M0L, and the degree of freezing point depression of a solution with an osmotic pressure of 108M0L is -1,858℃, so
By accurately measuring the freezing point of a solution, the osmotic pressure of that solution can be determined.

An example using the method for preserving microcapsules of the present invention is shown below.

Encapsulating fluorescent materials with phospholipids and cholesterol, antibodies or a portion of antibodies or antigen sources are sensitized to prepare liposomes for immunoassays. By measuring the osmotic pressure of the encapsulated fluorescent substance and adding Na (4!
The prepared liposomes were incubated for 4 hours and stored at 4°C. This storage method makes it possible to obtain a liposome immunoassay reagent that is stable for a long period of time and allows for reproducible measurements. Since the conventional buffer solution is used for the measurement, there is no need to adjust the osmotic pressure each time the measurement is performed.

(Function) Until now, microcapsules have been stored in a solution that is isotonic with the material in which they are encapsulated. but.

In the present invention, by storing the microcapsule at an osmotic pressure 3 to 50% higher than that of the encapsulated components, the components within the microcapsules are kept stably inside with a slight osmotic pressure difference, and the storage stability is achieved through isotonic multi-solution solutions. This can be improved by more than 5 times compared to storing data in storage.

(Example) The present invention will be explained in detail with reference to Examples below.

These Examples are not intended to limit the scope of the present invention in any way.

[Example 1] Storage stability of liposome immunoassay reagent for human A, FP analysis ((A)) Preparation of liposome immunoassay reagent Cholesterol, dipalmitoylphosphatidylethanolamine (
DPPE) and dithiothreitol (DTT) manufactured by Sigma were used. N-succinimidyl 3-(
2-pyridyldithio)propionate (SPDP) and Sephadex G-25 fine were purchased from Pharmacia. Other reagents were commercially available (special grade) and used without purification. Note that all water used was ion-exchanged water.

■ Preparation of immobilized liposomes a) DPPP-dithiopyridinate (Dl'PE-D
Preparation of TP) Transfer 70B of DPPE into an Erlenmeyer flask with a tightly stopper, and add 25
Dissolve mQ in chloroform/methanol (5:1) solution, 60 μl toluethanolamine and 50 g
After adding PDP, the atmosphere was replaced with nitrogen. After reacting at room temperature for 1 hour, the solvent was removed using a rotary evaporator. The dried product was dissolved in 5 m9 of chloroform/methanol (10:1) and purified using a silica gel column. The product surface was collected and immersed in an evaporator to about 5 mR. Yield was 80-95%. Storage was performed at F-20°C under nitrogen.

b) Preparation of liposomes All lipids used were dissolved in chloroform or chloroform/methanol (2/1).

First, 5 mQM D P P C (200u Q)
, 1061M cholesterol (100μQ) and 1 cod 1
) PPE-DTP (50 μQ) was placed in a 1ONQ eggplant-shaped flask, and 2 ml of chloroform was added and mixed well. The solvent was removed by rotary evaporation in water (approximately 50° C.). After adding 2 mg of chloroform again and stirring thoroughly, the solvent was evaporated again using a rotary evaporator. When this operation was repeated several times, a thin film was formed on the flask wall. The flask was transferred to a nassicator and absorbed for about 1 hour using a vacuum pump to completely remove the solvent.Then, 100μ2 of 0.2M carboxyfluorescein (hereinafter referred to as CF: manufactured by Eastman Kodak Company, pH 7.4) was added. ) was added, the inside of the flask was purged with nitrogen, the flask was tightly stoppered, and the flask was immersed in a water bath at about 60° C. for about 1 minute. Subsequently, using a Vor-tex mixer,
The flask was shaken vigorously until the lipid film on the wall completely disappeared. Through this operation, a liposome WA suspension was prepared. 0 for this. OIM HEPESI 1m liquid (0
, 85% NaCf1 content, PH7.45, hereinafter J(B
(abbreviated as S), completely transfer the liposome suspension to a centrifuge tube, and incubate at 4°C, 15,000 rpm for 20 min.
The operation of centrifugation for several minutes was repeated several times.

C) Modified antibody human AFP of anti-human AFT antibody (mouse curvature) 2n+g/2mQ
(After diluting with BS and adding 10μ2 of 10+oM N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP/ethanol solution) and reacting for 30 minutes at room temperature after replacing with nitrogen M, 0.1M Only the protein fraction was collected using a Sephadex G-25 fine column (gel volume: about 15 mN) equilibrated with an acetate buffer (containing 0.85% NaCj!, pH 4.5).

Approximately 2011 g of dithiothreitol (DTT) was added to this fraction.
), add nitrogen*X! After exchanging the mixture, the mixture was allowed to react at room temperature for 20 minutes. Again, unreacted DTT was removed by gel filtration using a Cetiphadex G-25 fine column equilibrated with HBS.
A protein fraction was collected.

d) Preparation of anti-human AEP antibody-immobilized liposomes Liposomes obtained as described above! @Fuchishi 1m1134℃1
The liposome sediment was centrifuged at 500 rpm for 20 minutes and the modified anti-human AFP antibody solution (0.1 g protein trade/+aQ
2 mg of the mixture was mixed, the atmosphere was replaced with nitrogen, and the mixture was sealed and reacted overnight at 20° C. with slow shaking.

