JP2000321269A - Drying solid phasing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To store an organization or a cell for a long time without damaging it by treating the organization that is subjected to solid phasing or the cell or the like with a low-concentration surface-active agent before drying and gradually unloading contents such as the organization or the cell or the like. SOLUTION: An organization or a cell is subjected to solid phasing to such carrier as a microplate well, glass, a plastic test tube, and slide glass via a bonding agent such as lectin, antibody, and positive reaction electric charge polymer being selected according to the characteristics. To unload the contents of the organization or the cell that has been subjected to solid phasing, isotonic liquid containing a low-concentration surface-active agent is operated. The isotonic liquid extremely gently damage one portion of the organization or the cell and cannot be released from a solid phase support due to the shock, and an antigen or the like existing in the organization or the cell cannot be modified. In this manner, the organization or the cell can be stored for a long time even at a room temperature, thus achieving an accurate assay.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for drying and fixing a solid-phased tissue or cell.

[0002]

2. Description of the Related Art Immobilized tissues or cells are frequently used in many clinical test kits such as blood type tests, but cells and marker substances such as antigens present on the cells are extremely easily denatured. Therefore, it is indispensable to perform a treatment which can withstand long-term storage.

[0003] For example, in Japanese Patent Application Laid-Open No. 2-126464, which discloses a method for immobilizing erythrocytes as they are on a support, erythrocytes are suspended in an Alsever solution or a MAP solution in order to preserve the erythrocytes. However, since red blood cells gradually lyse, cells fixed by this method are not always suitable for long-term storage.

On the other hand, Japanese Patent Application Laid-Open No. 2-151765 and Japanese Patent Application Laid-Open No. 5-142122 by the applicant of the present invention disclose a method of allowing cells to be solid-phased on a carrier, A dry solidification method for drying is disclosed. Cells immobilized in a dry state in this way can be stored at least several times longer than the above-described method of immobilizing cells as they are.

However, in the method of JP-A-2-151765, a monolayer of cells is formed on a solid support dyed with an organic dye having a net positive charge. Since the phase formation is inhibited by the presence of the protein, there is a major drawback that a protein for stabilizing antigenicity cannot be added to the drying solution added during the drying operation.

On the other hand, Japanese Patent Application Laid-Open No. 5-142122 uses a support such as a microplate as a carrier, and immobilizes a tissue or a cell on the support via a binding agent such as lectin. In such a solid phase immobilization method, the tissue or cell binding ability does not decrease due to the presence of the protein, and therefore, a protein having a stabilizing effect can be added to the drying solution. Furthermore, in these two methods,
In each case, since the operation of discharging the contents in the tissue or the cell is performed prior to the drying step, the tissue or the cell is not adversely affected by enzymes or the like present therein. For this reason, according to the method characterized by drying the cells and the like after the operation of discharging the contents of the cells, compared to other conventional techniques, preserved tissues or cells immobilized for a longer period of time. It becomes possible.

However, in this method, the contents of the tissue or the cells are discharged using a hypotonic solution that ruptures the cells and the like under physiologically hypotonic conditions. However, there is also a problem that cells and the like are detached from the solid support during the process.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in order to solve the problems in the prior art, and does not damage tissues or cells and substances such as antigens present thereon. Another object of the present invention is to provide a dry immobilization method for preparing a dry tissue or cell which can be stored for a long period of time.

[0009]

Means for Solving the Problems To solve the above problems, the present invention provides a method for treating a solid or immobilized tissue or cells with a low-concentration surfactant before performing a drying operation. Alternatively, there is provided a method for dry-solidifying a tissue or a cell, which comprises gently discharging contents such as a cell.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention comprises a step of eluting the contents of a tissue or cells immobilized on a carrier, a drying operation after adding a drying solution to the tissues or cells from which the contents have been eluted. Performing the following.

In the present invention, the carrier for immobilizing a tissue or a cell can be any carrier whose tissue or cell immobilization ability is not substantially reduced by the presence of a solution containing a protein. Preferred supports for use in the present invention are supports such as microplate wells, glass-plastic test tubes, glass slides, plastic, and the like.

In the present invention, a tissue or a cell is a lectin,
It is preferable that the solid phase is immobilized on a carrier via a binding agent such as an antibody or a positively charged polymer. The type of binder to be used is
Depending on the characteristics of the tissue or cells used, it may be selected,
For example, when erythrocytes are immobilized on a solid phase, a lectin capable of binding a specific sugar chain present on the erythrocytes can be used as a binder. As the lectin, wheat germ lectin (hereinafter referred to as WGA), ricin, lentil lectin and the like can be used, but are not limited thereto. Materials that can be used as binders include polyethylene imine and poly L lysine.

