JP2001356127A - Dipping solution facilitating uniform suspension - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a dipping solution capable of easily uniformly suspending a ligand-immobilized fine particle carrier. SOLUTION: This dipping solution for ligand-immobilized fine particle carrier is characterized by having a specific gravity equal to or higher than the specific gravity of the ligand-immobilized fine particle carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immersion for easily dispersing a fine particle carrier on which a substance (ligand) having a biological affinity such as an antibody, a receptor or a nucleic acid probe is immobilized. Liquid.

[0002]

2. Description of the Related Art A ligand-immobilized fine particle carrier is a fine particle on which a substance having biological affinity is immobilized. For example, known are those in which an antigen or an antibody used in a heterogeneous immunoassay is physically or chemically bound to an insoluble fine particle solid phase carrier, or a solid phase carrier bound to a nucleic acid probe used in a nucleic acid hybridization assay. Have been.

[0003] In general, heterogeneous immunoassays and hybridization assays are quantitative measurements and the like, and the amount of such carriers used must be strictly controlled to be constant per assay. When supplying these ligand-immobilized fine particle carriers in a solution immersion state, a method of dispensing a constant weight of the carrier is to uniformly suspend a solution containing the ligand-immobilized fine particle carriers, Is performed by dispensing. At this time, it is an indispensable requirement that the carrier be uniformly suspended in order to guarantee a constant weight. Therefore, when actually used, a method of creating a uniform suspension state by a mechanical method such as stirring by a stirrer that rotates or vibrates a storage container before use or a stirring rod placed in a liquid and stirred is generally used. is there.

[0004]

However, since the specific gravity of the solution for suspending the fine particle carrier, which is usually used, is smaller than the specific gravity of the carrier, when the mechanical stirring is stopped, the fine particle carrier rapidly precipitates. Therefore, the uniformity of the carrier cannot be guaranteed. Another problem is that once the ligand-immobilized fine particle carrier precipitates, it takes a long time to achieve a uniform suspension state even with the mechanical stirring described above. Furthermore, if the stirring is carried out violently in order to pursue uniformity, the carriers may collide with each other or with the stirrer, causing physical damage to the carriers and denaturation of ligands such as antibodies and receptors immobilized on the surface of the carriers. There is also a danger of being alive.

Accordingly, an object of the present invention is to provide an immersion liquid which enables uniform suspension of the fine particle carrier by gentle physical stirring without vigorous physical stirring, and which does not adversely affect the ligand activity. Is to do.

[0006]

Means for Solving the Problems According to the first aspect of the present invention, which has been made to achieve the above object, the specific gravity is equal to or higher than the specific gravity of the ligand-immobilized fine particle carrier. Immersion liquid for the ligand-immobilized fine particle carrier. The invention according to claim 2 of the present application is directed to the invention according to claim 1, wherein the specific gravity of the permeate is adjusted by adding a salt. The invention according to claim 3 of the present application relates to the invention according to claim 2, wherein the salt is a cesium salt. Hereinafter, the present invention will be described in detail.

[0007] The present invention is an immersion liquid having a composition such that the ligand-immobilized fine particle carrier does not precipitate, thereby facilitating uniform suspension of the fine particle carrier by gentle physical stirring. The above-mentioned problem has been solved by making it equal to or larger than. The specific gravity of the permeate can be quickly and easily adjusted to an arbitrary value by adding a salt. Although the specific gravity can be adjusted by adding a salt as described above, it is preferable to use a cesium salt which has little risk of impairing the biological activity of the ligand. As the cesium salt, for example, CsBr (bromine salt), CsCl (chlorine salt), CsI (iodine salt), Cs ₂
Although SO ₄ (sulfate) can be exemplified, CsCl (chlorine salt) is particularly preferred from the viewpoint of solubility.

Hereinafter, the ligand-immobilized fine particle carrier to which the present invention is applied will be described with reference to an example. Ligand-immobilized microparticle carriers in heterogeneous immunoassays and hybridization assays are generally mixed with a sample that is assumed to contain a substance that has affinity for the ligand, and then separated from those that did not bind to the ligand. , Through a so-called B / F separation step, used for quantification and detection of a substance having an affinity for a ligand. Accordingly, the carrier suspension is separated into a carrier and a solution by a treatment such as filtration or magnetic collection, and preferably in such a form that the separation can be completed in a short time. For example, in the case of filtration, a spherical carrier that has a uniform particle size and the like and does not easily cause clogging can be exemplified. Examples of a method that does not use filtration include magnetic collection using a carrier containing a magnetic substance.