After the reaction, add HBS, then gelatin veronal rice buffer solution (GV
Washing was repeated three times by centrifugation at 15,000 rpm for 20 minutes at 4° C. (abbreviated as B-). Furthermore, when the osmotic pressure of the CF encapsulated in the liposome reagent thus obtained was measured, it was 305 mOsmoQ, so GVB- for storage was diluted with NaCff so that the final osmotic pressure was 330 mOsmoQ. It was adjusted. As above °C
GVB- for preservation of 2@Q and 10% NaN3 of 20μ pupil were added to the washed liposome precipitate, thoroughly stirred, and then stored at 4°C.

Furthermore, as a comparative example, Lipotum reagent prepared in the same manner was suspended in the previously used unadjusted GVB-solution 211N, 20 μQ of 10% NaN was added, stirred thoroughly, and then stored at 4°C. did.

((B)) Human A using liposome immunoassay reagent
FP analysis and storage stability ■ AFP analysis in human serum AFP in human serum was measured using anti-human AFP antibody-immobilized liposomes and a rabbit-anti-human AFP antibody sandwich assay using the standard curve shown in Figure 2. When 10 human serum samples were measured, the results were as shown in Table 1.

This value is calculated for reagents stored in a storage solution with adjusted osmotic pressure (abbreviated below) and reagents stored in an unadjusted storage solution (abbreviated below).
There was no difference between the two.

■ Storage stability of liposome immunoassay reagents and ■ In order to examine the storage stability of reagents, 10.2
When the respective calibration curves were measured after 0.30, 50.80 days, the results were as shown in FIG.

As is clear from FIG. 1, the reagent according to the present invention was extremely stable, with the calibration curve maintaining its initial sensitivity even after more than 80 days from the sample.

[Example 2 Storage stability of microcapsule reagent ((A)) of microcapsule reagent! %1 stability and storage stability 5 m dissolved in chloroform:methanol = 2:1
1 dissolved in the same solvent as MD P P C (200 μQ)
Put ゜ into a 10m12 pear-shaped flask, and add 2mj2
of chloroform was added and mixed well. The solvent was removed using a rotary evaporator in an aqueous solution at 45°C to form a thin film on the wall of the flask. The flask was placed in a desiccator for about 1 hour to remove the solvent under vacuum using an absorption pump.

Next, 100 .mu.2 of 0.1M xylenol orange (XO) (manufactured by Isotope Institute) was added to this flask, the flask was tightly stoppered, and the flask was immersed in an aqueous solution at about 60.degree. C. for 1 minute. Next, Vort
The flask was shaken vigorously using an EX mixer until the thin film of fat on the walls completely disappeared.

Through this operation, microcapsules Mii[ were prepared. This is 0.01. MI (by adding a small amount of BS,
Microcapsule! @ Transfer the suspension completely to a centrifuge tube and centrifuge at 4℃, 15,000 rpm for 15 minutes.
Washed repeatedly. And when we measured the osmotic pressure of XO encapsulated in microcapsules, we found that it was 296 times ○5.
Since it was IaoI2, I gave Nac+ to HB S! After the washing, the microcapsule reagent was precipitated with HBS.
It was suspended in storage solution 2+a12 and stored at room temperature.

For comparison, 'I!' was similarly applied to HBS2mQ without adjustment of osmolality. The prepared microcapsule reagent was suspended and stored at room temperature.

Figure 3 shows the change over time in the concentration of XO leaking from the microcapsule reagent. As is clear from FIG. 3, the storage solution according to the present invention had almost no leakage and was proven to be a stable storage solution for microcapsule reagents.

〔Effect of the invention〕

According to the present invention, it is possible to provide a method for preserving microcapsules that allows microcapsules to be stably preserved over a long period of time.

[Brief explanation of drawings]

FIG. 1 is a graph showing storage stability, FIG. 2 is an AFP calibration curve, and FIG. 3 is a graph showing storage stability of other examples. Agent Patent Attorney Noriyuki Chika Yudo Kikuo Takehana (Japanese) Figure 3

Claims (4)

[Claims] (1) A method for preserving microcapsules, which comprises retaining microcapsules having an encapsulating member in a preservation solution having an osmotic pressure 3 to 50% higher than the osmotic pressure of the encapsulating member. (2) The method for preserving microcapsules according to claim 1, wherein the microcapsules are liposomes made of phospholipids and/or glycolipids and cholesterol. (3) The method for protecting microcapsules according to claim 1, item 1 or 2, wherein the osmotic pressure of the preservation solution is 3 to 30% higher than the osmotic pressure of the enclosing member in the microcapsule. (4) The method for preserving microcapsules according to claim 1, 2, or 3, wherein the encapsulating member is a hydrophilic light-absorbing substance or a fluorescent substance.