[0013] The tissue or cell to be bound to the carrier can be any tissue or cell.

An operation of discharging the contents of the immobilized tissue or cell is performed. This operation is for improving the preservability of the tissue or the cell by discharging the degrading enzyme or the like contained in the tissue or the cell.

In the present invention, the operation is performed by allowing a tissue or cell to act on a liquid having a low concentration of a surfactant, which is not less than isotonic, preferably an isotonic liquid. An isotonic solution containing a low concentration of a surfactant extremely gently destroys a part of a tissue or a cell. It does not peel off from the tissue, and uses a low concentration of a surfactant, so that antigens and the like present in tissues or cells are hardly denatured. It is preferable that conditions such as the concentration of the additive used in the content discharging operation be set to optimal conditions according to the types of target organisms, tissues, and cells within an effective range described later.

Surfactants that can be used in the present invention include: 1 polyoxysorbitan fatty acid monoester (Tween surfactant), 2 polyoxyethylene pt-octylphenyl ether (Triton surfactant and NP-40) 3, cholate, 4N-acyl N-methyl glycine and salts thereof (sarcosinates), and 5 octyl glycoside.
In order to destroy a tissue or a cell and maintain the reactivity of a substance existing in the tissue or the cell, the type of surfactant used and its concentration are extremely important. The type and concentration of the surfactant to be used will be described in detail in the following examples.

After the operation of discharging the contents of the tissue or the cell is performed, the operation of drying the tissue or the cell is performed. The drying operation includes adding a drying solution to a tissue or a cell, followed by vacuum drying, natural drying, and air drying.

It is preferable to add a protein and sugar to the drying solution, and it is preferable that the solution does not substantially contain a salt.

The protein in the drying solution plays a role not only to improve the preservability of cells and the like in a dry state, but also to reduce non-specific reactions during the assay. As used herein, “protein” includes peptides, cofactors,
Complex proteins to which coenzymes, sugar chains and the like are bound are also included.
Preferred proteins to be added to the drying solution are animal serum proteins, especially bovine serum albumin (hereinafter BS).
A), gelatin and its hydrolyzate. The most preferred protein is 0.05-0.5% by weight, especially about 0.2% by weight.
BSA.

The sugars to be added to the drying solution include:
Monosaccharides such as glucose and lactose; disaccharides such as saccharose and maltose; and polysaccharides are included. Particularly preferred sugars are glucose, lactose, saccharose, trehalose, maltose, fructose. Most preferred is 3-10% by weight, especially 5% by weight saccharose.

The dried solid-phased tissue or cell prepared by the above-mentioned method is most suitable for an agglutination immunoassay using magnetically encapsulated sensitized particles as disclosed in Japanese Patent Application Laid-Open No. H2-124464. It can also be used for various analysis methods (including biochemical analysis and genetic analysis) other than agglutination immunoassays such as the MPHA method, EIA method, RIA method, and fluorescence-labeled antibody method. When used in a method other than the agglutination analysis assay, the type of various additives and the conditions such as the concentration may be optimized within the effective range described later based on the analysis results.

Now, the present invention will be described in further detail with reference to Examples.

Example 1 This example describes the preparation of dried solid-phased red blood cells by the method of the present invention.

1. Preparation of microplate for immobilizing erythrocytes A 50 μL / well solution of WGA dissolved at 0.01 μM PBS pH = 7.0 dissolved at 10 μg / mL in a U-bottom plate (Maxisorp) of Nvnc. After incubation at room temperature for 30 minutes, the plate was washed 5 times with PBS.

2. Immobilization of erythrocytes O-type erythrocytes for screening with known antigenicity (Ortho Selectogen) are diluted with PBS so that the hemocyte concentration becomes about 0.7%, and 25 μL / well of the diluted erythrocytes is dispensed. Ten
After standing at room temperature for 3 minutes, the plate was washed three times with physiological saline, and the erythrocytes were solid-phased in a monolayer on the bottom of the microplate well.

3. Hemolysis and drying of erythrocytes In the well of the immobilized erythrocytes obtained in Step 2, 0.0125
Physiological saline solution containing 100% Triton X-100 was dispensed at 100 μL / well, and allowed to stand for 10 to 15 minutes to perform hemolysis. After hemolysis, the cells are washed twice with physiological saline to remove the surfactant and the lysate. Thereafter, 100 μL / well of a 5% sucrose aqueous solution containing 0.2% BSA is dispensed as a drying solution, and the mixture is incubated for 30 minutes. Thereafter, it was drained and dried under reduced pressure at room temperature using a vacuum dryer.