As the material of the carrier, various materials such as an organic polymer or an inorganic polymer, or a composite thereof can be exemplified, and the specific gravity is 1.9 g / cm ³ or less at 25 ° C., preferably 1.5 g / cm ^3. The present invention can be applied to those having a size of cm ³ or less. If the specific gravity is higher than this, it is difficult to adjust the specific gravity of the permeated liquid, and even if mechanical stirring is performed, the liquid immediately sediments and it becomes difficult to maintain uniformity.

The size of the carrier is not particularly limited. However, if the particle size of the spherical carrier is in the range of 0.1 μm to several tens of micrometers, particularly preferably 0.5 to 5.0 μm, the immersion liquid of the present invention can be used. It can easily be homogeneously suspended.

[0011] The pH of the carrier suspension is not particularly limited, either, but it does not affect the activity of the ligand.
In general, the pH is preferably in the range of pH 6 to pH 8. The immersion liquid of the present invention can be prepared with a buffer solution in an arbitrary pH range as appropriate. Examples of a buffer suitable for adjusting the immersion liquid of the present invention to the general pH range include aminoethanesulfonic acid or aminopropanesulfonic acid-based buffer, phosphate buffer, Tris
Buffer and the like.

In particular, when suspending a fine particle carrier on which an antibody is immobilized, a protein such as bovine serum albumin is added in an amount of 0.1% to several%.
It is common practice to include the protein in a certain degree, but the immersion liquid of the present invention may contain such a protein. In particular, when a fine particle carrier on which nucleic acids and the like are immobilized is suspended, it is basically the same as when a fine particle carrier on which antibodies and the like are immobilized is suspended. ED
It is preferable to add a chelating agent such as TA to a few mM.

[0013]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be further described by way of examples, but the present invention is not limited thereto.

Example 1 Preparation of Immunoreagent with Immobilized Antibody Commercially available streptavidin magnetic fine particles (Norway DY
Product name Dynabeads M-280 manufactured by NAL
streptavidin) (specific gravity 1.3 g / cm ³ ,
The average particle size is 2.8 μm) in 0.1 M phosphate buffer (pH
7.4) The liquid was replaced and washed three times to prepare a suspension having a concentration of 10 mg / ml. To 1 ml of this suspension, 46 μl of a biotin-labeled Fab′-conjugated antibody solution (concentration 2.2 mg / m 2)
After l) was added, the mixture was slowly stirred at room temperature for 1 hour. Subsequently, it was replaced and washed four times with a 0.1 M phosphate buffer (pH 7.4) solution to prepare a 10 mg / ml concentration antibody-immobilized magnetic particle suspension.

Cesium chloride was added to a 0.1 M phosphate buffer (pH 7.4) to prepare an immersion liquid having a specific gravity of 1.4 g / cm ³ . The procedure of collecting the antibody-immobilized magnetic fine particles prepared as described above with a magnet, removing the solution portion by suction, and resuspending in the immersion liquid was repeated several times, and replacing the antibody solid phase with the cesium-containing immersion liquid. Magnetic fine particle suspension (10mg
/ Ml). The suspension of the magnetic particles having the antibody immobilized thereon was placed in a test tube and allowed to stand at room temperature for 1 hour. For comparison, the antibody-immobilized magnetic microparticles prepared as described above were simultaneously added to a 0.1 M phosphate buffer (pH 7.4) solution.
The mixture was mixed at a rate of 0 mg / ml, placed in a test tube, and left at room temperature for 1 hour.

FIG. 1A shows the state after standing for one hour. On the right is the antibody-immobilized magnetic microparticles suspended in the immersion liquid of the present invention, and on the left is a 0.1 M phosphate buffer (pH 7.0) for comparison.
Fig. 4 shows a state of the antibody-immobilized magnetic fine particles suspended in 4). In the immersion liquid of the present invention, the antibody-immobilized magnetic fine particles floated on the liquid surface, and the phosphate buffer for comparison settled at the bottom of the tube.