4. Detection of Antibody 75 μL / well of LISS (low ionic strength medium) was dispensed to the dried red blood cell plate prepared in Step 3. As a positive sample, antiserum containing each anti-red blood cell antibody (anti-D, K,
E, Fya, Jka, c, Jra sera) diluted twice with negative serum, and 25 μg each of negative serum as negative samples
Dispense L / well, incubate at 37 ° C for 10 minutes,
Washed 6 times with saline. After washing, the anti-human IgG antibody-sensitized magnetic substance-encapsulated sensation reconstituted with the specified particle reconstitution solution (0.02 M phosphoric acid-0.01 M citrate isotonic buffer pH 6.8 containing 0.4% gelatin and 0.05% rabbit serum) The resulting particles (Olympus Optical Co., Ltd.) were dispensed, and a pattern was formed on a magnet for 3 minutes (DRY-M-MPHA).

As a control, using the same erythrocytes, Anti-R
Red blood cell antibodies were assayed by the BC-Olivio-MPHA method (M-MP
HA).

The results are shown in Table 1.

[0030]

[Table 1]

As is clear from Table 1, all the tested antibodies achieved the same or higher reactivity as the Anti-RBC-Olivio-MPHA method.

[Embodiment 2] In this embodiment, Triton X-10 is used.
The change of each antigen when various cells were hemolyzed using 0 was examined over time, and the procedure was performed in the same manner as in Example 1 except that the hemolysis time was changed.

Table 2 shows the results.

[0034]

[Table 2]

As is clear from the table, it was found that the hemolysis time affected the reaction results. As a result of examination using various hemolysis times, it was found that the detection sensitivity can be kept high if washing and the like are performed within 20 minutes, preferably 10 minutes from the time of hemolysis and the hemolysis treatment is terminated. The preferred hemolysis time for Triton X-100 has been found to be about 9-21 minutes, especially about 9-15 minutes.

Example 3 In this example, the effect of additives in the drying solution was examined.

As in Example 1, erythrocytes were immobilized on a microplate and lysed. Then, in the combinations shown in Table 3, sugar, BSA, and salt (NaCL) were added to the drying solution. Erythrocytes are dried using the drying solution, and anti-D
The reactivity with the antibody, the negative image and the BLK image were observed in the same manner as in Example 1.

[0038]

[Table 3]

As shown in Table 3, 5% saccharose-BSA gave the best results in both reactivity and negative image. However, even if the BSA concentration was constant, the reactivity decreased as NaCl was increased. With 0.9% NaCl-BSA in the absence of sugar, the negative image etc. were good, but the reactivity was reduced.

With only sugar (5% saccharose), the negative image is shaved or blurred.

That is, even if BSA is present, if a salt is contained in the drying solution, the reactivity is reduced, and the expected reaction titer cannot be achieved. Without BSA, the negative image would worsen.
It became clear that was necessary.

Example 4 In this example, erythrocytes were lysed with solutions containing various surfactants.

Erythrocytes were immobilized on a microplate in the same manner as in Example 1, and various concentrations of saline and the following surfactants: Triton X-100 (Sigma), NP-40 (Sigma), Tween 20 (Nacalai) Tesque), Sarcosinate LN (Nikko Chemicals),
A physiological saline solution containing sodium cholate (Nacalai Tesque) and octylglycoside (Dojindo Chemical) was prepared, and hemolysis and drying operations were performed as described in Example 1.

75 μL / well of a dry erythrocyte plate LISS (low ionic strength medium) was dispensed, and 25 μL / well of anti-erythrocyte antibody-positive specimens obtained by doubling the dilution of anti-D serum with negative serum and negative serum were added. The plate was incubated at 37 ° C. for 10 minutes and washed six times with physiological saline. After washing, anti-human IgG antibody-sensitized magnetic material-encapsulated sensitized particles (manufactured by Olympus Optical Co., Ltd.) reconstituted with a specified particle reconstituting solution were dispensed, and a pattern was formed on a magnet for 3 minutes.

Table 4 shows the results.

[0046]

[Table 4]

As shown in Table 4, the surfactant which can lyse erythrocytes (*) and gives a negative pattern (○) and the concentration of the surfactant are more than 0.25% by weight and 1% by weight.
Octyl glycosides of less than 0.125% by weight and less than 1% by weight of sodium cholate, more than 0.0063% by weight and less than 0.025% by weight of N
P-40 was Triton X-100 exceeding 0.0063% by weight and less than 0.025% by weight. NaCl solutions and other surfactants did not produce negative patterns when they reached hemolytic concentrations. However, Tween 20 did not show hemolysis for 10-15 minutes.
If left for a long time (1 hour), hemolysis is observed at 0.1% by weight or more,
And a negative pattern was obtained.

According to the present example, in order to obtain a good negative pattern in the dried solid-phased erythrocytes subjected to the hemolysis treatment, hemolysis was carried out using an isotonic solution containing an appropriate type of surfactant at an appropriate concentration. Has been shown to be extremely important.

Example 5 In this example, the preservability of dried solid-phased red blood cells at room temperature was examined.

In the same manner as in Example 1, a dried erythrocyte plate was prepared, stored at room temperature (25 to 30 ° C.), and the change in reactivity for 1 to 13 months was examined.

LISS was added to the prepared erythrocyte drying plate at 75 μL.
L / well dispensed, anti-D,
C, c, E, Fy ^a, those that have been byebye diluted Jr ^a serum with 2% BSA / PBS and negative serum was dispensed 25 [mu] L / well min, and incubated 37 ° C. 10 minutes, 6 times with saline wash did. After washing, anti-human IgG antibody-sensitized magnetic material-encapsulated sensitized particles (manufactured by Olympus Optical Co., Ltd.) reconstituted with a specified particle reconstituting solution were dispensed, and a pattern was formed on a magnet for 3 minutes. Table 5 shows the results.

[0052]

[Table 5]

As is clear from Table 5, the room temperature (25 to 30)
C) for 13 months, the reactivity with various antibodies hardly changed.

This example demonstrates that dried solid phase erythrocytes prepared by the method of the present invention can withstand long-term storage at room temperature.

[0055]

According to the method of the present invention, before drying a solid-phased tissue or cell, the contents of the tissue or cell are gently discharged with an isotonic solution containing a surfactant. By doing so, it becomes possible to prepare a dried solid-phased tissue or cell that can be stored for a long time at room temperature.

In the tissue or cells prepared as described above,
Since a substance such as an antigen is not denatured, an accurate assay can be performed.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/543 501 G01N 1/28 J F term (Reference) 2G045 AA24 BA14 BB02 BB22 BB29 BB32 BB49 CA01 CB01 FB02 FB03 4B033 NA16 NB25 NB33 NC04 NC18 ND05 NF02 NF05

Claims (9)

[Claims] The method comprises the steps of eluting the contents of a tissue or cells immobilized on a carrier, and performing a drying operation after adding a drying solution to the tissues or cells from which the contents have been eluted. In the method for drying a solid phase of a tissue or a cell, the step of eluting the contents of the tissue or the cell immobilized on the carrier is performed by contacting a liquid containing isotonic or more containing a surfactant. Features method. 2. The method according to claim 1, wherein the isotonic or more liquid containing the surfactant comprises: (1) a fatty acid having 12 to 18 carbon atoms. A certain polyoxysorbitan fatty acid monoester and a mixture thereof; (2) a polyoxyethylene pt-octylphenyl ether having 4 to 40 polyoxyethylene groups and a mixture thereof; (3) a cholate; A method comprising a physiological saline solution containing a surfactant selected from the group consisting of N-acyl N-methylglycine and salts thereof, and (5) octyl glycoside. 3. The method according to claim 2, wherein the physiological saline containing the surfactant comprises: (1) 0.1% by weight or more of polyoxysorbitan monolaurate. (2) more than 0.25% by weight and less than 1% by weight of octyl glycoside; (3) more than 0.125% by weight and less than 1% by weight of sodium cholate; (4) more than 0.0063% by weight and 0.025% by weight of polyoxyethylene pt-octylphenyl Surface activity selected from ether (number of polyoxyethylene groups = 9), or (5) polyoxyethylene pt-octylphenyl ether (number of polyoxyethylene groups = 9, 10) of more than 0.0063% by weight and 0.025% by weight A physiological saline solution containing the agent. 4. The method according to claim 1, wherein the drying solution contains a protein and a sugar. 5. The method according to claim 4, wherein the dry solution is substantially free of salt. 6. The method according to claim 1, wherein the drying operation includes vacuum drying, natural drying,
The method of claim 1, wherein the method is air-drying. 7. The method according to claim 1, wherein the carrier is a support. 8. The drying of a tissue or a cell according to claim 1, wherein the tissue or the cell is made into a carrier by a binding agent selected from the group consisting of a lectin, an antibody, and a positively charged polymer. Solid phase method. 9. The lectin is a wheat malt lectin,
The method of claim 8, wherein the method is lysine or lentil lectin.