FIG. 1 (2) shows a state after the two tubes are lightly hit once with a finger. In the immersion liquid of the present invention, the suspended antibody-immobilized magnetic fine particles began to be dispersed in the immersion liquid, but no change was observed in the phosphate buffer for comparison.

Next, FIG. 1 (3) shows a state after the two tubes are further lightly hit with a finger four times. In the immersion liquid of the present invention, the antibody-immobilized magnetic fine particles which were suspended were dispersed in the liquid, but no change was observed in the phosphate buffer for comparison.

As described above, the immersion liquid of the present invention is 0.1 M
It was confirmed that the antibody-immobilized magnetic fine particles could be more easily and uniformly suspended as compared with the phosphate buffer (pH 7.4) solution.

Example 2 The immersion liquid prepared in Example 1 and the antibody-immobilized magnetic fine particles were mixed at a ratio of 10 mg / ml, placed in a test tube, and allowed to stand at 4 ° C. overnight (hereinafter referred to as CsCl reagent). Described). For comparison, a 0.1 M phosphate buffer (p
H7.4) was mixed with the antibody-immobilized magnetic microparticles prepared in Example 1 at a rate of 10 mg / ml, placed in a test tube and allowed to stand at 4 ° C. overnight (hereinafter referred to as PB reagent).

After stirring the CsCl reagent and the PB reagent,
Reaction vessels (manufactured by Nunc, trade name: Nunc F16 BLACK MAXSORP F) which were each subjected to a blocking treatment with a solution containing 0.5% BSA, 50 mM Tris HCl, and 0.05% NaN ₃ (pH 7.0).
LUORONUNC), and 50 μl of an alkaline phosphatase anti-TSH Fab′-conjugated antibody conjugate solution (2 mA at an absorption value of 280 nm) was added thereto.
TSH standard sample solution (concentration 0 μIU / ml or 10 μIU
/ Ml) was dispensed and stirred at 37 ° C for 20 minutes to react.

A magnet is applied to the reaction vessel after the reaction to fix the magnetic fine particles to the inner wall of the vessel, and the residual liquid in the vessel is sucked out and washed with a washing solution (10 mM Tris buffer, 150 mM Na).
Cl, 1 mM MgCl ₂ , 0.05% Tween 20
And 0.0005% NaN ₃ ; pH 8.0), and the liquid in the container was removed four times.

Then, 50 μl of a luminescent substrate (Lumi-Phos 530, manufactured by Lumigen, USA) was dispensed and stirred, and then a luminescence measuring instrument (EG & G BE) at 37 ° C. was used.
(LB 96V, manufactured by R THOLD Co., Ltd.), and the luminescence was measured.

0 μIU of TSH of CsCl reagent and PB reagent
FIG. 2 shows the results of measurement using TSH1 / ml as a sample.
FIG. 3 shows the results of measurement using 0 μIU / ml as a sample. From these results, the CsCl reagent was found to be PB
It was shown to have the same reactivity as the reagent, indicating that addition of the cesium salt had no effect on the reactivity.

[0025]

According to the present invention, the suspension of the immobilized immunoreagent can be improved, and the mechanism for stirring or rotating the stirring rod or the container can be simplified. That is, the present invention contributes to improvement in operability in various measurements using a fine particle carrier such as an immunoassay and a nucleic acid measurement.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of Example 1.

FIG. 2 shows TSH0 μIU among the results of Example 2.
FIG. 4 is a diagram showing the relationship between the time after dispensing a luminescent substrate and the amount of luminescence when using / ml as a sample.

FIG. 3 shows TSH10 μI among the results of Example 2.
It is a figure which shows the relationship between the time after dispensing the luminescent substrate when U / ml is used for a sample, and the amount of luminescence.

Claims (3)

[Claims] (1) The specific gravity is equal to or higher than the specific gravity of the ligand-immobilized fine particle carrier,
Ligand-immobilized fine particle carrier immersion liquid. 2. The immersion liquid according to claim 1, wherein the permeation liquid has been adjusted in specific gravity by adding a salt. 3. The permeate of claim 2, wherein said salt is a cesium salt.